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PubMed2006

Indoxyl sulfate stimulates proliferation of rat vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of arteriosclerosis. Clinical studies show that uremic toxins deteriorate the arteriosclerosis in renal failure patients. Indoxyl sulfate (IS) is a strong protein-bound uremic toxin, but the effect of IS on VSMC proliferation has not been studied. We examined the effect of IS on rat VSMC proliferation, assessed by a cell counting kit (4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] assay) and by [(3)H]thymidine incorporation in vitro. We further evaluated a contribution of mitogen-activated protein kinase (MAPK; p44/42 MAPK) to VSMC proliferation by IS. Immunohistochemical staining was performed for VSMCs using antirat organic anion transporter (OAT)3 antibody. The mRNA expressions of platelet-derived growth factor (PDGF)-A and -C chains, and PDGF-beta receptor were evaluated by real-time PCR. IS stimulated the proliferation of VSMCs in a concentration-dependent manner and activated p44/42 MAPK. Concentration of IS needed to stimulate the proliferation of rat VSMC was about 250 microM, which is compatible with that in the serum of end-stage renal failure patients. PD98059 (10 microM), a selective inhibitor of MAPK/extracellular signal-regulated kinase, inhibited the IS-induced (250 microM) VSMC proliferation and phosphorylation of MAPK. Probenecid (0.5 mM), an inhibitor and substrate of OAT, inhibited the IS-induced (250 microM) VSMC proliferation. Rat OAT3 was detected in VSMCs. The mRNA expressions of PDGF-C chain and PDGF-beta receptor were significantly increased by IS. We conclude that IS directly stimulates rat VSMC proliferation and activates MAPK in vitro. This might be one of the mechanisms underlying the progression of atherosclerotic lesions in end-stage renal disease patients.

Animals↗
PubMed2003

Regulation of hypoxia-inducible factor-1alpha by cyclical mechanical stretch in rat vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) are exposed to hormonal and mechanical stress in vivo. Hormonal factors have been shown to affect hypoxia-inducible factor-1alpha (HIF-1alpha). How mechanical stress affects the regulation of HIF-1alpha in VSMCs has not been reported previously, and therefore we sought to investigate the regulation of HIF-1alpha by cyclical mechanical stretch in cultured rat VSMCs. Rat VSMCs grown on a flexible membrane base were stretched by vacuum to 20% of the maximum elongation at 60 cycles/min. The levels of HIF-1alpha protein began to increase as early as 2 h after stretch was applied and reached a maximum of 2.8-fold over the control by 4 h. Real-time PCR showed that the levels of HIF-1alpha mRNA increased 2.1-fold after cyclical stretch for 4 h. Cyclical mechanical stretch also increased the immunohistochemical labelling of HIF-1alpha in VSMCs after cyclical stretch for 4 h. The phosphorylation of p42/p44 mitogen-activated protein kinase (MAP kinase) increased after stretch and this was inhibited by the MAP kinase kinase inhibitors PD98059 and U0126. PD98059 and U0126 also blocked HIF-1alpha gene expression induced by cyclical stretch. In conclusion, cyclical mechanical stretch activates the gene expression of HIF-1alpha in cultured VSMCs and this mechanical effect is possibly mediated by the p42/p44 MAP kinase kinase pathway.

Animals↗
PubMed1999

Regulation of prostaglandin production by nitric oxide in rat smooth muscle myometrial cells.

Smooth muscle myometrial cells isolated by an enzymatic method from estrogenized rats were used after 7-10 days of culture. They were incubated for 24 h with two distinct competitive nitric oxide (NO) inhibitors: NG-monomethyl-L-arginine (L-NMMA: 300 microM) and L-nitro-arginine methylester (L-NAME: 600 microM, 5 mM and 10 mM). Afterwards, the supernatants were separated in order to measure nitrite production and prostaglandin PGE synthesis. In the present report, we demonstrate that myometrial cells from estrogenized rats are able to produce NO, since all the inhibitors significantly decrease the production of nitrites in the culture media. Furthermore, we report that both inhibitors inhibited PGE synthesis by myometrial cells. We also used a donor of NO in the incubation medium for 24 h, sodium nitroprusside (NP), obtaining an strong (P< 0.001) increase in both nitrite and PGE production. We conclude that myometrial cells can produce NO and that one possible role of the NO synthetized by this cells may be the modulation of PGE production.

Animals↗
PubMed2001

Down-regulation by antisense oligonucleotides establishes a role for the proline-rich tyrosine kinase PYK2 in angiotensin ii-induced signaling in vascular smooth muscle.

Abnormal vascular smooth muscle cell (VSMC) growth plays a key role in the pathogenesis of hypertension and atherosclerosis. Angiotensin II (Ang II) elicits a hypertrophic growth response characterized by an increase in protein synthesis in the absence of DNA synthesis and cell proliferation. Intracellular signaling mechanisms linking angiotensin type I receptor activation to protein synthesis in VSMC have not been fully characterized. The present study investigates the role of the nonreceptor proline-rich tyrosine kinase 2 (PYK2) in Ang II-induced VSMC protein synthesis and in the regulation of two signaling pathways that have been implicated in the control of protein synthesis, the extracellular signal-regulated kinase (ERK1/2) and the phosphatidylinositol 3-kinase/Akt pathways. PYK2 antisense oligonucleotides were used to down-regulate PYK2 expression in cultured VSMC. An 80% down-regulation in PYK2 expression resulted in an approximately 80% inhibition of ERK1/2 (3.8 +/- 1.3 versus 16.6 +/- 1.8), p70S6 kinase (1.03 +/- 0.03 versus 3.8 +/- 0.5), and Akt activation (3.0 +/- 0.8 versus 16.0 +/- 1.0) by Ang II. Furthermore, PYK2 down-regulation resulted in a complete inhibition of Ang II-induced VSMC protein synthesis. These data conclusively identify PYK2 as an upstream regulator of both the ERK1/2 and the phosphatidylinositol 3-kinase/Akt pathways that are involved in Ang II-induced VSMC protein synthesis.

Angiotensin II↗
PubMed2001

Contribution of the helix-loop-helix factor Id2 to regulation of vascular smooth muscle cell proliferation.

Smooth muscle cell (SMC) proliferation plays a key role in vascular proliferative disorders. The molecular mechanisms that control cell cycle entry of SMCs in response to vascular injury are not well understood. Id2 (inhibitor of DNA binding) is a member of the helix-loop-helix (HLH) family of transcription regulators that are known to promote cell cycle progression. Thus, we investigated the role of Id2 in SMC growth and cell cycle regulation. The results demonstrated that overexpression of Id2 resulted in a significant enhancement of SMC growth via increased S-phase entry. A possible mechanism of Id2-enhanced SMC growth is via regulation of p21 expression, as overexpression of Id2-inhibited transcriptional activity of a 2.3-kb p21 promoter/luciferase reporter construct as well as p21 protein levels. Id2 enhancement of SMC growth and inhibition of p21 expression were dependent on phosphorylation of Id2 by cyclin E/cdk2, as an Id2 cDNA containing a mutation in the cdk2 phosphorylation site (serine 5) failed to regulate SMC cell cycle progression or p21 promoter activity. The mechanism of cyclin E/cdk2 control of the Id2 effect may in part involve regulation of nuclear transport; unlike wild-type Id2, the Id2 mutant was not transported to the nucleus. Finally, in a rat carotid model of arterial injury, Id2 was expressed in a temporal pattern that parallels the kinetics of cellular proliferation. In summary, these results provide evidence that the Id2 protein is integrated into the cell cycle regulatory cascade that results in SMC proliferation following vascular injury and suggest that this effect is at least in part via a cdk2-dependent inhibition of p21 gene expression.

Adenoviridae↗
PubMed2004

Focal adhesion kinase (FAK)-dependent regulation of S-phase kinase-associated protein-2 (Skp-2) stability. A novel mechanism regulating smooth muscle cell proliferation.

Smooth muscle cell (SMC) proliferation is suppressed in intact blood vessels but stimulated in atherosclerosis, restenosis after angioplasty, and vein graft disease. The cyclin-dependent kinase inhibitors, including p27(Kip1), play important roles in maintaining SMC quiescence. Levels of p27(Kip1) are dependent on attachment to and the composition of the extracellular matrix (ECM). Here we sought to elucidate mechanisms underlying the ECM-dependent regulation of p27(Kip1) and hence, SMC proliferation. Serum stimulation decreased p27(Kip1) levels in isolated SMC but not in rat aorta. The effect was post-translational and mediated by proteasomal degradation. We studied the S-phase-associated kinase protein-2 (Skp-2), an F-box protein involved in ubiquitination and proteasome-mediated degradation. Skp-2 protein is strongly induced by serum from undetectable levels in isolated SMCs but remains undetectable in aorta; Skp-2 mRNA is also lower in aorta. Overexpression of wild-type Skp-2 in SMCs decreased p27(Kip1) levels, whereas dominant negative F-box deleted mutant (DeltaF-Skp-2) Skp-2 increased p27(Kip1) levels. Furthermore, hyperphosphorylation of retinoblastoma protein and SMC proliferation were also reciprocally affected by wild-type and dominant negative Skp-2. Skp-2 expression was absolutely dependent on cell attachment to the ECM and was inhibited by laminin and type-1 fibrillar collagen but increased by fibronectin. Expression of Skp-2 protein, but not mRNA, was associated with focal adhesion kinase (FAK) activity and inhibited by overexpression of FAK-related non-kinase and a dominant negative FAK(Y397F) mutant. Furthermore, the inhibition of Skp-2 expression by dominant negative FAK was reversed by the proteasome inhibitor MG-132. Taken together, these data demonstrate that the vascular ECM controls SMC proliferation via FAK-dependent regulation of Skp-2 protein stability.

Animals↗
PubMed2004

Regulation of cGMP-dependent protein kinase expression by soluble guanylyl cyclase in vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the alpha1 and beta1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro.

Adenoviridae↗
PubMed1983

Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells.

Isolated smooth muscle cells and cell fragments prepared by glycerination and subsequent homogenization will contract to one-third their normal length, provided Ca++ and ATP are present. Ca++-independent contraction was obtained by preincubation in Ca++ and ATP gamma S, or by addition of trypsin-treated myosin light chain kinase (MLCK) that no longer requires Ca++ for activation. In the absence of Ca++, myosin was rapidly lost from the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of this myosin is phosphorylated. The extent of myosin loss was ATP- and pH-dependent and occurred under conditions similar to those previously reported for the in vitro disassembly of gizzard myosin filaments. Ca++-dependent contraction was restored to extracted cells by addition of gizzard myosin under rigor conditions (i.e., no ATP), followed by addition of MLCK, calmodulin, Ca++, and ATP. Function could also be restored by adding all these proteins in relaxing conditions (i.e., in EGTA and ATP) and then initiating contraction by Ca++ addition. Incubation with skeletal myosin will restore contraction, but this was not Ca++-dependent unless the cells were first incubated in troponin and tropomyosin. These results strengthen the idea that contraction in glycerinated cells and presumably also in intact cells is primarily thick filament regulated via MLCK, that the myosin filaments are unstable in relaxing conditions, and that the spatial information required for cell length change is present in the thin filament-intermediate filament organization.

Adenosine Triphosphate↗
PubMed1997

Perlecan regulates Oct-1 gene expression in vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs) are very quiescent in the mature vessel and exhibit a remarkable phenotype-dependent diversity in gene expression that may reflect the growth responsiveness of these cells under a variety of normal and pathological conditions. In this report, we describe the expression pattern of Oct-1, a member of a family of transcription factors involved in cell growth processes, in cultured and in in vivo SMCs. Oct-1 mRNA was undetectable in the contractile-state in vivo SMCs; was induced upon disruption of in vivo SMC-extracellular matrix interactions; and was constitutively expressed by cultured SMCs. Oct-1 transcripts were repressed when cultured SMCs were plated on Engelbreth-Holm-Swarm tumor-derived basement membranes (EHS-BM) but were rapidly induced after disruption of SMC-EHS-BM contacts; reexpression was regulated at the transcriptional level. To identify the EHS-BM component involved in the active repression of Oct-1 mRNA expression, SMCs were plated on laminin, type IV collagen, fibronectin, or perlecan matrices. Oct-1 mRNA levels were readily detectable when SMCs were cultured on matrices composed of laminin, type IV collagen, or fibronectin but were repressed when SMCs were cultured on perlecan matrices. Finally, the Oct-1-suppressing activity of EHS-BM was sensitive to heparinase digestion but not to chondroitinase ABC or hyaluronidase digestion, suggesting that the heparan sulfate side chains of perlecan play a biologically important role in negatively regulating the expression of Oct-1 transcripts.

Animals↗
PubMed1992

Role of 17-kDa essential light chain isoforms of aorta smooth muscle myosin.

Aorta smooth muscle myosin contains two kinds of 17-kDa essential light chain, LC17nm (nonmuscle-type) and LC17gi (gizzard-type) [Hasegawa, Y., Ueda, Y., Watanabe, M., & Morita, F. (1992) J. Biochem. 111, 798-803]. The LC17 isoforms were released from porcine aorta myosin by incubation at 46 degrees C. The rate of release was 1.5 to 2 times higher with LC17gi than with LC17nm. Aorta myosins containing the two LC17 isoforms in various ratios could be reconstituted. The actin-activated ATPase activity was measured as a function of LC17nm content. The Vm value was lower with myosin which contained more LC17nm. The apparent dissociation constant for F-actin, Km, was 20-fold less with myosin which contained 81% LC17nm than myosin which contained 23% LC17nm. A similar difference in the dissociation constants of myosin for F-actin was observed in the presence of adenylyl imidodiphosphate. The role of LC17nm appears to be to make aorta myosin suitable for maintaining the muscle tension with a low expenditure of energy. The isoform-dependent difference in the F-actin-binding affinities of myosin seems partly due to the difference in the affinities of LC17 isoforms themselves for F-actin. We found that the isolated LC17nm itself could bind with F-actin with a dissociation constant of 64 microM, but LC17gi could not.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed2003

Pericellular plasmin induces smooth muscle cell anoikis.

Smooth muscle cell (SMC) rarefaction is involved in the development of several vascular pathologies. We suggest that the plasminogen activation system is a potential extracellular signal that can induce pericellular proteolysis and apoptosis of vascular SMCs. Using primary cultures of arterial SMCs, we show that plasmin generated from plasminogen on the cell surface induces cell retraction and fibronectin fragmentation, leading to detachment and morphological/biochemical changes characteristic of apoptosis (also called anoikis). The generation of cell-bound plasmin mediated by tissue-type plasminogen activator (t-PA), constitutively expressed by VSMCs, requires binding of plasminogen to the cell surface and is inhibited by epsilon-aminocaproic acid (IC50=0.9+/-0.2 mM), a competitor of plasminogen binding to membrane glycoproteins. Conversely, addition of alpha2-antiplasmin, which blocks free plasmin in the cell supernatant, could not fully prevent anoikis. Finally, an MMP inhibitor failed to prevent VSMC anoikis, arguing for a direct involvement of plasmin in this phenomenon. Indeed, similar changes are induced by plasmin directly added to VSMCs or to arterial rings, ex-vivo. We show for the first time that pathological anoikis can be triggered by a process that requires functional assembly of the plasminogen activation system on the surface of VSMCs.

Anoikis↗
PubMed1997

Differential signaling pathways in platelet-activating factor-induced proliferation and interleukin-6 production by rat vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs) can be induced to proliferate in response to several cytokines and growth factors, including interleukin (IL)-6. Platelet-activating factor (PAF) also has been shown to induce SMC proliferation. Because PAF can stimulate IL-6 production in monocytes, macrophages, and endothelial cells, our study was undertaken to determine whether PAF could induce IL-6 production by SMCs and to define the underlying signaling pathways. Exposure of rat aortic SMCs to picomolar concentrations of PAF resulted in enhanced production of IL-6. The effect was concentration dependent, selective for the active form of PAF, and mediated by specific PAF receptors. Pretreatment of the cells with Bordatella pertussis toxin (PTX) prevented the effect of PAF, suggesting the involvement of alpha i-type subunits of G proteins in the signal-transduction pathway. PAF-dependent IL-6 production was also prevented by inhibition of tyrosine kinases with genistein or erbstatin. Inhibition of eicosanoid production by blocking either phospholipase A2 or cyclooxygenase also abrogated the effect of PAF on IL-6 production. Moreover, inhibition of Ca2+-calmodulin activity with W7 or blocking of calcium channels with verapamil or nifedipine prevented PAF-mediated enhancement of IL-6 production. Whereas PAF-induced signal-transduction pathways leading to IL-6 production and SMC proliferation were partially common, they appeared to diverge downstream of PLA2 activation: inhibition of cyclooxygenase had no effect on proliferation, whereas augmentation of cyclic adenosine monophosphate (cAMP) levels or activation of protein kinase A inhibited proliferation, in contrast to IL-6 production. Our findings suggest a role for PAF in modulating vascular function by stimulating local production of IL-6 by SMCs and promoting their proliferation. The two effects are, however, associated with partially divergent signaling pathways and may not be causally related.

Animals↗
PubMed2003

Signaling through PI3K/Akt mediates stretch and PDGF-BB-dependent DNA synthesis in bladder smooth muscle cells.

PURPOSE: Smooth muscle cells (SMC) of the bladder undergo hypertrophy and hyperplasia following exposure to sustained mechanical overload. Although superficial similarities in the response of the heart and bladder to hypertrophic stimuli suggest that similar molecular mechanisms may be involved, this remains to be demonstrated. In this study we compared signal transduction pathway activation in primary culture bladder SMC and cardiac myofibroblasts in response to cyclic stretch. The effects of growth factor stimulation on pathway activation in bladder SMC were also investigated. MATERIALS AND METHODS: Primary culture rodent bladder SMC or cardiac myofibroblasts were subjected to cyclic stretch-relaxation in the absence or presence of pharmacologic inhibitors of the phosphoinositide-3-kinase, (PI3K)/Akt, extracellular signal-regulated kinase-mitogen activated protein kinase (Erk-MAPK) or the p38 stress-activated protein kinase-2 (SAPK2) pathways. In parallel experiments human bladder SMC were treated with platelet-derived growth factor-BB (PDGF-BB), heparin-binding EGF-like growth factor (HB-EGF) or fibroblast growth factor-2 (FGF-2). In each case the extent of DNA synthesis was determined by uptake of tritiated thymidine, and activation of specific signaling intermediates was determined by immunoblot analysis using antibodies to the non-phosphorylated and phosphorylated (activated) forms of Akt, p38 and Erk1/2. RESULTS: Akt and p38 were rapidly phosphorylated in stretched bladder SMC and cardiac myofibroblasts, and stretch-induced DNA synthesis in these cells was ablated with inhibitors of PI3K or p38 but not Erk-MAPK. Similarly, PDGF-BB up-regulated DNA synthesis in bladder SMC in a p38 and Akt-dependent manner. CONCLUSIONS: We conclude that distinct stimuli, such as mechanical stretch and PDGF-BB, promote DNA synthesis in bladder SMC through shared downstream signaling pathways. Furthermore, phenotypically similar cells from the bladder and heart show comparable pathway activation in response to stretch. These findings suggest that similar molecular mechanisms underlie the altered growth responses of the bladder and heart to mechanical overload. This study also provides the first report of Akt activation in bladder SMC and suggests that Akt, consistent with its pivotal role in cardiac hypertrophy, may also be a key regulator of remodeling in the SMC compartment of the bladder exposed to hypertrophic/hyperplastic stimuli in vivo.

Animals↗
PubMed2001

Role of blood shear stress in the regulation of vascular smooth muscle cell migration.

Smooth muscle cell (SMC) migration from the media to the intima of blood vessels contributes to neointimal formation and atherogenesis. Here, we demonstrate how blood shear stress regulates vascular SMC migration in the encapsulating tissue of a micro-cylinder implanted in the center of the rat vena cava with the micro-cylinder perpendicular to blood flow. In this model, the micro-cylinder was exposed to a laminar flow with a known shear stress field in the leading region and a vortex flow in the trailing region. After surgery, the micro-cylinder was encapsulated by a thrombus-like tissue within one day, followed by SMC migration from the vena cava to the encapsulating tissue from day 3 to 20. SMC migration was time-dependent with a peak migration speed at day 5. At each given time (excluding day 1), blood shear stress exerts an inhibitory effect on SMC migration with significantly suppressed SMC migration in the laminar flow region than in the stagnation, separation, and vortex flow regions. SMCs were relatively parallel to the shear stress direction in high shear stress regions, whereas perpendicular to the shear stress direction in low shear stress regions. These results suggest that blood shear stress plays a role in regulating SMC migration and orientation in this model.

Animals↗
PubMed2004

Mycophenolic acid inhibits platelet-derived growth factor-induced reactive oxygen species and mitogen-activated protein kinase activation in rat vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation is the major pathologic feature associated with chronic allograft nephropathy, and mycophenolic acid (MPA) inhibits VSMC proliferation. Since the role of inosine monophosphate dehydrogenase (IMPDH)-dependent de novo guanosine synthesis is limited in VSMCs, we examined the effects of MPA on platelet-derived growth factor (PDGF)-induced cellular ROS and mitogen-activated protein kinases (MAPK) activation in VSMCs. Primary cultured rat VSMCs were stimulated with PDGF-BB in the presence or absence of MPA. Cell proliferation was assessed by [3H]-thymidine incorporation, ROS by flow cytometry and MAPK activation by Western blot analysis. PDGF increased cell proliferation, cellular ROS and extracellular-regulated protein kinase (ERK) 1/2 and p38 MAPK activation by 3.4-, 1.6-, 3.3- and 3.9-fold, respectively. MPA at above 1 muM inhibited PDGF-induced cellular ROS and ERK 1/2 and p38 MAPK activation, as well as proliferation. Structurally different anti-oxidants and inhibitor of ERK or p38 MAPK blocked PDGF-induced proliferation. Anti-oxidants also inhibited ERK 1/2 and p38 MAPK activation. Exogenous guanosine partially recovered the inhibitory effect of MPA on VSMC proliferation. These results suggest that MPA may inhibit PDGF-induced VSMC proliferation partially through inhibiting cellular ROS, and subsequent ERK 1/2 and p38 MAPK activation in addition to inhibiting IMPDH.

Animals↗
PubMed1990

Role of G-proteins in muscarinic receptor inward and outward currents in rabbit jejunal smooth muscle.

1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of rabbit jejunum were held under voltage clamp using patch pipettes and membrane currents measured. The effects of carbachol or caffeine applied externally were examined in cells dialysed with normal pipette solutions or with a solution containing heparin (which blocks receptors for D-myo-inositol 1,4,5-trisphosphate, InsP3), guanosine 5-O-(gamma-thio)triphosphate (GTP gamma S) or guanosine 5-O-(beta-thio)diphosphate (GDP beta S). 2. Outward current in response to application of carbachol or caffeine was considered to represent the opening of calcium-activated potassium channels in response to a localized rise in the free ionized calcium concentration occasioned by the rapid discharge of stored calcium (Ca) by these agents. 3. Heparin included in the pipette solution blocked outward current to muscarinic receptor activation by carbachol but not that to caffeine, suggesting that receptor-evoked discharge of stored cellular Ca is caused by InsP3 action. However, heparin did not affect muscarinic-receptor inward current. 4. After dialysis with 0.1-0.5 mM-GTP gamma S, carbachol inward current was evoked in two out of three of the cells; after dialysis with 0.1-0.2 mM-GTP gamma S for an average of 7.7 min it was 80% of the normal response; after dialysis for an average of 8.6 min with 0.5 mM-GTP gamma S it was 31% of the normal response. In contrast, 0.1 mM-GTP gamma S reduced caffeine outward current by 93% after an average 4.5 min dialysis and spontaneous transient outward currents (STOCs) were abolished in 2.9 min on average. 5. Carbachol inward current (at -40 or -50 mV) and carbachol outward current (at 0 mV) in responding cells were reduced only by half after 8-10 min dialysis with 1 mM-GDP beta S which has been shown in portal vein cells to antagonize the depletion of Ca stores by intracellular GTP gamma S (Komori & Bolton, 1989). After 8-10 min dialysis with 5 mM-GDP beta S outward current was 27% of normal. However, if GDP beta S was present, outward current generally could not be evoked by a second application of carbachol. 6. The discharge of Ca stores by dialysis with 0.1 mM-GTP gamma S was prevented completely by heparin included in the pipette solution, suggesting that activation of a G-protein associated with phospholipase C (PLC) enzyme accelerates PLC activity. InsP3 production and depletion of Ca stores.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Protein kinase C requirement of Ca2+ channel stimulation by intracellular ATP in guinea-pig basilar artery smooth muscle cells.

1. Smooth muscle cells were isolated from guinea-pig basilar artery and conventional whole-cell recordings of Ca2+ channel activity were made at room temperature within 7 h of the isolation procedure. The purpose of the study was to investigate the mechanism of the stimulatory action of intracellular ATP on Ca2+ channels. 2. High (millimolar) concentrations of ATP were needed to produce stimulation of Ca2+ channels, and neither ADP nor AMP mimicked the action of ATP. 3. The ATP effect was not mimicked by stable ATP derivatives (AMP-PNP or AMP-PCP) and was abolished by incubation of cells in non-specific protein kinase inhibitors (staurosporine or H-7) or specific protein kinase C inhibitors (GF109203x, calphostin C or chelerythrine) but not by tyrosine kinase inhibitors (tyrphostin B42 and genistein). 4. The data suggest that ATP-induced stimulation of L-type Ca2+ channels requires functional activity of a protein kinase C isozyme.

Adenosine Diphosphate↗
PubMed2002

CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle.

In vascular smooth muscle (VSM) and many other cells, G protein receptor-coupled activation of mitogen-activated protein kinases has been linked, in part, to increases in free intracellular Ca(2+). Previously, we demonstrated that ionomycin-, angiotensin II-, and thrombin-induced activation of extracellular signal-regulated kinase (ERK)1/2 in VSM cells was attenuated by pretreatment with KN-93, a selective inhibitor of the multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaM kinase II). In the present study, we show that the Ca(2+)-dependent pathway leading to activation of ERK1/2 is preceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation and is attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibits Ca(2+)-dependent PYK2 activation and EGF receptor tyrosine phosphorylation in response to ionomycin, ATP, and platelet-derived growth factor but has no effect on phorbol 12,13-dibutyrate- or EGF-induced responses. The results implicate CaM kinase II as an intermediate in the Ca(2+)/calmodulin-dependent activation of PYK2.

Animals↗