PKC activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase

Normally, signaling mechanisms that activate large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channels in pulmonary vascular smooth muscle cause pulmonary vasodilatation. BK(Ca)-channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (decrease in the opening probability) of the BK(Ca) channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BK(Ca)-channel activity in pulmonary vascular smooth muscle. Accordingly, studies were done to determine the effect of PKC on BK(Ca)-channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMCs) of the Sprague-Dawley rat. The PKC activators phorbol myristate acetate (PMA) and thymeleatoxin opened BK(Ca) channels in single Sprague-Dawley rat PASMC. The activator response to both PMA and thymeleatoxin on BK(Ca)-channel activity was blocked by G?-6983, which selectively blocks PKC-alpha, -delta, -gamma, and -zeta, and by rottlerin, which selectively inhibits PKC-delta. In addition, the specific cyclic GMP-dependent protein kinase antagonist KT-5823 blocked the responses to PMA and thymelatoxin, whereas the specific cyclic AMP-dependent protein kinase blocker KT-5720 had no effect. In isolated pulmonary arterial vessels, both PMA and forskolin caused vasodilatation, which was inhibited by KT-5823, G?-6983, or the BK(Ca)-channel blocker tetraethylammonium. The results of this study indicate that activation of specific PKC isozymes increases BK(Ca)-channel activity in Sprague-Dawley rat PASMC via cyclic GMP-dependent protein kinase, which suggests a unique signaling mechanism for vasodilatation.     

Protein kinase C inhibits BKCa channel activity in pulmonary arterial smooth muscle

Signaling mechanisms that elevate cyclic AMP (cAMP) activate large-conductance, calcium- and voltage-activated potassium (BKCa) channels in pulmonary vascular smooth muscle and cause pulmonary vasodilatation. BKCa channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BKCa channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BKCa channel activity. Accordingly, studies were done to determine the effect of PKC activation on cAMP-induced BKCa channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 microM), a stimulator of adenylate cyclase and an activator of cAMP, opened BKCa channels in single FHR PASMC, which were blocked by the PKC activators phorbol 12-myristate 13-acetate (100 nM) and thymeleatoxin (100 nM). The inhibitory response by thymeleatoxin on forskolin-induced BKCa channel activity was blocked by G?-6983, which selectively blocks the alpha, beta, delta, gamma, and zeta PKC isozymes, and G?-6976, which selectively inhibits PKC-alpha, PKC-beta, and PKC-mu, but not by rottlerin, which selectively inhibits PKC-delta. Collectively, these results indicate that activation of specific PKC isozymes inhibits cAMP-induced activation of the BKCa channel in pulmonary arterial smooth muscle, which suggests a unique signaling pathway to modulate BKCa channels and subsequently cAMP-induced pulmonary vasodilatation.     

Testosterone-induced relaxation of coronary arteries: activation of BKCa channels via the cGMP-dependent protein kinase

Androgens are reported to have both beneficial and detrimental effects on human cardiovascular health. The aim of this study was to characterize nongenomic signaling mechanisms in coronary artery smooth muscle (CASM) and define the ionic basis of testosterone (TES) action. TES-induced relaxation of endothelium-denuded porcine coronary arteries was nearly abolished by 20 nM iberiotoxin, a highly specific inhibitor of large-conductance, calcium-activated potassium (BK(Ca)) channels. Molecular patch-clamp studies confirmed that nanomolar concentrations of TES stimulated BK(Ca) channel activity by ～100-fold and that inhibition of nitric oxide synthase (NOS) activity by N(G)-monomethyl-L-arginine nearly abolished this effect. Inhibition of nitric oxide (NO) synthesis or guanylyl cyclase activity also attenuated TES-induced coronary artery relaxation but did not alter relaxation due to 8-bromo-cGMP. Furthermore, we detected TES-stimulated NO production in porcine coronary arteries and in human CASM cells via stimulation of the type 1 neuronal NOS isoform. Inhibition of the cGMP-dependent protein kinase (PKG) attenuated TES-stimulated BK(Ca) channel activity, and direct assay determined that TES increased activity of PKG in a concentration-dependent fashion. Last, the stimulatory effect of TES on BK(Ca) channel activity was mimicked by addition of purified PKG to the cytoplasmic surface of a cell-free membrane patch from CASM myocytes (～100-fold increase). These findings indicate that TES-induced relaxation of endothelium-denuded coronary arteries is mediated, at least in part, by enhanced NO production, leading to cGMP synthesis and PKG activation, which, in turn, opens BK(Ca) channels. These findings provide a molecular mechanism that could help explain why androgens have been reported to relax coronary arteries and relieve angina pectoris.     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

Stretch activates myosin light chain kinase in arterial smooth muscle

Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.     

Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase

Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with a modulation of the vascular smooth muscle cell (SMC) phenotype from a contractile, differentiated to a synthetic, dedifferentiated state. We previously reported that acute hypoxia represses cGMP-dependent protein kinase (PKG) expression in ovine fetal pulmonary venous SMCs (FPVSMCs). Therefore, we tested if altered expression of PKG could explain SMC phenotype modulation after exposure to hypoxia. Hypoxia-induced reduction in PKG protein expression strongly correlated with the repressed expression of SMC phenotype markers, myosin heavy chain (MHC), calponin, vimentin, alpha-smooth muscle actin (alphaSMA), and thrombospondin (TSP), indicating that hypoxic exposure of SMC induced phenotype modulation to dedifferentiated state, and PKG may be involved in SMC phenotype modulation. PKG-specific small interfering RNA (siRNA) transfection in FPVSMCs significantly attenuated calponin, vimentin, and MHC expression, with no effect on alphaSMA and TSP. Treatment with 30 microM Drosophila Antennapedia (DT-3), a membrane-permeable peptide inhibitor of PKG, attenuated the expression of TSP, MHC, alphaSMA, vimentin, and calponin. The results from PKG siRNA and DT-3 studies indicate that hypoxia-induced reduction in protein expression was also similarly impacted by PKG inhibition. Overexpression of PKG in FPVSMCs by transfection with a full-length PKG construct tagged with green fluorescent fusion protein (PKG-GFP) reversed the effect of hypoxia on the expression of SMC phenotype marker proteins. These results suggest that PKG could be one of the determinants for the expression of SMC phenotype marker proteins and may be involved in the maintenance of the differentiated phenotype in pulmonary vascular SMCs in hypoxia.     

Vasoconstrictors inhibit ATP-sensitive K+ channels in arterial smooth muscle through protein kinase C

The effects of vasoconstrictor-receptor (neuropeptide Y, alpha- adrenergic, serotonergic, histaminergic) stimulation on currents through ATP-sensitive potassium (KATP) channels in arterial smooth muscle cells were examined. Whole-cell KATP currents, activated by the synthetic KATP channel opener pinacidil or by the endogenous vasodilator, calcitonin gene-related peptide, which acts through protein kinase A, were measured in smooth muscle cells isolated from mesenteric arteries of rabbit. Stimulation of NPY-, alpha 1-, serotonin (5-HT2)-, and histamine (H1)-receptors inhibited KATP currents by 40- 56%. The signal transduction pathway that links these receptors to KATP channels was investigated. An inhibitor of phospholipase C (D609) and of protein kinase C (GF 109203X) reduced the inhibitory effect of these vasoconstrictors on KATP currents from 40-56% to 11-23%. Activators of protein kinase C, a diacylglycerol analogue and phorbol 12-myristate 13- acetate (PMA), inhibited KATP currents by 87.3 and 84.2%, respectively. KATP currents, activated by calcitonin gene-related peptide, were also inhibited (47-87%) by serotonin, phenylephrine, and PMA. We propose that KATP channels in these arterial myocytes are subject to dual modulation by protein kinase C (inhibition) and protein kinase A (activation).     

Methacholine sensitivity and cAMP protein kinase in tracheal smooth muscle

We studied regional variation in canine trachealis smooth muscle sensitivity and responsiveness to methacholine as well as basal and methacholine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase activity. The trachea between the cricoid cartilage and the carina was divided into three segments of equal length (designated cervical, middle, and thoracic regions), each consisting of approximately 12-14 cartilage rings. Smooth muscle strips from each of the three regions were exposed to cumulative half-log increments of methacholine chloride. The sensitivity (-log EC50) and responsiveness (force per cross-sectional area and force per milligram protein) of the smooth muscle to methacholine in each region was determined from these data. Smooth muscle strips from cervical and thoracic regions were frozen before and after exposure to cumulative half-log increments of methacholine up to each region's previously determined EC50. Frozen samples were assayed for cAMP content or cAMP-dependent protein kinase activity. The relationship between resting tension and methacholine sensitivity and responsiveness were studied. For the size strips we used, 4 g resting tension set the average cervical and thoracic strips at 96 and 101% of their optimal length, respectively. The methacholine EC50 was not affected by a variation in resting tension. Sensitivity to methacholine was 7.1, 6.8, and 6.5 for cervical, middle, and thoracic regions, respectively. The responsiveness of the cervical and thoracic smooth muscle to methacholine was 16.4 and 16.3 g force/mm2, respectively, at an EC50 methacholine. Basal cAMP was lower in cervical smooth muscle than in thoracic. cAMP-dependent protein kinase activity ratios under both basal and EC50 methacholine-stimulated conditions were lower in cervical smooth muscle than in thoracic. We have observed in trachealis smooth muscle an inverse relationship between methacholine sensitivity and either cAMP or cAMP-dependent protein kinase activity. We suggest that cAMP and cAMP-dependent protein kinase play a role in the regulation of airway smooth muscle sensitivity to cholinergic agonists.     

Oxidation of cysteines activates cGMP-dependent protein kinase.

The functional significance of the oxidation/reduction state of sulfhydryl groups of cGMP-dependent protein kinase (cGMP kinase) was studied at 30 degrees C using different metal ions as oxidizing agents. Mn2+, Zn2+, Fe2+, Ni2+, and Co2+ failed to activate cGMP kinase, whereas Cu2+, Cu+, Fe3+, Hg2+, and Ag+ activated cGMP kinase by oxidation with an activity ratio (-cGMP/+cGMP) of about 0.7. The activation was not caused by degradation of the enzyme to a cGMP-independent constitutively active form. Reduction of the Cu(2+)-activated and gel-filtered enzyme with dithiothreitol lowered the activity ratio in the absence of cGMP to 0.17. Oxidation did not change the kinetic and binding parameters of cGMP kinase significantly but reduced the number of titratable sulfhydryl groups from 9.5 +/- 0.7 to 6.0 +/- 0.4 cysteines/75-kDa subunit. The free cysteinyl residues of the native and Cu(2+)-oxidized cGMP kinase were labeled with 4-dimethylaminoazobenzene-4'-iodoacetamide or N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Tryptic peptides of the labeled proteins were isolated and sequenced. The cysteinyl residues oxidized by Cu2+ were identified as disulfide bonds between Cys-117 and Cys-195 and Cys-312 and Cys-518, respectively. Cu2+ activation of cGMP kinase was prevented by mild carboxymethylation of the reduced enzyme with iodoacetamide, which apparently modified these four cysteinyl groups. The results show that cGMP kinase is activated by the formation of at least one intrachain disulfide bridge.     

Stimulation of arterial smooth muscle L-type calcium channels by hydrogen peroxide requires protein kinase C

Changes in intracellular calcium regulate countless biological processes. In arterial smooth muscle, voltage-dependent L-type calcium channels are major conduits for calcium entry with the primary function being determination of arterial diameter. Similarly, changes in intracellular redox status, either discrete controlled changes or global pathological perturbations, are also critical determinants of cell function. We recently reported that in arterial smooth muscle cells, local generation of hydrogen peroxide leads to colocalized calcium entry through L-type calcium channels. Here we extend our investigation into mechanisms linking hydrogen peroxide to calcium influx through L-type calcium channels by focusing on the role of protein kinase C (PKC). Our data indicate that stimulation of L-type calcium channels by hydrogen peroxide requires oxidant-dependent increases in PKC catalytic activity. This effect is independent of classical cofactor-dependent activation of PKC by diacylglycerol. These data provide additional experimental evidence supporting the concept of oxidative stimulation of L-type calcium channels.     

Downregulation of cGMP-dependent protein kinase expression by inflammatory cytokines in vascular smooth muscle cells

NO and cGMP have antigrowth and anti-inflammatory effects on the vessel wall in response to injury. It is well established that after vascular injury proinflammatory cytokines are involved in vascular wall remodeling. The purpose of this study was to ascertain the signaling mechanisms involved in cGMP-dependent protein kinase (PKG) suppression by inflammatory cytokines in primary bovine aortic vascular smooth muscle cells (VSMC). Interleukin (IL)-I, tumor necrosis factor (TNF)-, and LPS decreased the mRNA and protein levels of PKG in VSMC. IL-I, TNF-, and LPS increased inducible nitric oxide synthase (iNOS) expression and cGMP production. Treatment of cells with selective inhibitors of iNOS or soluble guanylate cyclase (sGC) reversed the downregulation of PKG expression induced by cytokines and LPS. The NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) and 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), a NO-independent sGC activator, decreased PKG mRNA and protein expression in bovine aortic VSMC. Cyclic nucleotide analogs [8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP) and 8-(4-chlorophenylthio)adenosine 3,5'-cyclic monophosphate (CPT-cAMP)] also suppressed PKG mRNA and protein expression. However, CPT-cAMP was more effective than CPT-cGMP in decreasing PKG mRNA levels. Selective inhibition of PKA with the Rp isomer of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphorothioate (Rp-8p-CPT cAMPS) prevented the downregulation of PKG by LPS. In contrast, the Rp isomer of 8-(4-chlorophenylthio)guanosine 3,5'-cyclic monophosphorothioate (Rp-8p-CPT cGMPS; inhibitor of PKG) had no effect on LPS-induced inhibition of PKG mRNA and protein expression. These studies suggest that cross-activation of PKA in response to iNOS expression by inflammatory mediators downregulates PKG expression in bovine aortic VSMC. vascular injury; nitric oxide; inflammation     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

Cyclic GMP-dependent protein kinase activates cloned BKCa channels expressed in mammalian cells by direct phosphorylation at serine 1072

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the alpha-subunit of the large conductance Ca2+-activated K+ channel, cslo-alpha, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-alpha exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) of cslo-alpha channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Ialpha to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-alpha in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-alpha by direct phosphorylation at serine 1072.     

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.     

Involvement of cGMP-dependent protein kinase in the relaxation of ovine pulmonary arteries to cGMP and cAMP.

Agonist-induced smooth muscle relaxation occurs following an increase in intracellular concentrations of cGMP or cAMP. However, the role of protein kinase G (PKG) and/or protein kinase A (PKA) in cGMP- or cAMP-mediated pulmonary vasodilation is not clearly elucidated. In this study, we examined the relaxation responses of isolated pulmonary arteries of lambs (age = 10 +/- 1 days), preconstricted with endothelin-1, to increasing concentrations of 8-bromo-cGMP (8-BrcGMP) or 8-BrcAMP (cell-permeable analogs), in the presence or absence of Rp-8-beta-phenyl-1,N(2)-etheno-bromoguanosine cyclic monosphordthioate (Rp-8-PET-BrcGMPS) or KT-5720, selective inhibitors of PKG and PKA, respectively. When examined for specificity, Rp-8-Br-PET-cGMPS abolished PKG, but not PKA, activity in pulmonary arterial extracts, whereas KT-5720 inhibited PKA activity only. 8-BrcGMP-induced relaxation was inhibited by the PKG inhibitor only, whereas 8-BrcAMP-induced relaxation was inhibited by both inhibitors. A nearly fourfold higher concentration of cAMP than cGMP was required to relax arteries by 50% and to activate PKG by 50%. Our results demonstrate that relaxation of pulmonary arteries is more sensitive to cGMP than cAMP and that PKG plays an important role in both cGMP- and cAMP-mediated relaxation.     

Site of action of fatty acids and other charged lipids on BKCa channels from arterial smooth muscle cells

Fattyacids and other negatively charged single-chain lipids increaselarge-conductance Ca²⁺-activated K⁺(BK_Ca) channel activity, whereas sphingosine and otherpositively charged single-chain lipids suppress activity. Because thesemolecules are effective on both inside-out and outside-out patches andbecause they can flip across the bilayer, the location of their site of action is unclear. To identify the site of action of charged lipids onthis channel, we used two compounds that are unlikely to flip acrossthe lipid bilayer. Palmitoyl coenzyme A (PCoA) was used to identify thesite of action of negatively charged lipids, and a positively chargedmyristoylated pentapeptide (myr-KPRPK) was used to investigate the siteof action of positively charged lipids. The effect of these compoundson channel activity was studied in excised patches using patch-clamptechniques. In "normal" ionic strength solutions and in experimentswhere high-ionic strength solutions were used to shield membranesurface charge, PCoA increased channel activity only when applied tooutside-out patches, suggesting that the site of action of negativelycharged lipids is located on the outer surface of the membrane. Adecrease in activity, similar to that of other positively chargedlipids, was observed only when myr-KPRPK was applied to outside-outpatches, suggesting that positively charged lipids suppress activity byalso acting on the outer membrane surface. Some channel blockadeeffects of myr-KPRPK and KPRPK are also described. The sidedness ofaction suggests that modulation of channel activity by single-chainlipids can occur by their interaction with the channel protein.

     

Selective phosphorylation of the IP3R-I in vivo by cGMP-dependent protein kinase in smooth muscle

This study examined the expression of inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) types and PKG isoforms in isolated gastric smooth muscle cells and determined the ability of PKG and PKA to phosphorylate IP(3)Rs and inhibit IP(3)-dependent Ca(2+) release, which mediates the initial phase of agonist-induced contraction. PKG-Ialpha and PKG-Ibeta were expressed in gastric smooth muscle cells, together with IP(3)-R-associated cG-kinase substrate, a protein that couples PKG-Ibeta to IP(3)R-I. IP(3)R-I and IP(3)R-III were also expressed, but only IP(3)R-I was phosphorylated by PKA and PKG in vitro and exclusively by PKG in vivo. Sequential phosphorylation by PKA and by PKG-Ialpha in vitro showed that PKA phosphorylated the same site as PKG (presumably S(1755)) and an additional PKA-specific site (S(1589)). In intact muscle cells, agents that activated PKG or both PKG and PKA induced IP(3)R-I phosphorylation that was reversed by the PKG inhibitor (8R,9S,11s)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced IP(3)R-I phosphorylation in permeabilized but not intact muscle cells, implying that PKA does not gain access to IP(3)R-I in intact muscle cells. The pattern of IP(3)R-I phosphorylation in vivo and in vitro was more consistent with phosphorylation by PKG-Ialpha. Phosphorylation of IP(3)R-I in microsomes by PKG, PKA, or a combination of PKG and PKA inhibited IP(3)-induced Ca(2+) release to the same extent, implying that inhibition was mediated by phosphorylation of the PKG-specific site. We conclude that IP(3)R-I is selectively phosphorylated by PKG-I in intact smooth muscle resulting in inhibition of IP(3)-dependent Ca(2+) release.