Cell membrane stretch activates intermediate-conductance Ca2+-activated K+ channels in arterial smooth muscle cells

The aim of this study is to determine the signal transduction of membrane stretch on intermediate-conductance Ca(2+)-activated K(+) (IKca) channels in rat aorta smooth muscle cells using the patch-clamp technique. To stretch the cell membrane, both suction to the rear end of patch pipette and hypotonic shock were used. In cell-attached and inside-out patch configurations, the open probability of IKca channels increased when 20- to 45-mmHg suction was applied. Hyposmotic swelling efficiently increased IKca channel current. When the Ca(2+)-free solution was superfused, the activation of IKca current by the hyposmotic swelling was reduced. Furthermore, gadolinium (Gd(3+)) attenuated the activation of IKca channels induced by hyposmotic swelling, whereas nicardipine did not. In the experiments with Ca(2+)-free bath solution, pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely abolished the stretch-induced activation of IKca currents. The stretch-induced activation of IKca channels was strongly inhibited by cytochalasin D, indicating a role for the F-actin in modulation of IKca channels by changes in cell stretching. These data suggest that cell membrane stretch activates IKca channels. In addition, the activation is associated with extracellular Ca(2+) influx through stretch-activated nonselective cation channels, and is also modulated by the F-actin cytoskeleton and the activation of PKC.     

Effects of 8-bromo-cGMP on Ca2+ levels in vascular smooth muscle cells: possible regulation of Ca2+-ATPase by cGMP-dependent protein kinase.

The effects of 8-bromo-cGMP on intracellular calcium concentrations in cultured rat aortic smooth muscle cells were studied. Both angiotensin II and depolarizing concentrations of K+ stimulated Ca2+ accumulation in the cytoplasm. The increase in Ca2+ due to angiotensin II was associated with an increase in inositol phosphates, while that due to K+ was not. Preincubation of cells with 8-bromo-cGMP (100 microM) caused an inhibition of peak Ca2+ accumulation to either angiotensin II or K+. To probe the mechanism of action of cGMP in vascular smooth muscle, the effects of cGMP-dependent protein kinase on Ca2+-ATPase from the cultured cell particulate material were investigated. Ca2+-activated ATPase was stimulated approximately equal to 2-fold by exogenous calmodulin and up to 4-fold by low concentrations of purified cGMP-dependent protein kinase. The inclusion of both calmodulin and cGMP-dependent protein kinase resulted in an additive stimulation of Ca2+-ATPase. Stimulation of Ca2+-ATPase activity was observed at all Ca2+ concentrations tested (0.01-1.0 microM). cAMP-dependent protein kinase catalytic subunit and protein kinase C were either ineffective or less effective than cGMP-dependent protein kinase in stimulating the Ca2+-ATPase from rat aortic smooth muscle cells. These results suggest a possible mechanism of action for cGMP in mediating decreases in cytosolic Ca2+ through activation of a Ca2+-ATPase and the subsequent removal of Ca2+ from the cell.     

Raf-1 kinase activation by angiotensin II in adrenal glomerulosa cells: roles of Gi, phosphatidylinositol 3-kinase, and Ca2+ influx

Little is known of the mechanisms leading to mitogen-activated protein kinase (MAPK) activation via Gq-coupled receptors. We therefore examined the pathways by which angiotensin II (Ang II) activates Raf-1 kinase, an upstream intermediate in the pathway to MAPK, via the Gq-coupled AT1 angiotensin receptor in bovine adrenal glomerulosa (BAG) cells. Ang II caused a rapid and transient activation of Raf-1 that reached a peak at 5-10 min. Ang II was a potent stimulus of Raf-1 activation with an ED50 of 10 pM and a maximal response at 1 nM, although higher Ang II concentrations elicited a submaximal response. Ang II-stimulated Raf-1 activity was unaffected by down-regulation of protein kinase C and intracellular Ca2+ chelation (using BAPTA) but was partially inhibited by pertussis toxin, and was abolished by manumycin A. Removal of extracellular Ca2+ (by EGTA) or blockade of L type Ca2+ channels (by nifedipine), as well as inhibition of MEK-1 kinase (by PD98059), enhanced Raf-1 activity, whereas wortmannin (100 nM) inhibited approximately one half of Ang II-stimulated Raf-1 activity. Hence, Raf-1 kinase activation by Ang II in BAG cells is dependent on Ras, is mediated in part via Gi and phosphatidylinositol 3-kinase, and is negatively regulated via Ca2+ influx and a downstream signaling element(s).     

Sarcosine1, glycine8 angiotensin II is a functional AT1 angiotensin receptor antagonist

Sarcosine¹, glycine⁸ angiotensin II (SG Ang II) displayed unusual characteristics in early pharmacological studies. It was a potent antagonist of the dipsogenic actions of intracerebroventricularly administered Ang II in the rat, but showed low affinity for bovine cerebellar Ang II receptors. It has recently been shown that SG Ang II binds preferentially to the AT₁ receptor, To determine if SG Ang II is a functional antagonist of the AT₁ receptor-mediated calcium signaling, CHO cells stably transfected with AT₁ receptors were exposed to Ang II in the presence and absence of SG Ang II. At concentrations of 10–100 nM, SG Ang II completely inhibited Ang II-stimulated intracellular Ca²⁺ mobilization in AT_1A and AT_1B receptor-transfected cells. SG Ang II and ¹²⁵I-SG Ang II bound to AT_1A and AT_1B receptor-transfected cells with K _D and K ₁ values of 2–4 nM. In contrast, SG Ang II bound to AT₂ transfected cells with a K ₁ value of 7.86 μM. These results demonstrate that SG Ang II is a selective and functional peptide antagonist of the AT₁ angiotensin II receptor subtype.     

Calcitonin gene-related peptide activates the K+ channels of vascular smooth muscle cells via adenylate cyclase

The aim of the present study was to examine the effects of calcitonin gene-related peptide (CGRP) on the K⁺ channels of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 nM CGRP activated two types of K⁺ channels the Ca²⁺-activated K⁺ channel (K_Ca channel) and the ATP-sensitive K⁺ channel (K_ATP channel) in cell-attached patch configurations. In cells pretreated with Rp-cAMPS, a membrane-permeable inhibitor of cAMP-dependent protein kinase (PKA), extracellular application of 100 nM CGRP could not activate the K_Ca or K_ATP channel, indicating that the activation of the K⁺ channels by CGRP occurs in connection with PKA. In the cell-attached patch configurations, extracellular application of 1 mM dibutyryl cAMP, a membrane permeable cAMP, activated K_Ca and K_ATP channels. In inside-out patch configurations, application of PKA to the cytosolic side activated both the K_Ca and K_ATP channels. These results indicate that CGRP modulates the K⁺ channels of vascular smooth muscle cells via adenylate cyclase, i.e., cAMP-PKA pathway, and contributes to control of vascular tone.     

Involvement of a GTP-binding protein in stimulating action of angiotensin II on calcium channels in vascular smooth muscle cells.

The possible involvement of a GTP-binding protein in the regulation of Ca2+ channels by angiotensin II (Ang II) in vascular muscle cells was investigated by the whole-cell voltage-clamp method. Single cells were freshly isolated from guinea pig portal vein. The pipette solution contained high Cs+ to inhibit K+ currents and thereby isolate the Ca2+ channel current. Ba2+ (2 mM) was in the bath solution as a charge carrier for the Ca2+ channel. Application of Ang II (0.1-100 nM) produced an increase in peak amplitude of the Ba2+ current, with a shift of the current-voltage curve in the negative direction. These effects were inhibited by pretreatment with an antagonist of the Ang II receptor, [Sar1,Ile8]-Ang II. Presence of 0.1 mM GTP in the pipette solution stabilized the Ang II action, but 0.3-1.0 mM GDP-beta-S and 1.0 mM GTP-gamma-S inhibited it. GTP-gamma-S alone produced a slowly progressing increase in the basal (unstimulated) current amplitude. Preincubation of muscle tissues with pertussis toxin (1 micrograms/ml, for up to 6 hours at 36 degrees C) or intracellular application of preactivated pertussis toxin (1 micrograms/ml) plus NAD (1 mM) did not inhibit the Ang II action. Cholera toxin (10 micrograms/ml) also had no effect on the Ang II action. These results suggest that the Ang II stimulation of Ca2+ channels in smooth muscle of guinea pig portal vein may be mediated by a G protein that is insensitive to both pertussis toxin and cholera toxin.     

Angiotensin selectively activates a subpopulation of postganglionic sympathetic neurons in mice

Angiotensin II (Ang II) increases renal sympathetic nerve activity in anesthetized mice before and after ganglionic blockade, suggesting that Ang II may directly activate postganglionic sympathetic neurons. The present study directly tested this hypothesis in vitro. Neurons were dissociated from aortic-renal and celiac ganglia of C57BL/6J mice. Cytosolic Ca(2+) concentration ([Ca(2+)](i)) was measured with ratio imaging using fura 2. Ang II increased [Ca(2+)](i) in a subpopulation of sympathetic neurons. At a concentration of 200 nmol/L, 14 (67%) of 21 neurons responded with a rise in [Ca(2+)](i). The Ang II type 1 (AT(1)) receptor blocker (losartan, 2 micromol/L) but not the Ang II type 2 (AT(2)) receptor blocker (PD123,319, 4 micromol/L) blocked this effect. The Ang II-induced [Ca(2+)](i) increase was abolished by removal of extracellular Ca(2+) but not altered by depletion of intracellular Ca(2+) stores with thapsigargin. Ang II no longer elicited a [Ca(2+)](i) increase in the presence of lanthanum (25 micromol/L). The specific N-type and L-type Ca(2+) channel blockers, omega-conotoxin GVIA and nifedipine, respectively, significantly inhibited the Ang II-induced [Ca(2+)](i) increase. The protein kinase C inhibitor H7 but not the protein kinase A inhibitor H89 blocked the response to Ang II. These results demonstrate that Ang II selectively activates a subpopulation of postganglionic sympathetic neurons in aortic-renal and celiac ganglia, triggering Ca(2+) influx through voltage-gated Ca(2+) channels. This effect is mediated through AT(1) receptors and requires the activation of protein kinase C. The activation of a subgroup of sympathetic neurons by Ang II may exert unique effects on kidney function in pathological states associated with elevated Ang II.     

Angiotensin II-activated protein kinase C targets caveolae to inhibit aortic ATP-sensitive potassium channels

OBJECTIVE: The vasoconstrictor angiotensin II (Ang II) acts at G(q/11)-coupled receptors to suppress ATP-sensitive potassium (K(ATP)) channel activity via activation of protein kinase C (PKC). The aim of this study was to determine the PKC isoforms involved in the Ang II-induced inhibition of aortic K(ATP) channel activity and to investigate potential mechanisms by which these isoforms specifically target these ion channels. METHODS AND RESULTS: We show that the inhibitory effect of Ang II on pinacidil-evoked whole-cell rat aortic K(ATP) currents persists in the presence of G?6976, an inhibitor of the conventional PKC isoforms, but is abolished by intracellular dialysis of a selective PKCepsilon translocation inhibitor peptide. This suggests that PKC-dependent inhibition of aortic K(ATP) channels by Ang II arises exclusively from the activation and translocation of PKCepsilon. Using discontinuous sucrose density gradients and Western blot analysis, we show that Ang II induces the translocation of PKCepsilon to cholesterol-enriched rat aortic smooth muscle membrane fractions containing both caveolin, a protein found exclusively in caveolae, and Kir6.1, the pore-forming subunit of the vascular K(ATP) channel. Immunogold electron microscopy of rat aortic smooth muscle plasma membrane sheets confirms both the presence of Kir6.1 in morphologically identifiable regions of the membrane rich in caveolin and Ang II-evoked migration of PKCepsilon to these membrane compartments. CONCLUSIONS: Ang II induces the recruitment of the novel PKC isoform, PKCepsilon, to arterial smooth muscle caveolae. This translocation allows PKCepsilon access to K(ATP) channels compartmentalized within these specialized membrane microdomains and highlights a potential role for caveolae in targeting PKC isozymes to an ion channel effector.     

Activation of angiotensin II type I receptor promotes protein kinase C translocation and cell proliferation in human cultured breast epithelial cells

The effect of angiotensin II (Ang II) on Ca(2+) signalling in human primary cultured breast epithelial cells was investigated by using fura-2 as the Ca(2+) probe. Ang II (0.1-1000 nM) induced an intracellular free calcium ([Ca(2+)](i)) transient peak which was unchanged by external Ca(2+ )removal. In Ca(2+)-free medium pretreatment with thapsigargin abolished Ang II-induced Ca(2+ )release. Suppression of 1,4,5-inositol trisphosphate formation by U73122, a phospholipase C inhibitor, blocked the Ang II-induced Ca(2+) response. Losartan (DuP753), an inhibitor of Ang II type I receptor (AT1), decreased the [Ca(2+)](i) increase evoked by Ang II, while CGP4221A, an inhibitor of Ang II type II receptor (AT2) did not. AT1 desensitisation was demonstrated with respect to the Ca(2+) response after subsequent exposure of cells to Ang II and also after pretreatment for 25 min with 1000 nM phorbol 12-myristate 13-acetate. Staurosporine, an inhibitor of protein kinases C (PKC), inhibited the AT1 desensitisation. Epithelial breast cells expressed PKC-alpha, -beta1, -delta and -zeta isozymes, and Ang II provoked translocation from the cytosol to the membranes of PKC-alpha, -beta1, and -delta (but not -zeta). Ang II was also able to stimulate cell proliferation in a dose-dependent manner; this effect was blocked by G? 6976, a specific inhibitor of PKC-alpha and -beta1, the Ca(2+)-dependent isozymes. The main conclusion of this study is that the the Ang II signalling mechanism in breast epithelial cells is based on the elevation of [Ca(2+)](i )released from intracellular stores through AT1 activation. In addition, Ang II stimulates cell proliferation by the activation of PKC isozymes.     

Angiotensin II signaling pathways mediate expression of cardiac T-type calcium channels

Recent studies indicate that cardiac T-type Ca2+ current (ICaT) reappears in hypertrophied ventricular cells. The aim of this study was to investigate the role of angiotensin II (Ang II), a major inducer of cardiac hypertrophy, in the reexpression of T-type channel in left ventricular hypertrophied myocytes. We induced cardiac hypertrophy in rats by abdominal aorta stenosis for 12 weeks and thereafter animals were treated for 2 weeks with losartan (12 mg/kg per day), an antagonist of type 1 Ang II receptors (AT1). In hypertrophied myocytes, we showed that the reexpressed ICaT is generated by the CaV3.1 and CaV3.2 subunits. After losartan treatment, ICaT density decreased from 0.40+/-0.05 pA/pF (n=26) to 0.20+/-0.03 pA/pF (n=27, P<0.01), affecting CaV3.1- and CaV3.2-related currents. The amount of CaV3.1 mRNA increased during hypertrophy and retrieved its nonhypertrophic level after losartan treatment, whereas the amount of CaV3.2 mRNA was unaffected by stenosis. In cultured newborn ventricular cells, chronic Ang II application (0.1 micromol/L) also increased ICaT density and CaV3.1 mRNA amount. UO126, a mitogen-activated protein kinase kinase-1/2 (MEK1/2) inhibitor, reduced Ang II-increased ICaT density and CaV3.1 mRNA amount. Bosentan, an endothelin (ET) receptor antagonist, reduced Ang II-increased ICaT density without affecting the amount of CaV3.1 mRNA. Finally, cotreatment with bosentan and UO126 abolished the Ang II-increased ICaT density. Our results show that AT1-activated MEK pathway and autocrine ET-activated independent MEK pathway upregulate T-type channel expression. Ang II-increased of ICaT density observed in hypertrophied myocytes may play a role in the pathogenesis of Ca2+ overload and arrhythmias seen in cardiac pathology.     

Serotonin potentiates angiotensin II--induced vascular smooth muscle cell proliferation.

Vascular smooth muscle cell (VSMC) proliferation is a key feature in the development of atherosclerosis and restenosis after angioplasty, which can occur in response to many different humoral and mechanical stimuli. We investigated the growth promoting activities of two potent vasoactive substances, angiotensin II (Ang II) and serotonin (5-HT), on cultured rabbit VSMCs. Growth-arrested VSMCs were incubated with serum-free medium containing different concentrations of Ang II in the presence or absence of 5-HT. [3H]thymidine incorporation into VSMC DNA was measured as an index of cell proliferation. Ang II and 5-HT stimulated DNA synthesis in a dose-dependent manner with a maximal effect at 1.75 microM for Ang II (202%) and 50 microM for 5-HT (205%). When added together, low concentrations of Ang II (1 microM) and 5-HT (5 microM) synergistically induced DNA synthesis (363%). Candesartan (1 microM), an AT(1) receptor antagonist, but not PD 123319 (1 microM), an AT(2) receptor antagonist, inhibited the mitogenic effect on Ang II and its interaction with 5-HT. Sarpogrelate (10 microM), a 5-HT(2A) receptor antagonist, and pertussis toxin (10 ng/ml) inhibited the mitogenic effect of 5-HT and its interaction with Ang II. The protein kinase C inhibitor Ro 31-8220 (0.1 microM), the Raf-1 inhibitor radicicol (10 microM), and the MAPK kinase inhibitor PD 098059 (10 microM) abolished mitogenic effects of Ang II and 5-HT, and also their synergistic interaction. The JAK2 inhibitor AG 490 (10 microM) had only a minimal inhibitory effect of Ang II-induced DNA synthesis but significantly inhibited the interaction of Ang II with 5-HT. The synergistic effect on Ang II (1 microM) with 5-HT (5 microM) on DNA synthesis was completely reversed by the combined use of both candesartan (1 microM) and sarpogrelate (10 microM). Our results suggest that Ang II and 5-HT exert a synergistic interaction on VSMC proliferation via AT(1) and 5-HT(2A) receptors. The activation of MAPK and JAK/STAT pathways may explain the synergistic interaction between Ang II and 5-HT.     

SNAP-25, a SNARE protein, inhibits two types of K channels in esophageal smooth muscle

BACKGROUND & AIMS: The plasma membrane-associated soluble N-ethylmaleimide-sensitive factors attachment protein receptors (SNAREs), synaptosome-associated protein of 25 kilodaltons (SNAP-25), and syntaxin 1A, have been found to physically interact with and functionally modify membrane-spanning ion channels. Studies were performed in cat esophageal body and lower esophageal sphincter (LES) smooth muscle to (1) show the presence of SNAP-25, and (2) determine whether SNAP-25 affects K+ channel activity. METHODS: Single circular muscle cells from the esophageal body and sphincter were studied. Cellular localization of SNAP-25 and K+ channel activity were assessed. RESULTS: SNAP-25 was found in the plasma membrane of all regions examined. Outward K+ currents in body circular muscle were mainly composed of large conductance Ca2+-activated channel currents (K(Ca), 40.1%) and delayed rectifier K+ channel currents (K(V), 54.2%). Microinjection of SNAP-25 into muscle cells caused a dose-dependent inhibition of both outward K+ currents, maximal 44% at 10(-8) mol/L. Cleavage of endogenous SNAP-25 by dialyzing botulinum neurotoxin A into the cell interior resulted in a 35% increase in outward currents. CONCLUSIONS: SNAP-25 protein is present in esophageal smooth muscle cells, and inhibits both K(V) and K(Ca) currents in circular muscle cells. The findings suggest a role for SNAP-25 in regulation of esophageal muscle cell excitability and contractility, and point to potential new targets for treatment of esophageal motor disorders.     

Modulation of cardiac Na+ channels expressed in a mammalian cell line and in ventricular myocytes by protein kinase C.

Cardiac rH1 Na+ channel alpha subunits were expressed in cells of the Chinese hamster lung 1610 cell line by transfection, and a stable cell line expressing cardiac Na+ channels (SNa-rH1) was isolated. Mean Na+ currents of 2.2 +/- 1.0 nA were recorded, which corresponds to a cell surface density of approximately 1-2 channels active at the peak of the Na+ current per micron2. The expressed cardiac Na+ current was tetrodotoxin resistant (Kd = 1.8 microM) and had voltage-dependent properties similar to those of the Na+ current in neonatal ventricular myocytes. Activation of protein kinase C by 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10 microM) decreased this current approximately 33% at a holding potential of -114 mV and 56% at -94 mV. This reduction in peak current was caused in part by an 8- to 14-mV shift of steady-state inactivation in the hyperpolarized direction. Na+ channel activation was unchanged. Effects of OAG in SNa-rH1 cells and in neonatal rat cardiac myocytes were similar, except that the time course of inactivation was slowed either transiently or persistently when protein kinase C was activated in myocytes bathed in low-Ca2+ (1 microM) or Ca(2+)-free solution but was unaffected in SNa-rH1 cells. The effects of OAG on cardiac Na+ current were blocked in cells that had been previously microinjected with a peptide inhibitor of protein kinase C but not with a peptide inhibitor of cAMP-dependent protein kinase, indicating that protein kinase C is responsible for the effect of OAG. Single-channel recordings from SNa-rH1 cells showed that the probability of channel opening was reduced by OAG, but the conductance was unaffected. OAG did not induce the late Na+ channel openings observed with PKC modulation of neuronal and skeletal muscle Na+ channels. Thus, the substantial reduction in Na+ current at normal diastolic depolarizations with 10 microM OAG is due to failure of channel opening in response to depolarization. Such Na+ current reductions may have profound effects on cardiac cell excitability.     

Role of Ca2+-activated K+ channels on adrenergic responses of human saphenous vein

BACKGROUND: We studied the participation of K(+) channels on the adrenergic responses in human saphenous veins as well as the intervention of dihydropyridine-sensitive Ca(2+) channels on modulation of adrenergic responses by K(+) channels blockade. METHODS: Saphenous vein rings were obtained from 40 patients undergoing coronary artery bypass surgery. The vein rings were suspended in organ bath chambers for isometric recording of tension. RESULTS: Iberiotoxin (10(-7) mol/L), an inhibitor of large conductance Ca(2+)-activated K(+) channels, and charybdotoxin (10(-7) mol/L), an inhibitor of both large and intermediate conductance Ca(2+)-activated K(+) channels, enhanced the contractions elicited by electrical field stimulation and produced a leftward shift of the concentration-response curve to norepinephrine. In contrast, the inhibitor of small conductance Ca(2+)-activated K(+) channels apamin (10(-6) mol/L) did not modify the contractile response to electrical field stimulation or norepinephrine. In the presence of the dihydropyridine Ca(2+)-channel blocker nifedipine (10(-6) mol/L), iberiotoxin and charybdotoxin failed to enhance the contractile responses to electrical field stimulation and norepinephrine. CONCLUSIONS: The results suggest that large conductance Ca(2+)-activated K(+) channels are activated by stimulation with norepinephrine to counteract the adrenergic-induced contractions of human saphenous vein. Thus, inhibition of these channels increases significantly the contraction, an effect that appears to be mediated by an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels.     

Evidence for the stimulatory effect of resveratrol on Ca(2+)-activated K+ current in vascular endothelial cells

OBJECTIVE: Resveratrol, a natural phytoalexin compound, is present in grapes and wine, and it can produce vasorelaxation. However, little is known of its mechanisms of action on ionic currents in endothelial cells. METHODS: The effect of resveratrol on Ca(2+)-activated K+ currents in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein was investigated with the aid of the patch-clamp technique. RESULTS: In the whole-cell configuration, resveratrol reversibly increased the amplitude of K+ outward currents. The increase in outward current caused by resveratrol was greatly inhibited by iberiotoxin (200 nM) or paxilline (1 microM), but not by glibenclamide (10 microM), tamoxifen (10 microM), or beta-bungarotoxin (200 nM). Thus, this outward current is believed to be Ca(2+)-activated K+ current (I K(Ca)). In the inside-out configuration, bath application of resveratrol (30 microM) caused no change in the single-channel conductance, but increased the activity of large-conductance Ca(2+)-activated K+ (BKCa) channels. Resveratrol enhanced the channel activity in a concentration-dependent manner. The EC50 value for resveratrol-induced channel activity was 20 microM. The resveratrol-stimulated increase in the channel activity was independent of internal Ca2+. Resveratrol (30 microM) also shifted the activation curve of BKCa channels to less positive membrane potentials. The change in the kinetic behavior of BKCa channels caused by resveratrol in these cells in due to an increase in mean open time and a decrease in mean closed time. In a pancreatic islet endothelial cell line (MS1), resveratrol (30 microM) also increased the activity of intermediate-conductance KCa channels. CONCLUSIONS: These results provide evidence that in addition to the presence of antioxidative activity, resveratrol can also stimulate KCa channels in endothelial cells. The direct stimulation of these KCa channels by resveratrol may be responsible for its effect on the functional activities of endothelial cells.     

Guanosine 5''-monophosphate modulates gating of high-conductance Ca2+-activated K+ channels in vascular smooth muscle cells.

Ca2+-activated K+ channels (PKCa channels) account for the predominant K+ permeability of many types of smooth muscle cells. When activated, they oppose depolarization due to Na+ and Ca2+ channel activity. Several vasodilatory agents that increase intracellular cGMP levels (e.g., nitroprusside, adenosine, and atrial natriuretic factor) enhance the activity of these high-conductance PKCa channels in on-cell patches of bovine aortic smooth muscle cells. In addition, dibutyryl-cGMP (1.0 mM) causes a similar increase in channel activity. To pursue the mechanism of channel modulation by these agents, a series of guanine and adenine nucleotides were evaluated by using inside-out excised patches. Whereas cAMP, AMP, ADP, and ATP were ineffective, all of the corresponding guanine nucleotides potentiated PKCa channel activity when tested at a high concentration (500 microM). However, only GMP consistently enhanced channel activity in the 1-100 microM range by increasing the percent open time and frequency of opening of these channels over a wide range of potentials and Ca2+ levels without affecting single-channel conductance. Thus, GMP is a potent modulator of PKCa channels and it, rather than cGMP, may mediate the action of the vasodilators examined in this study.     

Effects of angiotensin II on nitric oxide generation in proliferating and quiescent rat coronary microvascular endothelial cells.

Nitric oxide (NO) and the mitogenic peptide angiotensin II (Ang II) have been implicated in endothelial cell growth. However, the putative relationship between these two opposing agents with respect to endothelial cell growth remains unknown. In this study, proliferating and confluent rat coronary microvascular endothelial cells (CMEC) were treated with different doses of Ang II, Ca2+ ionophore A23187, or valsartan (an Ang II type 1 (AT1) receptor inhibitor) alone or in combination for 24 h before measuring the nitrite levels as an index of NO generation. NO production and endothelial NO synthase (eNOS) mRNA/protein expression were found to be 3-fold greater in proliferating vs. quiescent CMEC. Treatments of CMEC with Ang II or Ca2+ ionophore A23187 equally increased NO production without altering the fold-difference in the basal release of NO from proliferating vs. confluent CMEC. Valsartan abolished NO production in CMEC treated with Ang II but not Ca2+ ionophore A23187. Treatments of endothelium-intact vascular rings with Ang II (1 nmol/l to 10 micromol/l) plus valsartan or PD-123319, an Ang II type 2 (AT2) receptor inhibitor, attenuated vascular responses to acetylcholine in an Ang II dose-dependent manner. In these rings, phenylephrine produced significant increases in contractile responses only at nmol/l concentrations of Ang II. In contrast, pharmacological and mechanical inactivation of endothelium enhanced contractile responses to phenylephrine at micromol/I concentrations of Ang II. These data demonstrate that Ang II stimulates NO production in CMEC in both an AT1- and an AT2 receptor-regulated manner, and that this stimulation of NO may be beneficial in counterbalancing the direct vasoconstrictor effect of Ang II on underlying smooth muscle cells.     

The role of tyrosine kinases in capacitative calcium influx-mediated aldosterone production in bovine adrenal zona glomerulosa cells.

In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) involves the activation of a capacitative Ca(2+) influx through calcium release-activated calcium (CRAC) channels. In various mammalian cell systems, it has been shown that CRAC channel activation and Ca(2+) entry require tyrosine kinase activity. We have therefore examined in this work whether similar mechanisms contribute to Ang II-induced mineralocorticoid biosynthesis. In fluo-3-loaded isolated bovine glomerulosa cells, two inhibitors of tyrosine kinases, genistein and methyl-2, 5-dihydroxycinnamate (MDHC) (100 microM) prevented capacitative Ca(2+) entry elicited by Ang II (by 54 and 62% respectively), while the inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, lavendustin A, was without effect. Similar results were observed on Ca(2+) influx triggered by thapsigargin, an inhibitor of microsomal Ca(2+) pumps. The inhibitors blocked Ang II-stimulated pregnenolone and aldosterone production in the same rank order. In addition to its specific effect on capacitative Ca(2+) influx, genistein also affected the late steps of the steroidogenic pathway, as shown by experiments in which the rate-limiting step (intramitochondrial cholesterol transfer) was bypassed with 25-OH-cholesterol (25-OH-Chol), cytosolic calcium was clamped at stimulated levels or precursors of the late enzymatic steps were supplied. In contrast, genistin, a structural analogue of genistein devoid of tyrosine kinase inhibitory activity, was almost without effect on pregnenolone or 11-deoxycorticosterone (DOC) conversion to aldosterone. These results suggest that, in bovine adrenal glomerulosa cells, Ang II promotes capacitative Ca(2+) influx and aldosterone biosynthesis through tyrosine kinase activation.     

Angiotensin II stimulation of Na+/K+ATPase activity and cell growth by calcium-independent pathway in MCF-7 breast cancer cells

Here we demonstrated, by RT-PCR analysis, the expression of both angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in a breast cancer epithelial cell line, MCF-7. Ang II was not able to affect the intracellular Ca2+ concentration in Fura-2 loaded cells suggesting that AT1-mediated phospholipid hydrolysis is not involved in its intracellular transduction pathway. Ang II modulated the activity of the Na+/K+ATPase in a dose- and time-dependent manner and was mitogenic, with a dose-dependent (1-1000 nM) proliferative effect and a maximal response at 100 nM. Both Na+/K+ATPase activation and stimulation of proliferation were mediated by binding of Ang II to AT1, as the effects were completely blocked by DuP 753, a specific AT1 antagonist. CGP 42112, an AT2 antagonist, did not affect Ang II actions. The main conclusion of this study is that Ang II exerts its effects on cell proliferation and Na+/K+ATPase in breast cancer epithelial cells, MCF-7, via AT1 activation independently of the Ca(2+) signalling mechanism.