Smooth Muscle Intracellular Ca Research Articles

Activation of renal vascular smooth muscle TRPV4 channels by 5-hydroxytryptamine impairs kidney function in neonatal pigs.

Control of microvascular reactivity by 5-hydroxytryptamine (5-HT; serotonin) is complex and may depend on vascular bed type and 5-HT receptors. 5-HT receptors consist of seven families (5-HT1-5-HT7), with 5-HT2 predominantly mediating renal vasoconstriction. Cyclooxygenase (COX) and smooth muscle intracellular Ca2+ levels ([Ca2+]i) have been implicated in 5-HT-induced vascular reactivity. Although 5-HT receptor expression and circulating 5-HT levels are known to be dependent on postnatal age, control of neonatal renal microvascular function by 5-HT is unclear. In the present study, we demonstrate that 5-HT stimulated human TRPV4 transiently expressed in Chinese hamster ovary cells. 5-HT2A is the predominant 5-HT2 receptor subtype in freshly isolated neonatal pig renal microvascular smooth muscle cells (SMCs). HC-067047 (HC), a selective TRPV4 blocker, attenuated cation currents induced by 5-HT in the SMCs. HC also inhibited the 5-HT-induced increase in renal microvascular [Ca2+]i and constriction. Intrarenal artery infusion of 5-HT had minimal effects on systemic hemodynamics but reduced renal blood flow (RBF) and increased renal vascular resistance (RVR) in the pigs. Transdermal measurement of glomerular filtration rate (GFR) indicated that kidney infusion of 5-HT reduced GFR. HC and 5-HT2 receptor antagonist ritanserin attenuated 5-HT effects on RBF, RVR, and GFR. Moreover, the serum and urinary COX-1 and COX-2 levels in 5-HT-treated piglets were unchanged compared with the control. These data suggest that activation of renal microvascular SMC TRPV4 channels by 5-HT impairs kidney function in neonatal pigs independently of COX production.

Mechanical activation of angiotensin II type 1 receptors causes actin remodelling and myogenic responsiveness in skeletal muscle arterioles.

Candesartan, an inverse agonist of the type 1 angiotensin II receptor (AT1 R), causes a concentration-dependent inhibition of pressure-dependent myogenic tone consistent with previous reports of mechanosensitivity of this G protein-coupled receptor. Mechanoactivation of the AT1 R occurs independently of local angiotensin II production and the type 2 angiotensin receptor. Mechanoactivation of the AT1 R stimulates actin polymerization by a protein kinase C-dependent mechanism, but independently of a change in intracellular Ca2+ . Using atomic force microscopy, changes in single vascular smooth muscle cell cortical actin are observed to remodel following mechanoactivation of the AT1 R. The Gq/11 protein-coupled angiotensin II type 1 receptor (AT1 R) has been shown to be activated by mechanical stimuli. In the vascular system, evidence supports the AT1 R being a mechanosensor that contributes to arteriolar myogenic constriction. The aim of this study was to determine if AT1 R mechanoactivation affects myogenic constriction in skeletal muscle arterioles and to determine underlying cellular mechanisms. Using pressure myography to study rat isolated first-order cremaster muscle arterioles the AT1 R inhibitor candesartan (10-7 -10-5 m) showed partial but concentration-dependent inhibition of myogenic reactivity. Inhibition was demonstrated by a rightward shift in the pressure-diameter relationship over the intraluminal pressure range, 30-110mmHg. Pressure-induced changes in global vascular smooth muscle intracellular Ca2+ (using Fura-2) were similar in the absence or presence of candesartan, indicating that AT1 R-mediated myogenic constriction relies on Ca2+ -independent downstream signalling. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) reversed the inhibitory effect of candesartan, while this rescue effect was prevented by the protein kinase C (PKC) inhibitor GF 109203X. Both candesartan and PKC inhibition caused increased G-actin levels, as determined by Western blotting of vessel lysates, supporting involvement of cytoskeletal remodelling. At the single vascular smooth muscle cell level, atomic force microscopy showed that cell swelling (stretch) with hypotonic buffer also caused thickening of cortical actin fibres and this was blocked by candesartan. Collectively, the present studies support growing evidence for novel modes of activation of the AT1 R in arterioles and suggest that mechanically activated AT1 R generates diacylglycerol, which in turn activates PKC which induces the actin cytoskeleton reorganization that is required for pressure-induced vasoconstriction.

Purinergic modulation of smooth muscle intracellular Ca2+ concentration

Basal, steady‐state Ca2+ levels are determined by the balance between Ca2+ release/influx and uptake/extrusion mechanisms. Extrinsic factors can alter this balance to affect smooth muscle excitability. Extracellular ATP acts as an inhibitory neurotransmitter in gastrointestinal and vascular (portal vein) smooth muscle. This study examined the effect of extracellular ATP on the steady‐state intracellular Ca2+ concentration ([Ca2+]c). Extracellular ATP (1 mM; applied via picopritzer) reversibly decreased steady‐state [Ca2+]c (measured using fluo 4). To assess whether ATP functioned by altering influx or extrusion [Ca2+]c and inward currents were measured simultaneously in voltage‐clamped myocytes during a depolarizing pulse. ATP decreased the peak amplitude without altering the voltage‐dependence of the inward current. The P2 purinoceptor inhibitor, suramin, and the P2Y1 specific inhibitor, MRS2179, each prevented ATP inhibition of steady‐state [Ca2+]c and inward current. Interestingly, suramin and MRS2179 each also increased the magnitude of the inward current per se, indicating tonic ATP inhibition of Ca2+ channels by endogenous ATP. ATP did not alter Ca2+ uptake/extrusion mechanisms. ATP appears to influence Ca2+ influx mechanisms in smooth muscle via P2Y receptors to exert a negative regulation of excitability.

Hydrogen Sulfide Mediated Inhibitory Neurotransmission to the Pig Bladder Neck: Role of KATP Channels, Sensory Nerves and Calcium Signaling

Because neuronal released endogenous H2S has a key role in relaxation of the bladder outflow region, we investigated the mechanisms involved in H2S dependent inhibitory neurotransmission to the pig bladder neck. Bladder neck strips were mounted in myographs for isometric force recording and simultaneous measurement of intracellular Ca(2+) and tension. On phenylephrine contracted preparations electrical field stimulation and the H2S donor GYY4137 evoked frequency and concentration dependent relaxation, which was reduced by desensitizing capsaicin sensitive primary afferents with capsaicin, and the blockade of adenosine 5'-triphosphate dependent K(+) channels, cyclooxygenase and cyclooxygenase-1 with glibenclamide, indomethacin and SC560, respectively. Inhibition of vanilloid, transient receptor potential A1, transient receptor potential vanilloid 1, vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide and calcitonin gene-related peptide receptors with capsazepine, HC030031, AMG9810, PACAP6-38 and CGRP8-37, respectively, also decreased electrical field stimulation and GYY4137 responses. H2S relaxation was not changed by guanylyl cyclase, protein kinase A, or Ca(2+) activated or voltage gated K(+) channel inhibitors. GYY4137 inhibited the contractions induced by phenylephrine and by K(+) enriched (80 mM) physiological saline solution. To a lesser extent it decreased the phenylephrine and K(+) induced increases in intracellular Ca(2+). H2S produces pig bladder neck relaxation via activation of adenosine 5'-triphosphate dependent K(+) channel and by smooth muscle intracellular Ca(2+) desensitization dependent mechanisms. H2S also promotes the release of sensory neuropeptides and cyclooxygenase-1 pathway derived prostanoids from capsaicin sensitive primary afferents via transient receptor potential A1, transient receptor potential vanilloid 1 and/or related ion channel activation.

Another vascular TRP channel: functional expression and pharmacological properties of vanilloid receptor‐1 (TRPV1) in vascular smooth muscle

Transient receptor potential channels (TRP), in particular, TRPCs and TRPMs are recognized as important determinants of myogenic vascular tone and vascular diameter. Physiological and pharmacological properties of an other TRP (vanilloid receptor-1, TRPV1) were studied. It was found that (1) TRPV1 is expressed in vascular smooth muscle cells; (2) stimulation of vascular TRPV1 results in a substantial constriction in the gracilis artery of the rat and mouse; (3) this effect is missing in TRPV1 knock out mice or in the presence of TRPV1 specific inhibitors; (4) constriction is mediated by TRPV1 dependent increase in smooth muscle intracellular Ca2+ concentrations; (5) pharmaceutical properties of vascular TRPV1 are different from the sensory neuron located TRPV1. Our data suggests that vascular TRPV1 may be a new pharmaceutical target in the regulation of tissue perfusion, and may also represent an undesirable target for antinociceptive TRPV1 antagonists being developed.

Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization.

Intracerebral (parenchymal) arterioles are morphologically and physiologically unique compared with pial arteries and arterioles. The ability of subarachnoid hemorrhage (SAH) to induce vasospasm in large-diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is controversial. Currently, little is known regarding the impact of SAH on parenchymal arterioles, which are critical for regulation of local and global cerebral blood flow. Here diameter, smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)), and membrane potential measurements were used to assess the function of intact brain parenchymal arterioles isolated from unoperated (control), sham-operated, and SAH model rats. At low intravascular pressure (5 mmHg), membrane potential and [Ca(2+)](i) were not different in arterioles from control, sham-operated, and SAH animals. However, raising intravascular pressure caused significantly greater membrane potential depolarization, elevation in [Ca(2+)](i), and constriction in SAH arterioles. This SAH-induced increase in [Ca(2+)](i) and tone occurred in the absence of the vascular endothelium and was abolished by the L-type voltage-dependent calcium channel (VDCC) inhibitor nimodipine. Arteriolar [Ca(2+)](i) and tone were not different between groups when smooth muscle membrane potential was adjusted to the same value. Protein and mRNA levels of the L-type VDCC Ca(V)1.2 were similar in parenchymal arterioles isolated from control and SAH animals, suggesting that SAH did not cause VDCC upregulation. We conclude that enhanced parenchymal arteriolar tone after SAH is driven by smooth muscle membrane potential depolarization, leading to increased L-type VDCC-mediated Ca(2+) influx.

Disruption of vasoconstrictor responses in isolated cerebral arteries following treatment with siRNA against TRPM4

Membrane depolarization, Ca2+ influx, and activation of the Ca2+‐calmodulin pathway for smooth muscle contraction regulate arterial tone. The molecular mechanisms responsible for membrane depolarization in response to vasoconstrictor stimuli have not been fully characterized. We recently demonstrated that the melastatin transient receptor potential (TRP) channel TRPM4 is critical for membrane potential depolarization in response to increases in intraluminal pressure in cerebral arteries. In the current study, we further characterized the role of TRPM4 in intraluminal pressure‐ and agonist‐induced vasoconstriction using a newly developed RNA interference technique. Treatment of intact cerebral arteries with TRPM4 siRNA decreased mRNA expression by ~55% compared with controls. Using simultaneous video dimensional analysis and ratiometric Ca2+ imaging , we found that arteries treated with TRPM4 siRNA (n=7) developed less myogenic tone and had lower smooth muscle intracellular Ca2+ levels compared with controls (n=5). KCl (60 mM)‐induced constriction did not differ between groups. In addition, we found that TRPM4 siRNA‐treated arteries pressurized to 10 mmHg were less sensitive to the receptor‐dependent vasoconstrictor agonist uridine triphoshphate (UTP). These data suggest that TRPM4 plays a central role in regulating responses to vasoconstrictor stimuli in cerebral arteries. AHA0535226N

Exercise training prevents Ca2+ dysregulation in coronary smooth muscle from diabetic dyslipidemic yucatan swine

Aerobic exercise training is known to have profound cardioprotective effects in disease, yet cellular mechanisms remain largely undefined. We tested the hypothesis that increased sarcoplasmic reticulum Ca(2+) buffering and increased voltage-gated Ca(2+) channel density underlie coronary smooth muscle intracellular Ca(2+) (Ca(2+)(i)) dysregulation in diabetic dyslipidemia and that exercise training would prevent these increases. Yucatan swine were maintained in 1) control, 2) alloxan-induced hyperglycemic, 3) high fat/cholesterol fed, 4) hyperglycemic plus high fat/cholesterol fed (diabetic dyslipidemic), and 5) diabetic dyslipidemic plus exercise-trained (treadmill running) conditions. After 20 wk, the heart was removed and smooth muscle cells isolated from the right coronary artery. We utilized fura-2 imaging of Ca(2+)(i) levels to separate the functional role of the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) from the Na(+)-Ca(2+) exchanger and the plasmalemmal Ca(2+)-ATPase, and whole-cell patch clamp to examine voltage-gated Ca(2+) channel current density (i.e., Ca(2+) influx). Results indicated that diabetic dyslipidemia impaired plasmalemmal Ca(2+) efflux, increased basal Ca(2+)(i) levels, increased SERCA protein and sarcoplasmic reticulum Ca(2+)(i) buffering, and elicited an approximately 50% decrease in voltage-gated Ca(2+) channel current density. Exercise training concurrent with the diabetic dyslipidemic state restored plasmalemmal Ca(2+) efflux, SERCA protein, sarcoplasmic reticulum Ca(2+)(i) buffering, and voltage-gated Ca(2+) channel current density to control levels. Interestingly, basal Ca(2+)(i) levels were significantly lower in the exercise-trained group compared with control. Collectively, these results demonstrate a crucial role for exercise in the prevention of diabetic dyslipidemia-induced Ca(2+)(i) dysregulation.

5,6-EET-induced contraction of intralobar pulmonary arteries depends on the activation of Rho-kinase

The mechanism mediating epoxyeicosatrienoic acid (EET)-induced contraction of intralobar pulmonary arteries (PA) is currently unknown. EET-induced contraction of PA has been reported to require intact endothelium and activation of the thromboxane/endoperoxide (TP) receptor. Because TP receptor occupation with the thromboxane mimetic U-46619 contracts pulmonary artery via Rho-kinase activation, we examined the hypothesis that 5,6-EET-induced contraction of intralobar rabbit pulmonary arteries is mediated by a Rho-kinase-dependent signaling pathway. In isolated rings of second-order intralobar PA (1-2 mm OD) at basal tension, 5,6-EET (0.3-10 microM) induced increases in active tension that were inhibited by Y-27632 (1 microM) and HA-1077 (10 microM), selective inhibitors of Rho-kinase activity. In PA in which smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased with KCl (25 mM) to produce a submaximal contraction, 5,6-EET (1 microM) induced a contraction that was 7.0 +/- 1.6 times greater than without KCl. 5,6-EET (10 microM) also contracted beta-escin permeabilized PA in which [Ca(2+)](i) was clamped at a concentration resulting in a submaximal contraction. Y-27632 inhibited the 5,6-EET-induced contraction in permeabilized PA. 5,6-EET (10 microM) increased phosphorylation of myosin light chain (MLC), increasing the ratio of phosphorylated MLC/total MLC from 0.10 +/- 0.03 to 0.30 +/- 0.02. Y-27632 prevented this increase in MLC phosphorylation. These data suggest that 5,6-EET induces contraction in intralobar PA by increasing Rho-kinase activity, phosphorylating MLC, and increasing the Ca(2+) sensitivity of the contractile apparatus.

Ion channels in smooth muscle: regulators of intracellular calcium and contractility

Smooth muscle (SM) is essential to all aspects of human physiology and, therefore, key to the maintenance of life. Ion channels expressed within SM cells regulate the membrane potential, intracellular Ca2+ concentration, and contractility of SM. Excitatory ion channels function to depolarize the membrane potential. These include nonselective cation channels that allow Na+ and Ca2+ to permeate into SM cells. The nonselective cation channel family includes tonically active channels (Icat), as well as channels activated by agonists, pressure-stretch, and intracellular Ca2+ store depletion. Cl--selective channels, activated by intracellular Ca2+ or stretch, also mediate SM depolarization. Plasma membrane depolarization in SM activates voltage-dependent Ca2+ channels that demonstrate a high Ca2+ selectivity and provide influx of contractile Ca2+. Ca2+ is also released from SM intracellular Ca2+ stores of the sarcoplasmic reticulum (SR) through ryanodine and inositol trisphosphate receptor Ca2+ channels. This is part of a negative feedback mechanism limiting contraction that occurs by the Ca2+-dependent activation of large-conductance K+ channels, which hyper polarize the plasma membrane. Unlike the well-defined contractile role of SR-released Ca2+ in skeletal and cardiac muscle, the literature suggests that in SM Ca2+ released from the SR functions to limit contractility. Depolarization-activated K+ chan nels, ATP-sensitive K+ channels, and inward rectifier K+ channels also hyperpolarize SM, favouring relaxation. The expression pattern, density, and biophysical properties of ion channels vary among SM types and are key determinants of electrical activity, contractility, and SM function.

Calcium, mitochondria and oxygen sensing in the pulmonary circulation

A key event in hypoxic pulmonary vasoconstriction (HPV) is the elevation in smooth muscle intracellular Ca 2+ concentration. However, there is controversy concerning the source of this Ca 2+, the signal transduction pathways involved, and the identity of the oxygen sensor. Although there is wide support for the hypothesis that hypoxia elicits depolarisation via inhibition of K + channels, and thus promotes Ca 2+ entry through L-type channels, a significant number of studies are inconsistent with this mechanism being either the sole or even major means by which Ca 2+ is elevated during HPV. There is strong evidence that intracellular Ca 2+ stores play a critical role, and voltage-independent Ca 2+ entry mechanisms including capacitative Ca 2+ entry (CCE) have also been implicated. There is renewed interest in the role of mitochondria in HPV, both in terms of modulators of Ca 2+homeostasis per se and as oxygen sensors. There is however considerable uncertainty concerning the mechanisms involved in the latter, with proposals for changes in redox couples and both an increase and decrease in mitochondrial production of reactive oxygen species (ROS). In this article we review the evidence for and against involvement of such mechanisms in HPV, and propose a model for the regulation of intracellular [Ca 2+] in pulmonary artery during hypoxia in which the mitochondria play a central role.

Intraluminal pressure stimulates MAPK phosphorylation in arterioles: temporal dissociation from myogenic contractile response.

Members of the MAPK family of enzymes, p42/44 and p38, have been implicated in both the regulation of contractile function and growth responses in vascular smooth muscle. We determined whether such kinases are activated during the arteriolar myogenic response after increases in intraluminal pressure. Particular emphasis was placed on temporal aspects of activation to determine whether such phosphorylation events parallel the known time course for myogenic contraction. Experiments used single cannulated arterioles isolated from the cremaster muscle of rats with some vessels loaded with the fluorescent Ca2+-sensitive dye fura 2 (2 microM). The p42/44 inhibitor PD-98059 (50 microM) caused vasodilation but did not prevent pressure-induced myogenic constriction. The vasodilator response was accompanied by decreased smooth muscle intracellular Ca2+. Western blotting revealed a significant increase in the level of phosphorylation of p42/44 15 min after the application of a 30- to 100-mmHg pressure step. Phosphorylation of p42/44 was a late event that appeared to be temporally dissociated from contraction, which was complete within 1-5 min. EGF (80 nM) caused marked phosphorylation of p42/44 but only acted as a weak vasoconstrictor. The p38 inhibitor SB-203580 (10 microM) did not alter baseline diameter, nor did it prevent myogenic vasoconstriction. Consistent with these observations, SB-203580 did not cause a measurable change in intracellular Ca2+. The results demonstrate activation of the p42/44 class of MAPK resulting from increased transmural pressure. Such activation is, however, dissociated from the acute pressure-induced vasoconstrictor response in terms of time course and may represent the activation of compensatory, but parallel, pathways, including those related to growth and remodeling.

Different roles of PKC and MAP kinases in arteriolar constrictions to pressure and agonists.

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinases have been implicated in the modulation of agonist-induced contractions of large vessels. However, their role in pressure- and agonist-induced constrictions of skeletal muscle arterioles, which have a major role in regulating peripheral resistance, is not clearly elucidated. Thus constrictions of isolated rat gracilis muscle arterioles (approximately 80 microm in diameter) to increases in intraluminal pressure and to norepinephrine (NE) or angiotensin II (ANG II) were assessed in the absence or presence of chelerythrine, PD-98058, and SB-203580 (inhibitors of PKC, p42/44 and p38 MAP kinase pathways, respectively). Arteriolar constriction to NE and ANG II were significantly reduced by chelerythrine (by approximately 90%) and unaffected by SB-203580, whereas PD-98058 decreased only ANG II-induced constrictions (by approximately 60%). Pressure-induced increases in wall tension (from 0.1 to 0.7 N/m) resulted in significant arteriolar constrictions (50% maximum) that were abolished by chelerythrine without altering smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) (fura 2 microfluorimetry). PD-98058 and SB-203580 significantly decreased the magnitude of myogenic tone (by 20% and 60%, respectively) and reduced the sensitivity of the myogenic mechanism to wall tension, causing a significant rightward shift in the wall tension-myogenic tone relationship without affecting smooth muscle [Ca(2+)i]. MAP kinases were demonstrated with Western blotting. Thus in skeletal muscle arterioles 1) PKC is involved in both myogenic and agonist-induced constrictions, 2) PD-98058-sensitive p42/44 MAP kinases modulate both wall tension-dependent and ANG II-induced constrictions, whereas 3) a SB-203580-sensitive p38 MAP kinase pathway seems to be specifically involved in the mechanotransduction of wall tension.

The mechanism of excitation-contraction coupling in phenylephrine-stimulated human saphenous vein.

The human saphenous vein (HSV) is the most widely used graft in coronary artery revascularization procedures and is susceptible to spasm perioperatively. The aim of this study is to elucidate the mechanism(s) of agonist-induced excitation-contraction coupling in this vessel. Isometric contraction experiments were combined with in situ smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) imaging by confocal microscopy of intact undistended HSV segments during activation with phenylephrine (PE; 50 microM). Stimulation with PE produced a sustained contraction. Preincubation with 5 microM nifedipine, a blocker of the L-type voltage-operated Ca(2+) channel, or 50 microM SKF-96365, a blocker of both the voltage- and receptor-operated channels, reduced force generation by 25-30%. Ca(2+) imaging revealed that PE elicited only a transient rise in [Ca(2+)](i), suggesting that Ca(2+) plays only a minor role. However, a requirement for basal Ca(2+) levels was demonstrated when PE contractions could not be maintained in Ca(2+)-free medium. In light of the transient Ca(2+) response, it appears that signals other than Ca(2+) must maintain the tonic contraction elicited by PE, such as those that sensitize the myofilaments to Ca(2+). Application of HA-1077 (a Rho kinase inhibitor) at the peak of the contraction completely abolished the plateau phase of the response, whereas application of genistein (a tyrosine kinase inhibitor) reduced this phase by approximately 50%. The foregoing results suggest that, whereas the transient Ca(2+) signal can contribute to the development of force, maintenance of the plateau phase of the PE contraction in the HSV is the result of myofilament Ca(2+) sensitization by Rho kinase and tyrosine phosphorylation. The elucidation of the mechanisms of excitation-contraction coupling in the HSV may be useful for the development of therapeutic strategies for the alleviation of vein graft spasm.

Endothelium-derived mediators and hypoxic pulmonary vasoconstriction.

The vascular endothelium synthesises, metabolises or converts a multitude of vasoactive mediators, and plays a vital role in the regulation of pulmonary vascular resistance. Its role in hypoxic pulmonary vasoconstriction (HPV) is however controversial. Although HPV has been demonstrated in both pulmonary arteries where the endothelium has been removed and isolated pulmonary artery smooth muscle cells, many reports have shown either partial or complete dependence on an intact endothelium for sustained HPV (> approximately 20 min). However, despite many years of study no known endothelium-derived mediator has yet been unequivocally shown to be essential for HPV, although several may either facilitate the response or act as physiological brakes to limit the extent of HPV. In this article we review the evidence for and against the role of specific endothelium-derived mediators in HPV. We make the case for a facilitatory or permissive function of the endothelium, that in conjunction with a rise in smooth muscle intracellular Ca(2+) initiated by a mechanism intrinsic to smooth muscle, allows the development of sustained HPV. In particular, we propose that in response to hypoxia the pulmonary vascular endothelium releases an as yet unidentified agent that causes Ca(2+) sensitisation in the smooth muscle.

Pregnancy enhances endothelium-dependent relaxation of ovine uterine artery: role of NO and intracellular Ca(2+).

The present study tested the hypothesis that the pregnancy-associated increase in endothelium-dependent relaxation of the uterine artery was mediated primarily by an increase in nitric oxide (NO) release, resulting in a reduction in smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)). Uterine arteries obtained from nonpregnant and near-term (140 days gestation) pregnant sheep were used. The Ca(2+) ionophore A23187 induced endothelium-dependent relaxations in both nonpregnant and pregnant uterine arteries, with an increased relaxation in the pregnant tissue. In contrast, endothelium-independent relaxations induced by sodium nitroprusside were the same in nonpregnant and pregnant arteries. In addition, removal of the endothelium significantly increased noradrenaline-induced contractions in pregnant, but not nonpregnant, uterine arteries. In accordance, pregnancy increased both basal and A23187-stimulated NO releases in the uterine artery. Simultaneous measurement of tension and [Ca(2+)](i) in the smooth muscle demonstrated a linear correlation with the slope of unity between A23187-induced relaxation and the reduction of [Ca(2+)](i) in both nonpregnant and pregnant uterine arteries. The A23187-induced reduction of [Ca(2+)](i) was significantly enhanced in pregnant, compared with nonpregnant, uterine arteries. The results indicate that pregnancy increases NO release, which, through decreasing [Ca(2+)](i) in the smooth muscle, accounts for the increased endothelium-dependent relaxation of the uterine artery. Signal transduction pathways distal to NO production are not changed by pregnancy.

Increased Ca2+ sensitivity as a key mechanism of PKC-induced constriction in pressurized cerebral arteries.

The effects of activating protein kinase C (PKC) with indolactam V (Indo-V) and 1,2-dioctanoyl-sn-glycerol (DOG) on smooth muscle intracellular Ca2+ concentrations ([Ca2+]i) and arterial diameter were determined using ratiometric Ca2+ imaging and video edge detection of pressurized rat posterior cerebral arteries. Elevation of intraluminal pressure from 10 to 60 mmHg resulted in an increase in [Ca2+]i from 74 +/- 5 to 219 +/- 8 nM and myogenic constriction. Application of Indo-V (0.01-3 microM) or DOG (0.1-30 microM) induced constriction and decreased [Ca2+]i to 140 +/- 11 and 127 +/- 12 nM, respectively, at the highest concentrations used. In the presence of Indo-V, the dihydropyridine Ca2+-channel-blocker nisoldipine produced nearly maximum dilation and decreased [Ca2+]i to 97 +/- 7 nM. In alpha-toxin-permeabilized arteries, the constrictor effects of Indo-V and DOG were not observed in the absence of Ca2+. Both PKC activators significantly increased the degree of constriction of permeabilized arteries at different [Ca2+]i. We conclude that 1) Indo-V- or DOG-induced constriction of pressurized arteries requires Ca2+ influx through voltage-dependent Ca2+ channels, and 2) PKC-induced constriction of pressurized rat cerebral arteries is associated with a decrease in [Ca2+]i, suggesting an increase in the Ca2+ sensitivity of the contractile process.

Effect of acidosis on tension and [Ca2+]i in rat cerebral arteries: is there a role for membrane potential?

The cellular mechanism responsible for the reduction of tension in cerebral small arteries to acidosis is not known. In this study the role of smooth muscle intracellular Ca2+ concentration ([Ca2+]i) and membrane potential for the relaxation to acidosis was investigated in isolated rat cerebral small arteries. Isometric force was measured simultaneously with [Ca2+]i (fura 2) or with membrane potential (intracellular microelectrodes), and acidosis was induced by increasing PCO2 or reducing HCO3- of the bathing solution. Both hypercapnic and normocapnic acidosis were associated with a reduction of intracellular pH [measured with 2',7'-bis-(carboxyethyl)-5 (and -6)-carboxyfluorescein], caused relaxation, and reduced [Ca2+]i. However, whereas hypercapnic acidosis caused hyperpolarization, normocapnic acidosis was associated with depolarization. It is concluded that a reduction of [Ca2+]i is in part responsible for the direct effect of the acidosis on the vascular smooth muscle both during normo- and hypercapnia. The mechanism responsible for the reduction of [Ca2+]i differs between the hypercapnic and normocapnic acidosis, being partly explained by hyperpolarization during hypercapnic acidosis, whereas it is seen despite depolarization during normocapnic acidosis.