AJP: Cell Physiology current issue

Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

Zhang, M., Maddala, R., Rao, P. V. — 2008-11-13

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells.

Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways

Jing, M., Cheruvu, V. K, Ismail-Beigi, F. — 2008-11-13

AMP-activated protein kinase (AMPK) plays a critical role in the stimulation of glucose transport in response to hypoxia and inhibition of oxidative phosphorylation. In the present study, we examined the signaling pathway(s) mediating the glucose transport response following activation of AMPK. Using mouse fibroblasts of AMPK wild type and AMPK knockout, we documented that the expression of AMPK is essential for the glucose transport response to both azide and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). In Clone 9 cells, the stimulation of glucose transport by a combination of azide and AICAR was not additive, whereas there was an additive increase in the abundance of phosphorylated AMPK (p-AMPK). In Clone 9 cells, AMPK wild-type fibroblasts, and H9c2 heart cells, azide or hypoxia selectively increased p-ERK1/2, whereas, in contrast, AICAR selectively stimulated p-p38; phosphorylation of JNK was unaffected. Azide's effect on p-ERK1/2 abundance and glucose transport in Clone 9 cells was partially abolished by the MEK1/2 inhibitor U0126. SB 203580, an inhibitor of p38, prevented the phosphorylation of p38 and the glucose transport response to AICAR and, unexpectedly, to azide. Hypoxia, azide, and AICAR all led to increased phosphorylation of Akt substrate of 160 kDa (AS160) in Clone 9 cells. Employing small interference RNA directed against AS160 did not inhibit the glucose transport response to azide or AICAR, whereas the content of P-AS160 was reduced by ~80%. Finally, we found no evidence for coimmunoprecipitation of Glut1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway.

Electron probe X-ray microanalysis of intact pathway for human aqueous humor outflow

2008-11-13

Intraocular pressure (IOP) is regulated by the resistance to outflow of the eye's aqueous humor. Elevated resistance raises IOP and can cause glaucoma. Despite the importance of outflow resistance, its site and regulation are unclear. The small size, complex geometry, and relative inaccessibility of the outflow pathway have limited study to whole animal, whole eye, or anterior-segment preparations, or isolated cells. We now report measuring elemental contents of the heterogeneous cell types within the intact human trabecular outflow pathway using electron-probe X-ray microanalysis. Baseline contents of Na⁺, K⁺, Cl^–, and P and volume (monitored as Na+K contents) were comparable to those of epithelial cells previously studied. Elemental contents and volume were altered by ouabain to block Na⁺-K⁺-activated ATPase and by hypotonicity to trigger a regulatory volume decrease (RVD). Previous results with isolated trabecular meshwork (TM) cells had disagreed whether TM cells express an RVD. In the intact tissue, we found that all cells, including TM cells, displayed a regulatory solute release consistent with an RVD. Selective agonists of A₁ and A₂ adenosine receptors (ARs), which exert opposite effects on IOP, produced similar effects on juxtacanalicular (JCT) cells, previously inaccessible to functional study, but not on Schlemm's canal cells that adjoin the JCT. The results obtained with hypotonicity and AR agonists indicate the potential of this approach to dissect physiological mechanisms in an area that is extremely difficult to study functionally and demonstrate the utility of electron microprobe analysis in studying the cellular physiology of the human trabecular outflow pathway in situ.

Early mechanical dysfunction of the diaphragm in the muscular dystrophy with myositis (Ttnmdm) model

Lopez, M. A., Pardo, P. S., Cox, G. A., Boriek, A. M. — 2008-11-13

A complex rearrangement mutation in the mouse titin gene leads to an in-frame 83-amino acid deletion in the N2A region of titin. Autosomal recessive inheritance of the titin muscular dystrophy with myositis (Ttn^mdm/mdm) mutation leads to a severe early-onset muscular dystrophy and premature death. We hypothesized that the N2A deletion would negatively impact the force-generating capacity and passive mechanical properties of the mdm diaphragm. We measured in vitro active isometric contractile and passive length-tension properties to assess muscle function at 2 and 6 wk of age. Micro-CT, myosin heavy chain Western blotting, and histology were used to assess diaphragm structure. Marked chest wall distortions began at 2 wk and progressively worsened until 5 wk. The percentage of myofibers with centrally located nuclei in mdm mice was significantly (P < 0.01) increased at 2 and 6 wk by 4% and 17%, respectively, compared with controls. At 6 wk, mdm diaphragm twitch stress was significantly (P < 0.01) reduced by 71%, time to peak twitch was significantly (P < 0.05) reduced by 52%, and half-relaxation time was significantly (P < 0.05) reduced by 57%. Isometric tetanic stress was significantly (P < 0.05) depressed in 2- and 6-wk mdm diaphragms by as much as 64%. Length-tension relationships of the 2- and 6-wk mdm diaphragms showed significantly (P < 0.05) decreased extensibility and increased stiffness. Slow myosin heavy chain expression was aberrantly favored in the mdm diaphragm at 6 wk. Our data strongly support early contractile and passive mechanical aberrations of the respiratory pump in mdm mice.

Connexin 37 profoundly slows cell cycle progression in rat insulinoma cells

Burt, J. M., Nelson, T. K., Simon, A. M., Fang, J. S. — 2008-11-13

In addition to providing a pathway for intercellular communication, the gap junction-forming proteins, connexins, can serve a growth-suppressive function that is both connexin and cell-type specific. To assess its potential growth-suppressive function, we stably introduced connexin 37 (Cx37) into connexin-deficient, tumorigenic rat insulinoma (Rin) cells under the control of an inducible promoter. Proliferation of these iRin37 cells, when induced to express Cx37, was profoundly slowed: cell cycle time increased from 2 to 9 days. Proliferation and cell cycle time of Rin cells expressing Cx40 or Cx43 did not differ from Cx-deficient Rin cells. Cx37 suppressed Rin cell proliferation irrespective of cell density at the time of induced expression and without causing apoptosis. All phases of the cell cycle were prolonged by Cx37 expression, and progression through the G₁/S checkpoint was delayed, resulting in accumulation of cells at this point. Serum deprivation augmented the effect of Cx37 to accumulate cells in late G₁. Cx43 expression also affected cell cycle progression of Rin cells, but its effects were opposite to Cx37, with decreases in G₁ and increases in S-phase cells. These effects of Cx43 were also augmented by serum deprivation. Cx-deficient Rin cells were unaffected by serum deprivation. Our results indicate that Cx37 expression suppresses cell proliferation by significantly increasing cell cycle time by extending all phases of the cell cycle and accumulating cells at the G₁/S checkpoint.

Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

Kruchten, A. E., Krueger, E. W., Wang, Y., McNiven, M. A. — 2008-11-13

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

MDA-MB-231 produces ATP-mediated ICAM-1-dependent facilitation of the attachment of carcinoma cells to human lymphatic endothelial cells

Kawai, Y., Kaidoh, M., Ohhashi, T. — 2008-11-13

We examined the effects of supernatants of culture media of MDA-MB-231 and MCF-7 cells on the expression of adhesion molecules on human lymphatic endothelial cells (LECs) and evaluated whether the overexpression of adhesion molecules facilitated the attachment of carcinoma cells to LECs. The 48-h stimulation of MDA-MB-231, but not MCF-7, supernatant produced a significant expression of ICAM-1 on human LECs but little or no expression of E-selectin. Chemical treatment with dialyzed substances of <1,000 molecular weight (MW) caused a complete reduction of the supernatant-mediated response. In contrast, pretreatment with heating, digestion with protease, or chemical treatment with dialyzed substances of <500 MW produced no significant effect on the supernatant-mediated response. ATP (10^–7 M) caused overexpression of ICAM-1 on human LECs similar to that produced by the supernatant of MDA-MB-231. The ATP- and MDA-MB-231 supernatant-mediated responses were significantly reduced by treatment with 10^–6 M suramin (a purinergic P2X and P2Y receptor antagonist). In attachment assays, 10^–7 M ATP or MDA-MB-231 supernatant produced a significant increase in the attachment of carcinoma cells to human LECs. The treatment with 10^–6 M suramin caused a significant reduction of ATP- and supernatant-mediated facilitation of the attachment responses. Additional treatment with anti-ICAM-1 antibody also caused a significant reduction of ATP- and supernatant-mediated facilitation of the attachment responses. The experimental findings suggest that MDA-MB-231 may release or leak ATP, which produces the overexpression of ICAM-1 on human LECs through activation of purinergic P2X and/or P2Y receptors and then facilitates ICAM-1-mediated attachment of carcinoma cells to LECs.

How strict is the correlation between STIM1 and Orai1 expression, puncta formation, and ICRAC activation?

Gwozdz, T., Dutko-Gwozdz, J., Zarayskiy, V., Peter, K., Bolotina, V. M. — 2008-11-13

Stromal interaction molecule 1 (STIM1) and Orai1 have been identified as crucial elements of the store-operated Ca²⁺ entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIM1 and Orai1 as the most straightforward mechanism for signal transduction from the stores to the plasma membrane. To test some of the predictions of a conformational coupling model, we assessed how tight the relationships are between STIM1 and Orai1 expression, puncta formation, and SOCE activation. Here we present evidence that STIM1 accumulates in puncta equally well in the presence or absence of Orai1 expression, that STIM1 accumulation is not sufficient for Orai1 accumulation in the same areas, and that normal Ca²⁺ release-activated Ca²⁺ current (I_CRAC) can be activated in STIM1-deficient cells. These data challenge the idea of direct conformational coupling between STIM1 and Orai1 as a viable mechanism of puncta formation and SOCE activation and uncover greater complexity in their relationship, which may require additional intermediate elements.

GATA-6 mediates transcriptional activation of aquaporin-5 through interactions with Sp1

2008-11-13

We investigated mechanisms underlying GATA-6-mediated transcriptional activation of the alveolar epithelial type I cell-enriched gene aquaporin-5 (AQP5). GATA-6 expression increases in alveolar epithelial cells in primary culture, concurrent with upregulation of AQP5 and transition to a type I cell-like phenotype. Cotransfections in MLE-15 and NIH 3T3 cells demonstrated trans-activation by GATA-6 of a rat 1,716-bp-AQP5-luciferase (–1716-AQP5-Luc) reporter. Electrophoretic mobility shift assay and chromatin immunoprecipitation identified an interaction between GATA-6 and putative binding sites in the AQP5 promoter. However, mutation of these sites did not reduce GATA-6-mediated activation, implicating mechanisms in addition to direct binding of GATA-6 to DNA. A 5'-deletion construct, –358-AQP5-Luc, that does not encompass GATA motifs was still activated by GATA-6 by as much as 50% relative to –1716-AQP5-Luc. Internal deletion of the –358/–173 GC-rich domain, which includes several putative Sp1 consensus sites, reduced trans-activation by ~60%, suggesting importance of this region for GATA-mediated activity. –358-AQP5-Luc was similarly activated by both GATA-6 and a GATA DNA-binding defective mutant, whereas cotransfections in Schneider S2 cells demonstrated dose-dependent trans-activation of –358-AQP5-Luc by Sp1. Activation of –358-AQP5-Luc by GATA-6 was dramatically reduced by Sp1 small-interfering RNA, and –358-AQP5-Luc was activated synergistically by GATA-6 and Sp1 in NIH 3T3 cells. Furthermore, association between endogenous GATA-6 and Sp1 was demonstrated by coimmunoprecipitation. These results suggest that transcriptional activation of AQP5 by GATA-6 is mediated at least in part through cooperative interactions with Sp1 occurring at the proximal promoter.

The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F

Gorges, L. L., Lents, N. H., Baldassare, J. J. — 2008-11-13

The retinoblastoma protein pRb plays a pivotal role in G₁- to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and COOH-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the NH₂-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using "reverse mutational analysis" of Rb^CDK (a dominantly active repressive allele of Rb), we have previously shown that restoration of Thr-373 is sufficient to render Rb^CDK sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the NH₂-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we have expanded this analysis to include additional residues in the NH₂-terminal region of pRb and further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further have found that removal of the COOH-terminal domain of either RbCDK^+T373 or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Our data demonstrate a novel function for the NH₂-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.

Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA

Qiao, J., Holian, O., Lee, B.-S., Huang, F., Zhang, J., Lum, H. — 2008-11-13

The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDI showed two potential protein kinase A (PKA) phosphorylation motifs, Ser¹⁷⁴ and Thr¹⁸². Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDI-GST fusion protein and PKA motif-containing GDI peptide at Ser¹⁷⁴, but not Thr¹⁸². Transfection of COS-7 cells with mutated full-length GDI at Ser¹⁷⁴ to Ala¹⁷⁴ (GDI-Ser^174A) abrogated the ability of cAMP to phosphorylate GDI. However, mutation of Thr¹⁸² to Ala¹⁸² (GDI-Thr^182A) did not abrogate, and cAMP increased phosphorylation of GDI to a similar extent as wild-type GDI transfectants. The mutant GDI-Ser^174A, but not GDI-Thr^182A, was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDI-Ser^174A was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDI binding with RhoA, suggesting that phosphorylation of Ser¹⁷⁴ regulated preformed GDI-RhoA complexes. The findings provide strong support that the selective phosphorylation at Ser¹⁷⁴ by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury.

MAP kinase/phosphatase pathway mediates the regulation of ACE2 by angiotensin peptides

Gallagher, P. E., Ferrario, C. M., Tallant, E. A. — 2008-11-13

Angiotensin-converting enzyme 2 (ACE2) catalyzes the conversion of the vasoconstrictor angiotensin II (ANG II) to the vasodilatory peptide angiotensin-(1-7) [ANG-(1-7)]. We showed that treatment of hypertensive rats with the AT₁ receptor antagonist olmesartan increased ACE2 mRNA and protein in the thoracic aorta, suggesting that endogenous ANG II tonically reduces the enzyme. We now report that ANG II downregulates ACE2 activity and mRNA in rat aortic vascular smooth muscle cells (VSMCs) to reduce the conversion of ANG II to ANG-(1-7). Although ANG-(1-7) alone had no effect on the regulation of ACE2 mRNA, the heptapeptide prevented the ANG II-mediated reduction in ACE2 mRNA, an effect blocked by the selective ANG-(1-7) receptor antagonist [d-Ala⁷]-ANG-(1-7). The reduction in ACE2 mRNA by ANG II was also prevented by the mitogen-activated protein (MAP) kinase kinase inhibitor PD98059. Treatment of VSMCs with ANG II increased ERK1/ERK2 activity, which was significantly reduced by pretreatment with ANG-(1-7). Blockade of the ANG II-mediated reduction in ACE2 mRNA and increase in MAP kinase activity by ANG-(1-7) was prevented by pretreatment with sodium vanadate, a tyrosine phosphatase inhibitor, or okadaic acid, a serine-threonine phosphatase inhibitor, suggesting that the heptapeptide activates a MAP kinase phosphatase. This study is the first to show that the MAP kinase-phosphatase pathway is a primary molecular mechanism for regulating ACE2 to maintain the balance between ANG II and ANG-(1-7). The modulatory role of ANG-(1-7) in the regulation of ACE2 by ANG II suggests a complex interplay between the two peptides that is mediated by specific receptors to activate distinct signaling pathways.

Kruppel-like factor 4, Elk-1, and histone deacetylases cooperatively suppress smooth muscle cell differentiation markers in response to oxidized phospholipids

Yoshida, T., Gan, Q., Owens, G. K. — 2008-11-13

Phenotypic switching of vascular smooth muscle cells (SMCs), such as increased proliferation, enhanced migration, and downregulation of SMC differentiation marker genes, is known to play a key role in the development of atherosclerosis. However, the factors and mechanisms controlling this process are not fully understood. We recently showed that oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), which accumulate in atherosclerotic lesions, are potent repressors of expression of SMC differentiation marker genes in cultured SMCs as well as in rat carotid arteries in vivo. Here, we examined the molecular mechanisms whereby POVPC induces suppression of SMC differentiation marker genes in cultured SMCs. Results showed that POVPC induced phosphorylation of ERK1/2 and Elk-1. The MEK inhibitors U-0126 and PD-98059 attenuated POVPC-induced suppression of smooth muscle (SM) -actin and SM-myosin heavy chain. POVPC also induced expression of Krüppel-like factor 4 (Klf4). Chromatin immunoprecipitation assays revealed that POVPC caused simultaneous binding of Elk-1 and Klf4 to the promoter region of the SM -actin gene. Moreover, coimmunoprecipitation assays showed a physical interaction between Elk-1 and Klf4. Results in Klf4-null SMCs showed that blockade of both Klf4 induction and Elk-1 phosphorylation completely abolished POVPC-induced suppression of SMC differentiation marker genes. POVPC-induced suppression of SMC differentiation marker genes was also accompanied by hypoacetylation of histone H4 at the SM -actin promoter, which was mediated by the recruitment of histone deacetylases (HDACs), HDAC2 and HDAC5. Coimmunoprecipitation assays showed that Klf4 interacted with HDAC5. Results provide evidence that Klf4, Elk-1, and HDACs coordinately mediate POVPC-induced suppression of SMC differentiation marker genes.

Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells

2008-11-13

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC). In isolated porcine pulmonary artery segments (PAS), UA (7.5 mg/dl) inhibits acetylcholine-induced vasodilation. The incubation of PAEC with UA caused a dose-dependent decrease in NO and cGMP production stimulated by bradykinin or Ca²⁺-ionophore A23187. We explored cellular mechanisms by which UA might cause reduced NO production focusing on the effects of UA on the l-arginine-endothelial NO synthase (eNOS) and l-arginine-arginase pathways. Incubation of PAEC with different concentrations of UA (2.5–15 mg/dl) for 24 h did not affect l-[³H]arginine uptake or activity/expression of eNOS. However, PAEC incubated with UA (7.5 mg/dl; 24 h) released more urea in culture media than control PAEC, suggesting that arginase activation might be involved in the UA effect. Kinetic analysis of arginase activity in PAEC lysates and rat liver and kidney homogenates demonstrated that UA activated arginase by increasing its affinity for l-arginine. An inhibitor of arginase (S)-(2-boronoethyl)-l-cysteine prevented UA-induced reduction of A23187-stimulated cGMP production by PAEC and abolished UA-induced inhibition of acetylcholine-stimulated vasodilation in PAS. We conclude that UA-induced arginase activation is a potential mechanism for reduction of NO production in PAEC.

Proinflammatory cytokines tumor necrosis factor-{alpha} and interferon-{gamma} alter tight junction structure and function in the rat parotid gland Par-C10 cell line

2008-11-13

Sjögren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor- (TNF-) and interferon- (IFN-) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF- and IFN- decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I_sc) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y₂ nucleotide receptor agonist. In contrast, TNF- and IFN- had no effect on agonist-induced increases in the intracellular calcium concentration [Ca²⁺]_i in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF- and IFN- increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-, but not IFN-, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN- causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.

Ankyrin facilitates intracellular trafficking of {alpha}1-Na+-K+-ATPase in polarized cells

Stabach, P. R., Devarajan, P., Stankewich, M. C., Bannykh, S., Morrow, J. S. — 2008-11-13

Defects in ankyrin underlie many hereditary disorders involving the mislocalization of membrane proteins. Such phenotypes are usually attributed to ankyrin's role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We found in Madin-Darby canine kidney cells and in other cultured cells that the 25-residue ankyrin-binding sequence of ₁-Na⁺-K⁺-ATPase facilitates the entry of ₁,β₁-Na⁺-K⁺-ATPase into the secretory pathway and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrin dependency on the endoplasmic reticulum (ER) to Golgi trafficking of VSV-G. Expression of the ankyrin-binding sequence of ₁-Na⁺-K⁺-ATPase alone as a soluble cytosolic peptide acts in trans to selectively block ER to Golgi transport of both wild-type ₁-Na⁺-K⁺-ATPase and a VSV-G fusion protein that includes the ankyrin-binding sequence, whereas the trafficking of other proteins remains unaffected. Similar phenotypes are also generated by small hairpin RNA-mediated knockdown of ankyrin R or the depletion of ankyrin in semipermeabilized cells. These data indicate that the adapter protein ankyrin acts not only at the plasma membrane but also early in the secretory pathway to facilitate the intracellular trafficking of ₁-Na⁺-K⁺-ATPase and presumably other selected proteins. This novel ankyrin-dependent assembly pathway suggests a mechanism whereby hereditary disorders of ankyrin may be manifested as diseases of membrane protein ER retention or mislocalization.

Molecular profile of endothelial invasion of three-dimensional collagen matrices: insights into angiogenic sprout induction in wound healing

Su, S.-C., Mendoza, E. A., Kwak, H.-i., Bayless, K. J. — 2008-11-13

Sprouting angiogenesis is a multistep process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and sprout stabilization. Such complexity is consistent with a requirement for orchestration of individual gene expression alongside multiple signaling pathways. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to develop multicellular invading sprouts, we analyzed differential gene expression with time using a defined in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate, basic FGF, and VEGF. Gene expression changes were confirmed by real-time PCR and Western blot analyses. A cohort of cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated, whereas a set of genes associated with tight junctions were downregulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, a disintegrin and metalloproteinase with thrombospondin-type repeats-1 (ADAMTS1), decreased invasion responses, confirming a role for ADAMTS1 in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt and Notch pathways was observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. This study has enhanced the molecular road map of gene expression changes that occur during endothelial invasion and highlighted the utility of three-dimensional models to study EC morphogenesis.

Glucagon receptor recycling: role of carboxyl terminus, {beta}-arrestins, and cytoskeleton

Krilov, L., Nguyen, A., Miyazaki, T., Unson, C. G., Bouscarel, B. — 2008-11-13

Glucagon receptor (GR) activity and expression are altered in several diseases, including Type 2 diabetes. Previously, we investigated the mechanism of GR desensitization and internalization. The present study focused on the fate of internalized GR. Using both hamster hepatocytes and human embryonic kidney (HEK)-293 cells, we showed that internalized GR recycled to the plasma membrane within 30–60 min following stimulation of the cells with 100 nM glucagon. In HEK-293 cells and during recycling, GR colocalized with Rab4, Rab11, β-arrestin1, β-arrestin2, and actin filaments, in the cytosolic and/or perinuclear domains. Glucagon treatment triggered redistribution of actin filaments from the plasma membrane to the cytosol. GR coimmunoprecipitated with β-actin in both hepatocytes and HEK-293 cells. Downregulation of β-arrestin1 and β-arrestin2 or disruption of the cytoskeleton inhibited recycling, but not internalization of GR. Deletion of the GR carboxyl-terminal 70 amino acids abolished internalization of GR in response to glucagon while deletion of the last 40 amino acids only did not affect GR internalization and recycling. After exposure of the cells to either high concentrations or prolonged duration of glucagon, GR colocalized with lysosomes. GR degradation was inhibited by lysosomal, but not proteosomal, inhibitors. In conclusion, GR recycles through Rab4- and Rab11- positive vesicles. The actin cytoskeleton, β-arrestin1, β-arrestin2, and the receptor's carboxyl terminus are involved in recycling. Prolonged stimulation with glucagon targets GR for degradation in lysosomes. Therefore, the present study provides a better understanding of the GR recycling mechanism, which could become useful in the treatment of certain diseases, including diabetes.

Age-dependent FOXO regulation of p27Kip1 expression via a conserved binding motif in rat muscle precursor cells

Lees, S. J., Childs, T. E., Booth, F. W. — 2008-11-13

Previously, we have demonstrated that forkhead box O3a (FOXO3a) overexpression increased p27^Kip1 promoter activity and protein expression, whereas it decreased proliferation in muscle precursor cells (MPCs). The objectives of the present study were to 1) locate and identify FOXO regulatory elements in the rat p27^Kip1 promoter using deletion analysis of a promoter/reporter construct and 2) determine if age-related differences exist in FOXO-induced p27^Kip1 expression. The full-length (–4.0/+0.4 kb) rat p27^Kip1 promoter construct revealed that both FOXO1 and FOXO3a induced an increase in transcriptional activity. Interestingly, MPCs isolated from old animals exhibited an increased FOXO3a-induced p27^Kip1 promoter activity compared with MPCs isolated from young animals. Deletion of a 253-bp portion of the 5'-untranslated region (UTR) resulted in a significant decrease in FOXO-induced p27^Kip1 promoter expression. Site-specific mutation of a daf-16 family protein-binding element (DBE) within this 253-bp portion of the 5'-UTR also demonstrated a decrease in FOXO-induced p27^Kip1 promoter expression. These data suggest that a putative FOXO regulatory element located in the 5'-UTR of the rat p27^Kip1 gene plays a role in the age-dependent differences in FOXO3a-dependent p27^Kip1 promoter expression. These findings have implications for developing treatment strategies aimed at increasing the proliferation of MPCs and regenerative capacity of aged skeletal muscle.

Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria

Baggaley, E. M., Elliott, A. C., Bruce, J. I. E. — 2008-11-13

Impairment of the normal spatiotemporal pattern of intracellular Ca²⁺ ([Ca²⁺]_i) signaling, and in particular, the transition to an irreversible "Ca²⁺ overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca²⁺ overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H₂O₂) evokes a Ca²⁺ overload response and inhibition of plasma membrane Ca²⁺-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938–C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca²⁺]_i clearance assay in which mitochondrial Ca²⁺ uptake was blocked with Ru-360, H₂O₂ (50 µM–1 mM) markedly inhibited the PMCA activity. This H₂O₂-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H₂O₂-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca²⁺ handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca²⁺ overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.

Vasoconstrictive effect of hydrogen sulfide involves downregulation of cAMP in vascular smooth muscle cells

Lim, J. J., Liu, Y.-H., Khin, E. S. W., Bian, J.-S. — 2008-11-13

Hydrogen sulfide (H₂S), a new endogenous mediator, produces both vasorelaxation and vasoconstriction. This study was designed to examine whether cAMP mediates the vasoconstrictive effect of H₂S. We found that NaHS at a concentration range of 10–100 µM (yields ~3–30 µM H₂S) concentration-dependently reversed the vasodilation caused by isoprenaline and salbutamol, two β-adrenoceptor agonists, and forskolin, a selective adenylyl cyclase activator, in phenylephrine-precontracted rat aortic rings. Pretreatment with NaHS (10–100 µM) for 5 min also significantly attenuated the vasorelaxant effect of salbutamol and forskolin. More importantly, NaHS (5–100 µM) significantly reversed forskolin-induced cAMP accumulation in vascular smooth muscle cells. However, NaHS produced significant, but weaker, vasoconstriction in the presence of N^G-nitro-l-arginine methyl ester (100 µM), a nitric oxide synthase inhibitor, or in endothelium-denuded aortic rings. Blockade of ATP-sensitive potassium channels with glibenclamide (10 µM) failed to attenuate the vasoconstriction induced by H₂S. Taken together, we demonstrated for the first time that the vasoconstrictive effect of H₂S involves the adenyly cyclase/cAMP pathway.

In vitro neovasculogenic potential of resident adipose tissue precursors

Madonna, R., De Caterina, R. — 2008-11-13

Adipose tissue development is associated with neovascularization, which might be exploited therapeutically. We investigated the neovasculogenesis antigenic profile and kinetics in adipose tissue-derived stromal cells (ADSCs) to understand the potential of ADSCs to generate new vessels. Murine and human visceral adipose tissues were processed with collagenase to obtain ADSCs from the stromal vascular fraction. Freshly isolated murine and human ADSCs featured the expression of early markers of endothelial differentiation [uptake of DiI-labeled acetylated LDL, CD133, CD34, kinase insert domain receptor (KDR)], but not markers for more mature endothelial cells (CD31 and von Willebrand factor). In methylcellulose medium, multilocular cells positive for Oil Red O staining appeared after 6 days. After 10 days, clusters of ADSCs spontaneously formed branched tubelike structures, which were strongly positive for CD34 and CD31, while losing their ability to undergo adipocyte differentiation. In Matrigel, in the presence of endothelial growth factors ADSCs formed branched tubelike structures. By clonal assays in methylcellulose we also determined the frequency of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) colony-forming units from ADSCs, compared with bone marrow-derived stromal cells (BMSCs) used as a positive control. After 4–14 days, BMSCs formed 8 ± 3 BFU-E and 40 ± 10 CFU-GM, while ADSCs never produced colonies of myeloid progenitors. The developing adipose tissue has neovasculogenic potential, based on the recruitment of local rather than circulating progenitors. Adipose tissue might therefore be a viable autonomous source of cells for postnatal neovascularization.

Activation of Hsp90-eNOS and increased NO generation attenuate respiration of hypoxia-treated endothelial cells

Presley, T., Vedam, K., Velayutham, M., Zweier, J. L., Ilangovan, G. — 2008-11-13

Hypoxia induces various adoptive signaling in cells that can cause several physiological changes. In the present work, we have observed that exposure of bovine aortic endothelial cells (BAECs) to extreme hypoxia (1–5% O₂) attenuates cellular respiration by a mechanism involving heat shock protein 90 (Hsp90) and endothelial nitric oxide (NO) synthase (eNOS), so that the cells are conditioned to consume less oxygen and survive in prolonged hypoxic conditions. BAECs, exposed to 1% O₂, showed a reduced respiration compared with 21% O₂-maintained cells. Western blot analysis showed an increase in the association of Hsp90-eNOS and enhanced NO generation on hypoxia exposure, whereas there was no significant accumulation of hypoxia-inducible factor-1 (HIF-1). The addition of inhibitors of Hsp90, phosphatidylinositol 3-kinase, and NOS significantly alleviated this hypoxia-induced attenuation of respiration. Thus we conclude that hypoxia-induced excess NO and its derivatives such as ONOO^– cause inhibition of the electron transport chain and attenuate O₂ demand, leading to cell survival at extreme hypoxia. More importantly, such an attenuation is found to be independent of HIF-1, which is otherwise thought to be the key regulator of respiration in hypoxia-exposed cells, through a nonphosphorylative glycolytic pathway. The present mechanistic insight will be helpful to understand the difference in the magnitude of endothelial dysfunction.

Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

2008-11-13

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

Anisotropic diffusion of fluorescently labeled ATP in rat cardiomyocytes determined by raster image correlation spectroscopy

Vendelin, M., Birkedal, R. — 2008-11-13

A series of experimental data points to the existence of profound diffusion restrictions of ADP/ATP in rat cardiomyocytes. This assumption is required to explain the measurements of kinetics of respiration, sarcoplasmic reticulum loading with calcium, and kinetics of ATP-sensitive potassium channels. To be able to analyze and estimate the role of intracellular diffusion restrictions on bioenergetics, the intracellular diffusion coefficients of metabolites have to be determined. The aim of this work was to develop a practical method for determining diffusion coefficients in anisotropic medium and to estimate the overall diffusion coefficients of fluorescently labeled ATP in rat cardiomyocytes. For that, we have extended raster image correlation spectroscopy (RICS) protocols to be able to discriminate the anisotropy in the diffusion coefficient tensor. Using this extended protocol, we estimated diffusion coefficients of ATP labeled with the fluorescent conjugate Alexa Fluor 647 (Alexa-ATP). In the analysis, we assumed that the diffusion tensor can be described by two values: diffusion coefficient along the myofibril and that across it. The average diffusion coefficients found for Alexa-ATP were as follows: 83 ± 14 µm²/s in the longitudinal and 52 ± 16 µm²/s in the transverse directions (n = 8, mean ± SD). Those values are ~2 (longitudinal) and ~3.5 (transverse) times smaller than the diffusion coefficient value estimated for the surrounding solution. Such uneven reduction of average diffusion coefficient leads to anisotropic diffusion in rat cardiomyocytes. Although the source for such anisotropy is uncertain, we speculate that it may be induced by the ordered pattern of intracellular structures in rat cardiomyocytes.

Activation of Na+/H+ and K+/H+ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

Ortiz-Acevedo, A., Rigor, R. R., Maldonado, H. M., Cala, P. M. — 2008-11-13

Alteration in cell volume of vertebrates results in activation of volume-sensitive ion flux pathways. Fine control of the activity of these pathways enables cells to regulate volume following osmotic perturbation. Protein phosphorylation and dephosphorylation have been reported to play a crucial role in the control of volume-sensitive ion flux pathways. Exposing Amphiuma tridactylu red blood cells (RBCs) to phorbol esters in isotonic medium results in a simultaneous, dose-dependent activation of both Na⁺/H⁺ and K⁺/H⁺ exchangers. We tested the hypothesis that in Amphiuma RBCs, both shrinkage-induced Na⁺/H⁺ exchange and swelling-induced K⁺/H⁺ exchange are activated by phosphorylation-dependent reactions. To this end, we assessed the effect of calyculin A, a phosphatase inhibitor, on the activity of the aforementioned exchangers. We found that exposure of Amphiuma RBCs to calyculin-A in isotonic media results in simultaneous, 1–2 orders of magnitude increase in the activity of both K⁺/H⁺ and Na⁺/H⁺ exchangers. We also demonstrate that, in isotonic media, calyculin A-dependent increases in net Na⁺ uptake and K⁺ loss are a direct result of phosphatase inhibition and are not dependent on changes in cell volume. Whereas calyculin A exposure in the absence of volume changes results in stimulation of both the Na⁺/H⁺ and K⁺/H⁺ exchangers, superimposing cell swelling or shrinkage and calyculin A treatment results in selective activation of K⁺/H⁺ or Na⁺/H⁺ exchange, respectively. We conclude that kinase-dependent reactions are responsible for Na⁺/H⁺ and K⁺/H⁺ exchange activity, whereas undefined volume-dependent reactions confer specificity and coordinated control.

Renal proximal tubules from old Fischer 344 rats grow into epithelial cells in cultures and exhibit increased oxidative stress and reduced D1 receptor function

Asghar, M., Chillar, A., Lokhandwala, M. F. — 2008-11-13

Earlier we reported defects in D1 receptor function in renal proximal tubules (RPTs) of aged Fischer 344 (F344) and obese Zucker rats. However, the defects in the receptor function in RPTs of obese Zucker rats do not pass onto primary cultures of RPTs from these animals. Here, we determined whether the defects in D1 receptor function in RPTs of aged F344 rats pass onto the primary cultures. RPTs from aged (24-mo) and adult (6-mo) F344 rats were grown into primary cultures. The microscopic studies showed that cells in cultures from adult and old rats were healthy as determined by the shape and size of the cells and nuclei. D1 receptor agonist SKF-38393 produced inhibition of ⁸⁶Rb (rubidium) uptake, index of Na-K-ATPase activity, in cells from adult rats, but this was reduced in old rats. Also, SKF-38393 increased the [³⁵S]GTPS binding, index of receptor activation, in the membranes of cells from adult rats but to a lesser extent from old rats. Furthermore, there was a downward trend in the levels of D1 receptor numbers and in the receptor proteins in old rats. Interestingly, gp^91phox subunit of NADPH oxidase and cellular protein carbonyl levels (oxidative stress marker) were higher in cultures from old rats. These results show that RPTs from adult and old F344 rats grow into epithelial cells in cultures. Furthermore, cells in cultures from old rats are at a higher level of oxidative stress, which may be contributing to the reduced D1 receptor function in the cells from old compared with adult rats.

Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes

Mertl, M., Daniel, H., Kottra, G. — 2008-11-13

The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of ~5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-l-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed when endocytosis was stimulated by the activation of PKC. Cytochalasin D inhibited all changes evoked by either dipeptide or PKC stimulation, whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substrate-dependent retrieval of rPEPT1. Coexpression experiments with human Na⁺-glucose transporter 1 (hSGLT1) revealed that substrate exposure selectively affected PEPT1 but not the activity of hSGLT1.

Stimulation of {beta}3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca2+-activated K+ channels

Hristov, K. L., Cui, X., Brown, S. M., Liu, L., Kellett, W. F., Petkov, G. V. — 2008-11-13

We investigated the role of large-conductance Ca²⁺-activated K⁺ (BK) channels in β3-adrenoceptor (β3-AR)-induced relaxation in rat urinary bladder smooth muscle (UBSM). BRL 37344, a specific β3-AR agonist, inhibits spontaneous contractions of isolated UBSM strips. SR59230A, a specific β3-AR antagonist, and H89, a PKA inhibitor, reduced the inhibitory effect of BRL 37344. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration response curves for contraction amplitude, net muscle force, and tone to the right. Freshly dispersed UBSM cells and the perforated mode of the patch-clamp technique were used to determine further the role of β3-AR stimulation by BRL 37344 on BK channel activity. BRL 37344 increased spontaneous, transient, outward BK current (STOC) frequency by 46.0 ± 20.1%. In whole cell mode at a holding potential of V_h = 0 mV, the single BK channel amplitude was 5.17 ± 0.28 pA, whereas in the presence of BRL 37344, it was 5.55 ± 0.41 pA. The BK channel open probability was also unchanged. In the presence of ryanodine and nifedipine, the current-voltage relationship in response to depolarization steps in the presence and absence of BRL 37344 was identical. In current-clamp mode, BRL 37344 caused membrane potential hyperpolarization from –26.1 ± 2.1 mV (control) to –29.0 ± 2.2 mV. The BRL 37344-induced hyperpolarization was eliminated by application of iberiotoxin, tetraethylammonium or ryanodine. The data indicate that stimulation of β3-AR relaxes rat UBSM by increasing the BK channel STOC frequency, which causes membrane hyperpolarization and thus relaxation.

Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

Mitchell, T., Lo, A., Logan, M. R., Lacy, P., Eitzen, G. — 2008-11-13

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (≤10 µM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca²⁺ ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca²⁺ ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR

2008-11-13

Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study, we developed a bicistronic construct called R-Split CFTR, which encodes the front and back halves of CFTR as separate polypeptides without the RD. These fragments assemble to form a constitutively active CFTR channel. Coexpression of the third fragment corresponding to the missing RD restores regulation by PKA, and this is associated with dramatically enhanced binding of the phosphorylated RD. In the present study, we examined the effect of PKC phosphorylation on this PKA-induced interaction. We report here that PKC alone enhanced association of the RD with R-Split CFTR and that binding was further enhanced when the RD was phosphorylated by both kinases. Mutation of all seven PKC consensus sequences on the RD (7CA-RD) did not affect its association under basal (unphosphorylated) conditions but abolished phosphorylation-induced binding by both kinases. Iodide efflux responses provided further support for the essential role of RD binding in channel regulation. The basal activity of R-Split/7CA-RD channels was similar to that of R-Split/wild type (WT)-RD channels, whereas cAMP-stimulated iodide efflux was greatly diminished by removal of the PKC sites, indicating that 7CA-RD binding maintains channels in an inactive state that is unresponsive to PKA. These results suggest a novel mechanism for CFTR regulation in which PKC modulates PKA-induced domain-domain interactions.

Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca2+ signals, and vasoconstriction in cerebral arteries

Zhao, G., Adebiyi, A., Blaskova, E., Xi, Q., Jaggar, J. H. — 2008-11-13

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP₃R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP₃R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP₃R blockers, reduced Ca²⁺ wave activation and global intracellular Ca²⁺ ([Ca²⁺]_i) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP₃R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP₃R1 being the most abundant isoform at 82% of total IP₃R message. IP₃R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca²⁺ wave frequency and global [Ca²⁺]_i but abolished UTP-induced Ca²⁺ wave activation and reduced the UTP-induced global [Ca²⁺]_i elevation by ~61%. Antibodies targeting IP₃R1 and IP₃R1 knockdown reduced UTP-induced nonselective cation current (I_cat) activation. IP₃R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca²⁺ by ~45%. These data indicate that IP₃R1 is the predominant IP₃R isoform expressed in rat cerebral artery smooth muscle cells. IP₃R1 stimulation contributes to UTP-induced I_cat activation, Ca²⁺ wave generation, global [Ca²⁺]_i elevation, and vasoconstriction. In addition, IP₃R1 activation constricts cerebral arteries in the absence of SR Ca²⁺ release by stimulating plasma membrane I_cat.

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

Perry, C., Quissell, D. O., Reyland, M. E., Grichtchenko, I. I. — 2008-11-13

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na⁺-HCO₃^– cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO₃^– across the BLM, thus supporting HCO₃^– luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.

Impact of overexpression of metallothionein-1 on cell cycle progression and zinc toxicity

Smith, P. J., Wiltshire, M., Furon, E., Beattie, J. H., Errington, R. J. — 2008-11-13

Metallothioneins (MTs) have an important role in zinc homeostasis and may counteract the impact of oversupply. Both intracellular zinc and MT expression have been implicated in proliferation control and resistance to cellular stress, although the interdependency is unclear. The study addresses the consequences of a steady-state overexpression of MT-1 for intracellular zinc levels, cell cycle progression, and protection from zinc toxicity using a panel of cell lines with differential expression of MT-1. The panel comprised parental Chinese hamster ovary-K1 cells with low endogenous expression of MT and transfectants with enhanced expression of mouse MT-1 on an autonomously replicating expression vector with a noninducible promoter. Cell cycle progression, determined by flow cytometry and time-lapse microscopy, revealed that enhanced cytoplasmic expression of MT-1 does not impact on normal cell cycle operation, suggesting that basal levels of MT-1 expression are not limiting for background levels of oxidative stress. MT-1 overexpression correlated with a steady-state increase in cytoplasmic free Zn²⁺, assessed using the fluorescent zinc-sensor Zinquin, particularly at high levels of overexpression, further suggesting that zinc availability is normally not limiting for cell cycle progression. Enhanced MT-1 expression, over a 10-fold range, had a clear impact on resistance to Cd²⁺ and Zn²⁺ toxicity. In the case of Zn²⁺, the degree of protection afforded was less, indicating that MT-1 has a limited range and saturable capacity for effecting resistance. The results have implications for the use of cellular stress responses to exogenously supplied zinc and zinc-based systemic therapies.

Regulation of cell proliferation by intermediate-conductance Ca2+-activated potassium and volume-sensitive chloride channels in mouse mesenchymal stem cells

Tao, R., Lau, C.-P., Tse, H.-F., Li, G.-R. — 2008-11-13

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine; however, their cellular physiology is not fully understood. The present study aimed at exploring the potential roles of the two dominant functional ion channels, intermediate-conductance Ca²⁺-activated potassium (IK_Ca) and volume-sensitive chloride (I_Cl.vol) channels, in regulating proliferation of mouse MSCs. We found that inhibition of IK_Ca with clotrimazole and I_Cl.vol with 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) reduced cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific short interference (si)RNAs significantly reduced IK_Ca or I_Cl.vol density and channel protein and produced a remarkable suppression of cell proliferation (by 24.4 ± 9.6% and 29.5 ± 7.2%, respectively, P < 0.05 vs. controls). Flow cytometry analysis showed that mouse MSCs retained at G₀/G₁ phase (control: 51.65 ± 3.43%) by inhibiting IK_Ca or I_Cl.vol using clotrimazole (2 µM: 64.45 ± 2.20%, P < 0.05) or NPPB (200 µM: 82.89 ± 2.49%, P < 0.05) or the specific siRNAs, meanwhile distribution of cells in S phase was decreased. Western blot analysis revealed a reduced expression of the cell cycle regulatory proteins cyclin D1 and cyclin E. Collectively, our results have demonstrated that IK_Ca and I_Cl.vol channels regulate cell cycle progression and proliferation of mouse MSCs by modulating cyclin D1 and cyclin E expression.

The monomeric G proteins AGS1 and Rhes selectively influence G{alpha}i-dependent signaling to modulate N-type (CaV2.2) calcium channels

Thapliyal, A., Bannister, R. A., Hanks, C., Adams, B. A. — 2008-11-13

Activator of G protein Signaling 1 (AGS1) and Ras homologue enriched in striatum (Rhes) define a new group of Ras-like monomeric G proteins whose signaling properties and physiological roles are just beginning to be understood. Previous results suggest that AGS1 and Rhes exhibit distinct preferences for heterotrimeric G proteins, with AGS1 selectively influencing Gi and Rhes selectively influencing Gs. Here, we demonstrate that AGS1 and Rhes trigger nearly identical modulation of N-type Ca²⁺ channels (Ca_V2.2) by selectively altering Gi-dependent signaling. Whole-cell currents were recorded from HEK293 cells expressing Ca_V2.2 and Gi- or Gs-coupled receptors. AGS1 and Rhes reduced basal current densities and triggered tonic voltage-dependent (VD) inhibition of Ca_V2.2. Additionally, each protein attenuated agonist-initiated channel inhibition through Gi-coupled receptors without reducing channel inhibition through a Gs-coupled receptor. The above effects of AGS1 and Rhes were blocked by pertussis toxin (PTX) or by expression of a Gβ-sequestering peptide (masGRK3ct). Transfection with HRas, KRas2, Rap1A-G12V, Rap2B, Rheb2, or Gem failed to duplicate the effects of AGS1 and Rhes on Ca_V2.2. Our data provide the first demonstration that AGS1 and Rhes exhibit similar if not identical signaling properties since both trigger tonic Gβ signaling and both attenuate receptor-initiated signaling by the Gβ subunits of PTX-sensitive G proteins. These results are consistent with the possibility that AGS1 and Rhes modulate Ca²⁺ influx through Ca_V2.2 channels under more physiological conditions and thereby influence Ca²⁺-dependent events such as neurosecretion.

Essential role of EP3 subtype in prostaglandin E2-induced adhesion of mouse cultured and peritoneal mast cells to the Arg-Gly-Asp-enriched matrix

Sakanaka, M., Tanaka, S., Sugimoto, Y., Ichikawa, A. — 2008-11-13

Accumulating evidence has indicated that mast cells can modulate a wide variety of immune responses. Migration and adhesion play a critical role in regulation of tissue mast cell function, in particular, under inflammatory conditions. We previously demonstrated that prostaglandin (PG) E₂ stimulates adhesion of a mouse mastocytoma cell line, P-815, to the Arg-Gly-Asp (RGD)-enriched matrix through cooperation between two PGE₂ receptor subtypes: EP3 and EP4 (Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. J Biol Chem 278: 17977–17981, 2003). We here investigated PGE₂-induced adhesion of IL-3-dependent bone marrow-derived cultured mast cells (BMMCs). In contrast to the elevated cAMP-dependent adhesion of P-815 cells, EP3-mediated Ca²⁺ mobilization plays a pivotal role in PGE₂-induced adhesion of BMMCs. Adhesion and Ca²⁺ mobilization induced by PGE₂ were abolished in the Ptger3^–/– BMMCs and were significantly suppressed by treatment with pertussis toxin, a phospholipase C inhibitor, U-73122, and a store-operated Ca²⁺ channel inhibitor, SKF 36965, indicating the involvement of G_i-mediated Ca²⁺ influx. We then investigated PGE₂-induced adhesion of peritoneal mast cells to the RGD-enriched matrix. EP3 subtype was found to be the dominant PGE receptor that expresses in mouse peritoneal mast cells. PGE₂ induced adhesion of the peritoneal mast cells of the Ptger3^+/+ mice, but not that of the Ptger3^–/– mice. In rat peritoneal mast cells, PGE₂ or an EP3 agonist stimulated both Ca²⁺ mobilization and adhesion to the RGD-enriched matrix. These results suggested that the EP3 subtype plays a pivotal role in PGE₂-induced adhesion of murine mast cells to the RGD-enriched matrix through Ca²⁺ mobilization.

Human neutrophil surface protrusion under a point load: location independence and viscoelasticity

Xu, G., Shao, J.-Y. — 2008-11-13

Mechanical properties of neutrophils have been recognized as key contributors to stabilizing neutrophil rolling on the endothelium during the inflammatory response. In particular, accumulating evidence suggests that surface protrusion and tether extraction from neutrophils facilitate stable rolling by relieving the disruptive forces on adhesive bonds. Using a customized optical trap setup, we applied piconewton-level pulling forces on targeted receptors that were located either on the microvillus tip (CD162) or intermicrovillus surface of neutrophils (CD18 and CD44). Under a constant force-loading rate, there always occurred an initial tent-like surface protrusion that was terminated either by rupture of the adhesion or by a "yield" or "crossover" to tether extraction. The corresponding protrusional stiffness of neutrophils was found to be between 0.06 and 0.11 pN/nm, depending on the force-loading rate and the cytoskeletal integrity, but not on the force location, the medium osmolality, nor the temperature increase from 22°C to 37°C. More importantly, we found that neutrophil surface protrusion was accompanied by force relaxation and hysteresis. In addition, the crossover force did not change much in the range of force-loading rates studied, and the protrusional stiffness of lymphocytes was similar to that of neutrophils. These results show that neutrophil surface protrusion is essentially viscoelastic, with a protrusional stiffness that stems primarily from the actin cortex, and the crossover force is independent of the receptor-cytoskeleton interaction.

The use of BeWo cells as an in vitro model for placental iron transport

2008-11-13

BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 ± 0.5% of the total dose was transported after 8 h, equivalent to 38.8 ± 2.1 pmol·cm^–2·h^–1. Transfer of Tf across the cell layer was much more limited; 2.4 ± 0.2% of the total dose was transported after 8 h, equivalent to 5.0 ± 0.4 pmol·cm^–2·h^–1. Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta.

Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and {beta}2-integrin surface expression in human neutrophils

2008-11-13

Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pH_i) have on surface expression of adhesion molecules on neutrophils. When human neutrophils were exposed to pH 6 lactate, there was a marked decrease in surface L-selectin (CD62L) levels, and the decrease was significantly enhanced by inclusion of Na⁺/H⁺ exchanger (NHE) inhibitor 5-(N,N-hexamethylene)amiloride (HMA). Similar effects were observed when pH_i was reduced while maintaining normal extracellular pH, by using an NH₄Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors [HMA, cariporide, or 5-(N,N-dimethyl)amiloride (DMA)]. The amount of L-selectin shedding induced by different concentrations of NH₄Cl in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, β₂-integrin (CD11b and CD18) was only slightly upregulated in the low-pH_i condition and was enhanced by NHE inhibition to a much lesser extent. L-selectin shedding was prevented by treating human neutrophils with inhibitors of extracellular metalloproteases (RO-31-9790 and KD-IX-73-4) or with inhibitors of intracellular signaling via p38 MAP kinase (SB-203580 and SB-239063), implying a transmembrane effect of pH_i. Taken together, these data suggest that the ability of NHE inhibitors such as HMA to reduce ischemia-reperfusion injury may be related to the nearly complete removal of L-selectin from the neutrophil surface.

Measuring ion transport activities in Xenopus oocytes using the ion-trap technique

Blanchard, M. G., Longpre, J.-P., Wallendorff, B., Lapointe, J.-Y. — 2008-11-13

The ion-trap technique is an experimental approach allowing measurement of changes in ionic concentrations within a restricted space (the trap) comprised of a large-diameter ion-selective electrode apposed to a voltage-clamped Xenopus laevis oocyte. The technique is demonstrated with oocytes expressing the Na⁺/glucose cotransporter (SGLT1) using Na⁺- and H⁺-selective electrodes and with the electroneutral H⁺/monocarboxylate transporter (MCT1). In SGLT1-expressing oocytes, bath substrate diffused into the trap within 20 s, stimulating Na⁺/glucose influx, which generated a measurable decrease in the trap Na⁺ concentration ([Na⁺]_T) by 0.080 ± 0.009 mM. Membrane hyperpolarization produced a further decrease in [Na⁺]_T, which was proportional to the increased cotransport current. In a Na⁺-free, weakly buffered solution (pH 5.5), H⁺ drives glucose transport through SGLT1, and this was monitored with a H⁺-selective electrode. Proton movements can also be clearly detected on adding lactate to an oocyte expressing MCT1 (pH 6.5). For SGLT1, time-dependent changes in [Na⁺]_T or [H⁺]_T were also detected during a membrane potential pulse (150 ms) in the presence of substrate. In the absence of substrate, hyperpolarization triggered rapid reorientation of SGLT1 cation binding sites, accompanied by cation capture from the trap. The resulting change in [Na⁺]_T or [H⁺]_T is proportional to the pre-steady-state charge movement. The ion-trap technique can thus be used to measure steady-state and pre-steady-state transport activities and provides new opportunities for studying electrogenic and electroneutral ion transport mechanisms.