Tyrosine kinase participates in vasoconstriction through a Ca(2+)- and myosin light chain phosphorylation-independent pathway

This study was undertaken to determine the role of tyrosine kinase on intracellular Ca(2+) ([Ca(2+)](i)), myosin light chain (MLC) phosphorylation, and contraction caused by norepinephrine (NE) in rat aorta. NE induced a sustained contraction with an increase of [Ca(2+)](i). On the other hand, NE increased the phosphorylation of the 20 kDa MLC transiently. Pretreatment with genistein and tyrophostin 25, tyrosine kinase inhibitors, significantly inhibited NE-induced contraction, but did not affect the increase of [Ca(2+)](i) and MLC phosphorylation. These results suggest that tyrosine kinase may regulate the NE-mediated contraction without altering [Ca(2+)](i) and MLC phosphorylation in rat aorta.     

Cerebral artery sarcoplasmic reticulum Ca(2+) stores and contractility: changes with development

To test the hypothesis that sarcoplasmic reticulum (SR) Ca(2+) stores play a key role in norepinephrine (NE)-induced contraction of fetal and adult cerebral arteries and that Ca(2+) stores change with development, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, we measured NE-induced contraction and intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence and presence of different blockers. In adult MCA, after thapsigargin (10(-6) M), the NE-induced responses of tension and [Ca(2+)](i) were 37 +/- 5 and 47 +/- 7%, respectively, of control values (P < 0.01 for each). In the fetal artery, in contrast, this treatment resulted in no significant changes from control. When this was repeated in the absence of extracellular Ca(2+), adult MCA increases in tension and [Ca(2+)](i) were 32 +/- 5 and 13 +/- 3%, respectively, of control. Fetal cerebral arteries, however, showed essentially no response. Ryanodine (RYN, 3 x 10(-6) to 10(-5) M) resulted in increases in tension and [Ca(2+)](i) in both fetal and adult MCA similar to that seen with NE. For both adult and fetal MCA, the increased tension and [Ca(2+)](i) responses to RYN were essentially eliminated in the presence of zero extracellular Ca(2+). These findings provide evidence that in fetal MCA, in contrast to those in the adult, SR Ca(2+) stores are of less importance in NE-induced contraction, with such contraction being almost wholly dependent on Ca(2+) flux via plasma membrane L-type Ca(2+) channels. In addition, they suggest that in both adult and fetal MCA, the RYN receptor is coupled to the plasma membrane Ca(2+)-activated K(+) channel and/or L-type Ca(2+) channel.     

Impaired Ca2+ handling in penile arteries from prediabetic Zucker rats: involvement of Rho kinase

Diabetes is associated with an increased vascular tone usually involved in the pathogenesis of diabetic cardiovascular complications such as hypertension, stroke, coronary artery disease, or erectile dysfunction (ED). Enhanced contractility of penile erectile tissue has been associated with augmented activity of the RhoA/Rho kinase (RhoK) pathway in models of diabetes-associated ED. The present study assessed whether abnormal vasoconstriction in penile arteries from prediabetic obese Zucker rats (OZRs) is due to changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and/or in myofilament Ca(2+) sensitivity. Penile arteries from OZRs and lean Zucker rats (LZRs) were mounted on microvascular myographs for simultaneous measurements of [Ca(2+)](i) and tension. The relationships between [Ca(2+)](i) and contraction for the α(1)-adrenergic vasoconstrictor phenylephrine (PE) were left shifted and steeper in OZRs compared with LZRs, although the magnitude of the contraction was similar in both groups. In contrast, the vasoconstriction induced by the thromboxane A(2) receptor agonist U-46619 was augmented in arteries from OZRs, and this increase was associated with an increase in both the sensitivity and maximum responses to Ca(2+). The RhoK inhibitor Y-27632 (10 μM) reduced the vasoconstriction induced by PE to a greater extent in OZRs than in LZRs, without altering Ca(2+). Y-27632 inhibited with a greater potency the contraction elicited by high KCl in arteries from OZRs compared with LZRs without changing [Ca(2+)](i). RhoK-II expression was augmented in arteries from OZRs. These results suggest receptor-specific changes in the Ca(2+) handling of penile arteries under conditions of metabolic syndrome. Whereas augmented vasoconstriction upon activation of the thromboxane A(2) receptor is coupled to enhanced Ca(2+) entry, a RhoK-mediated enhancement of myofilament Ca(2+) sensitivity is coupled with the α(1)-adrenergic vasoconstriction in penile arteries from OZRs.     

Dual role of PKC in modulating pharmacomechanical coupling in fetal and adult cerebral arteries

This study tested the hypothesis that protein kinase C (PKC) has dual regulation on norepinephrine (NE)-mediated inositol 1,4, 5-trisphosphate [Ins (1,4,5)P(3)] pathway and vasoconstriction in cerebral arteries from near-term fetal ( approximately 140 gestational days) and adult sheep. Basal PKC activity values (%membrane bound) in fetal and adult cerebral arteries were 38 +/- 4% and 32 +/- 4%, respectively. In vessels of both age groups, the PKC isoforms alpha, beta(I), beta(II), and delta were relatively abundant. In contrast, compared with the adult, cerebral arteries of the fetus had low levels of PKC-epsilon. In response to 10(-4) M phorbol 12,13-dibutyrate (PDBu; PKC agonist), PKC activity in both fetal and adult cerebral arteries increased 40-50%. After NE stimulation, PKC activation with PDBu exerted negative feedback on Ins(1,4,5)P(3) and intracellular Ca(2+) concentration ([Ca(2+)](i)) in arteries of both age groups. In turn, PKC inhibition with staurosporine resulted in augmented NE-induced Ins(1,4,5)P(3) and [Ca(2+)](i) responses in adult, but not fetal, cerebral arteries. In adult tissues, PKC stimulation by PDBu increased vascular tone, but not [Ca(2+)](i). In contrast, in the fetal artery, PKC stimulation was associated with an increase in both tone and [Ca(2+)](i). In the presence of zero extracellular [Ca(2+)], these PDBu-induced responses were absent in the fetal vessel, whereas they remained unchanged in the adult. We conclude that, although basal PKC activity was similar in fetal and adult cerebral arteries, PKC's role in NE-mediated pharmacomechanical coupling differed significantly in the two age groups. In both fetal and adult cerebral arteries, PKC modulation of NE-induced signal transduction responses would appear to play a significant role in the regulation of vascular tone. The mechanisms differ in the two age groups, however, and this probably relates, in part, to the relative lack of PKC-epsilon in fetal vessels.     

L-type Ca(2+) channels, resting [Ca(2+)](i), and ET-1-induced responses in chronically hypoxic pulmonary myocytes

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca(2+) channels contributes to 1) maintenance of resting intracellular Ca(2+) concentration ([Ca(2+)](i)), 2) increased [Ca(2+)](i) in response to ET-1 (10(-8) M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca(2+)](i) in PASMCs from intrapulmonary arteries of rats exposed to 10% O(2) for 21 days was 293.9 +/- 25.2 nM (vs. 153.6 +/- 28.7 nM in normoxia). Resting [Ca(2+)](i) was decreased after extracellular Ca(2+) removal but not with nifedipine (10(-6) M), an L-type Ca(2+) channel antagonist. After CH, the ET-1-induced increase in [Ca(2+)](i) was reduced and was abolished after extracellular Ca(2+) removal or nifedipine. Removal of extracellular Ca(2+) reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca(2+)](i) in PASMCs from chronically hypoxic rats does not require activation of L-type Ca(2+) channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca(2+)](i).     

Enhanced [Ca2+]i in renal arterial smooth muscle cells of pregnant rats with reduced uterine perfusion pressure

Reduction of uterine perfusion pressure (RUPP) during late pregnancy has been suggested to trigger increases in renal vascular resistance and lead to hypertension of pregnancy. We investigated whether the increased renal vascular resistance associated with RUPP in late pregnancy reflects increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and contraction of renal arterial smooth muscle. Single smooth muscle cells were isolated from renal interlobular arteries of normal pregnant Sprague-Dawley rats and a rat model of RUPP during late pregnancy. The cells were loaded with fura 2 and both cell length and [Ca(2+)](i) were measured. In cells of normal pregnant rats incubated in Hanks' solution (1 mM Ca(2+)), ANG II (10(-7) M) caused an initial increase in [Ca(2+)](i) to 414 +/- 13 nM, a maintained increase to 149 +/- 8 nM, and 21 +/- 1% cell contraction. In RUPP rats, the initial ANG II-induced [Ca(2+)](i) (431 +/- 18 nM) was not different from pregnant rats, but both the maintained [Ca(2+)](i) (225 +/- 9 nM) and cell contraction (48 +/- 2%) were increased. Membrane depolarization by 51 mM KCl and the Ca(2+) channel agonist BAY K 8644 (10(-6) M), which stimulate Ca(2+) entry from the extracellular space, caused maintained increases in [Ca(2+)](i) and cell contraction that were greater in RUPP rats than control pregnant rats. In Ca(2+)-free (2 mM EGTA) Hanks' solution, the ANG II- and caffeine (10 mM)-induced [Ca(2+)](i) transient and cell contraction were not different between normal pregnant and RUPP rats, suggesting no difference in Ca(2+) release from the intracellular stores. The enhanced maintained ANG II-, KCl- and BAY K 8644-induced [Ca(2+)](i) and cell contraction in RUPP rats compared with normal pregnant rats suggest enhanced Ca(2+) entry mechanisms of smooth muscle contraction in resistance renal arteries and may explain the increased renal vascular resistance associated with hypertension of pregnancy.     

Cerebral artery K(ATP)- and K(Ca)-channel activity and contractility: changes with development

The present study was designed to test the hypothesis that in cerebral arteries of the fetus, ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) channels (K(Ca)) play an important role in the regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) and that this differs significantly from that of the adult. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, simultaneously we measured norepinephrine (NE)-induced responses of vascular tension and [Ca(2+)](i) in the absence and presence of selective K(+)-channel openers/blockers. In fetal MCA, in a dose-dependent manner, both the K(ATP)-channel opener pinacidil and the K(Ca)-channel opener NS 1619 significantly inhibited NE-induced tension [negative logarithm of the half-maximal inhibitory concentration (pIC(50)) = 5.0 +/- 0.1 and 8.2 +/- 0.1, respectively], with a modest decrease of [Ca(2+)](i). In the adult MCA, in contrast, both pinacidil and NS 1619 produced a significant tension decrease (pIC(50) = 5.1 +/- 0.1 and 7.6 +/- 0.1, respectively) with no change in [Ca(2+)](i). In addition, the K(Ca)-channel blocker iberiotoxin (10(-7) to 10(-6) M) resulted in increased tension and [Ca(2+)](i) in both adult and fetal MCA, although the K(ATP)-channel blocker glibenclamide (10(-7) to 3 x 10(-5) M) failed to do so. Of interest, administration of 10(-7) M iberiotoxin totally eliminated vascular contraction and increase in [Ca(2+)](i) seen in response to 10(-5) M ryanodine. In precontracted fetal cerebral arteries, activation of the K(ATP) and K(Ca) channels significantly decreased both tension and [Ca(2+)](i), suggesting that both K(+) channels play an important role in regulating L-type channel Ca(2+) flux and therefore vascular tone in these vessels. In the adult, K(ATP) and the K(Ca) channels also appear to play an important role in this regard; however, in the adult vessel, activation of these channels with resultant vasorelaxation can occur with no significant change in [Ca(2+)](i). These channels show differing responses to inhibition, e.g., K(Ca)-channel inhibition, resulting in increased tension and [Ca(2+)](i), whereas K(ATP)-channel inhibition showed no such effect. In addition, the K(Ca) channel appears to be coupled to the sarcoplasmic reticulum ryanodine receptor. Thus differences in plasma membrane K(+)-channel activity may account, in part, for the differences in the regulation of contractility of fetal and adult cerebral arteries.     

Interaction between norepinephrine, NPY and VIP in the ovarian artery.

The in vitro effect and the interaction between norepinephrine (NE), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were studied in dissected segments of the rabbit ovarian artery. In addition, the structural requirement of the NPY receptor was investigated using NPY peptide analogs. NE induced a dose-dependent vasoconstriction with an Emax of 131.4 +/- 2.9% of K(+)-induced constriction. The vasoconstrictor effect of NPY was less than 5% of K(+)-induced vasoconstriction. Incubation of the artery with 10(-7) M NPY for 4 min induced a significant potentiation of NE-induced contractions. The selective NPY Y1 receptor agonist [Leu31, Pro34]NPY was also able to potentiate the NE response at the half-maximum contraction level, but not NPY(11-36), an NPY peptide fragment predominantly stimulating the NPY Y2 receptor. NPY exerted a dose-dependent vasoconstrictor effect on vessels contracted for 20 min with 10(-6) M NE. VIP induced a dose-dependent relaxation of vessels contracted with 10(-6) M NE. The VIP-induced relaxation could be reversed by NPY. In conclusion, receptors capable of interacting with NPY, presumably of the Y1 type, and VIP are present in the rabbit ovarian artery, and activation of these receptors may profoundly influence the response of the artery to norepinephrine.     

Ca(2+)](i) signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS

Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg. kg(-1). day(-1) for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca(2+)), the basal [Ca(2+)](i) was 86 +/- 6 nM. Phenylephrine (Phe, 10(-5) M) caused a transient increase in [Ca(2+)](i) to 417 +/- 11 nM and maintained an increase to 183 +/- 8 nM and 32 +/- 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca(2+) entry from the extracellular space, caused maintained increase in [Ca(2+)](i) to 292 +/- 12 nM and 31 +/- 2% contraction. The maintained Phe- and KCl-induced [Ca(2+)](i) and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca(2+)](i) were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca(2+)-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca(2+) release from the intracellular stores, caused a transient increase in [Ca(2+)](i) and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca(2+)](i) signaling that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca(2+)](i) in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca(2+)](i) during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.     

ANG II AT2 receptor modulates AT1 receptor-mediated descending vasa recta endothelial Ca2+ signaling

We tested whether the respective angiotensin type 1 (AT(1)) and 2 (AT(2)) receptor subtype antagonists losartan and PD-123319 could block the descending vasa recta (DVR) endothelial intracellular calcium concentration ([Ca(2+)](i)) suppression induced by ANG II. ANG II partially reversed the increase in [Ca(2+)](i) generated by cyclopiazonic acid (CPA; 10(-5) M), acetylcholine (ACh; 10(-5) M), or bradykinin (BK; 10(-7) M). Losartan (10(-5) M) blocked that effect. When vessels were treated with ANG II before stimulation with BK and ACh, concomitant AT(2) receptor blockade with PD-123319 (10(-8) M) augmented the suppression of endothelial [Ca(2+)](i) responses. Similarly, preactivation with the AT(2) receptor agonist CGP-42112A (10(-8) M) prevented AT(1) receptor stimulation with ANG II + PD-123319 from suppressing endothelial [Ca(2+)](i). In contrast to endothelial [Ca(2+)](i) suppression by ANG II, pericyte [Ca(2+)](i) exhibited typical peak and plateau [Ca(2+)](i) responses that were blocked by losartan but not PD-123319. DVR vasoconstriction by ANG II was augmented when AT(2) receptors were blocked with PD-123319. Similarly, AT(2) receptor stimulation with CGP-42112A delayed the onset of ANG II-induced constriction. PD-123319 alone (10(-5) M) showed no AT(1)-like action to constrict microperfused DVR or increase pericyte [Ca(2+)](i). We conclude that ANG II suppression of endothelial [Ca(2+)](i) and stimulation of pericyte [Ca(2+)](i) is mediated by AT(1) or AT(1)-like receptors. Furthermore, AT(2) receptor activation opposes ANG II-induced endothelial [Ca(2+)](i) suppression and abrogates ANG II-induced DVR vasoconstriction.     

Chronic hypoxia upregulates pulmonary arterial ASIC1: a novel mechanism of enhanced store-operated Ca2+ entry and receptor-dependent vasoconstriction

Acid-sensing ion channel 1 (ASIC1) is a newly characterized contributor to store-operated Ca(2+) entry (SOCE) in pulmonary vascular smooth muscle (VSM). Since SOCE is implicated in elevated basal VSM intracellular Ca(2+) concentration ([Ca(2+)](i)) and augmented vasoconstriction in chronic hypoxia (CH)-induced pulmonary hypertension, we hypothesized that ASIC1 contributes to these responses. To test this hypothesis, we examined effects of the specific pharmacologic ASIC1a inhibitor, psalmotoxin 1 (PcTX1), on vasoconstrictor and vessel wall [Ca(2+)](i) responses to UTP and KCl (depolarizing stimulus) in fura-2-loaded, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) Wistar rats. PcTX1 had no effect on basal vessel wall [Ca(2+)](i), but attenuated vasoconstriction and increases in vessel wall [Ca(2+)](i) to UTP in arteries from control and CH rats; normalizing responses between groups. In contrast, responses to the depolarizing stimulus, KCl, were unaffected by CH exposure or PcTX1. Upon examining potential Ca(2+) influx mechanisms, we found that PcTX1 prevented augmented SOCE following CH. Exposure to CH resulted in a significant increase in pulmonary arterial ASIC1 protein. This study supports a novel role of ASIC1 in elevated receptor-stimulated vasoconstriction following CH which is likely mediated through increased ASIC1 expression and SOCE.     

Mibefradil improves beta-adrenergic responsiveness and intracellular Ca(2+) handling in hypertrophied rat myocardium

The present study investigated the effects of mibefradil, a novel T-type channel blocker, on ventricular function and intracellular Ca(2+) handling in normal and hypertrophied rat myocardium. Ca(2+) transient was measured with the bioluminescent protein, aequorin. Mibefradil (2 microM) produced nonsignificant changes in isometric contraction and peak systolic intracellular Ca(2+) concentration ([Ca(2+)](i)) in normal rat myocardium. Hypertrophied papillary muscles isolated from aortic-banded rats 10 weeks after operation demonstrated a prolonged duration of isometric contraction, as well as decreased amplitudes of developed tension and peak Ca(2+) transient compared with the sham-operated group. Additionally, diastolic [Ca(2+)](i) increased in hypertrophied rat myocardium. The positive inotropic effect of isoproterenol stimulation was blunted in hypertrophied muscles despite a large increase in Ca(2+) transient amplitude. Afterglimmers and corresponding aftercontractions were provoked with isoproterenol (10(-5) and 10(-4) M) stimulation in 4 out of 16 hypertrophied muscles, but were eliminated in the presence of mibefradil (2 microM). In addition, hypertrophied muscles in the presence of mibefradil had a significant improvement of contractile response to isoproterenol stimulation and a reduced diastolic [Ca(2+)](I), although a mild decrease of peak Ca(2+)-transient was also shown. However, verapamil (2 microM) did not restore the inotropic and Ca(2+) modulating effects of isoproterenol in hypertrophied myocardium. Mibefradil partly restores the positive inotropic response to beta-adrenergic stimulation in hypertrophied myocardium from aortic-banded rats, an effect that might be useful in hypertrophied myocardium with impaired [Ca(2+)](i) homeostasis.     

Effect of timosaponin A-III,from Anemarrhenae asphodeloides Bunge (Liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension

The effects of timosaponin A-III (TA-III), from Rhizoma Anemarrhenae, on Ca(2+) mobilization in vascular endothelial cells and smooth muscle cells and on vascular tension have been explored. TA-III increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) in endothelials cells at a concentration larger than 5 microM with an EC(50) of 15 microM, and increased [Ca(2+)](i) in smooth muscle cells at a concentration larger than 1 microM with an EC(50) of 8 microM. Within 5 min, the [Ca(2+)](i) signal was composed of a gradual rise, and the speed of rising depended on the concentration of TA-III. The [Ca(2+)](i) signal was abolished by removing extracellular Ca(2+) and was recovered after reintroduction of Ca(2+). The TA-III-induced [Ca(2+)](i) increases in smooth muscle cells were partly inhibited by 10 microM nifedipine or 50 microM La(3+), but was insensitive to 10 microM verapamil and diltiazem. TA-III (10-100 microM) inhibited 0.3 microM phenylephrine-induced vascular contraction, which was abolished by pretreatment with 100 microM N(omega)-nitro-L-arginine (L-NNA) or by denuding the aorta. TA-III also increased [Ca(2+)](i) in renal tubular cells with an EC(50) of 8 microM. Collectively, the results show for the first time that TA-III causes [Ca(2+)](i) increases in the vascular system. TA-III acted by causing Ca(2+) influx without releasing intracellular Ca(2+). TA-III induced relaxation of phenylephrine-induced vascular contraction via inducing release of nitric oxide from endothelial cells.     

Mechanism of cGMP contribution to the vasodilator response to NO in rat middle cerebral arteries

This study examined the mechanism by which cGMP contributes to the vasodilator response to nitric oxide (NO) in rat middle cerebral arteries (MCA). Administration of a NO donor, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate), or 8-bromo-cGMP (8-BrcGMP) increased the diameter of serotonin-preconstricted MCA by 79 +/- 3%. The response to DEA-NONOate, but not 8-BrcGMP, was attenuated by iberiotoxin (10(-7) M) or a 80 mM high-K(+) media, suggesting that activation of K(+) channels contributes to the vasodilator response to NO but not 8-BrcGMP. The effects of NO and cGMP on the vasoconstrictor response to Ca(2+) were also studied in MCA that were permeabilized with alpha-toxin and ionomycin. Elevations in bath Ca(2+) from 10(-8) to 10(-5) M decreased the diameter of permeabilized MCA by 76 +/- 5%. DEA-NONOate (10(-6) M) and 8-BrcGMP (10(-4) M) blunted this response by 60%. Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (10(-5) M) blocked the inhibitory effect of the NO donor, but not 8-BrcGMP, on Ca(2+)-induced vasoconstriction. 8-BrcGMP (10(-4) M) had no effect on intracellular Ca(2+) concentration ([Ca(2+)](i)) in control, serotonin-stimulated, or alpha-toxin- and ionomycin-permeabilized vascular smooth muscle cells isolated from the MCA. These results indicate that the vasodilator response to NO in rat MCA is mediated by activation of Ca(2+)-activated K(+) channels via a cGMP-independent pathway and that cGMP also contributes to the vasodilator response to NO by decreasing the contractile response to elevations in [Ca(2+)](i).     

48-h Hypoxic exposure results in endothelium-dependent systemic vascular smooth muscle cell hyperpolarization

Chronic hypoxia (CH) results in reduced sensitivity to vasoconstrictors in conscious rats that persists upon restoration of normoxia. We hypothesized that this effect is due to endothelium-dependent hyperpolarization of vascular smooth muscle (VSM) cells after CH. VSM cell resting membrane potential was determined for superior mesenteric artery strips isolated from CH rats (PB = 380 Torr for 48 h) and normoxic controls. VSM cells from CH rats studied under normoxia were hyperpolarized compared with controls. Resting vessel wall intracellular Ca(2+) concentration ([Ca(2+)](i)) and pressure-induced vasoconstriction were reduced in vessels isolated from CH rats compared with controls. Vasoconstriction and increases in vessel wall [Ca(2+)](i) in response to the alpha(1)-adrenergic agonist phenylephrine (PE) were also blunted in resistance arteries from CH rats. Removal of the endothelium normalized resting membrane potential, resting vessel wall [Ca(2+)](i), pressure-induced vasoconstrictor responses, and PE-induced constrictor and Ca(2+) responses between groups. Whereas VSM cell hyperpolarization persisted in the presence of nitric oxide synthase inhibition, heme oxygenase inhibition restored VSM cell resting membrane potential in vessels from CH rats to control levels. We conclude that endothelial derived CO accounts for persistent VSM cell hyperpolarization and vasoconstrictor hyporeactivity after CH.     

Ionizing radiation alters myofilament calcium sensitivity in vascular smooth muscle: potential role of protein kinase C

Radiation exposure increases vascular responsiveness, and this change involves endothelial damage, as well as direct effects on vascular smooth muscle. In this study, we tested the hypothesis that myofilament Ca(2+) sensitivity in vascular smooth muscle is increased from single whole body gamma irradiation (6 Gy). We measured contractile responses from intact and permeabilized rat thoracic aortic rings combined with cytosolic Ca(2+) ([Ca(2+)](i)) measurements. The sensitivity to KCl and phenylephrine increased significantly in tissues from animals on the 9th and 30th days postirradiation compared with control. Irradiation also significantly increased Ca(2+) sensitivity in beta-escin permeabilized smooth muscle on the 9th and 30th days postirradiation. Inhibitors of protein kinase C, chelerythrine, and staurosporine, had no effect on the pCa-tension curves in control permeabilized tissues but significantly decreased Ca(2+) sensitivity in permeabilized tissues on the 9th and 30th days postirradiation. Phorbol dibutyrate (PDBu, 10(-7) M) increased Ca(2+) sensitivity in control skinned smooth muscle but was without effect in irradiated vascular rings. Simultaneous measurement of contractile force and [Ca(2+)](i) showed that myofilament Ca(2+) sensitivity defined as the ratio of force change to [Ca(2+)](i) significantly increased following gamma-irradiation. PDBu (10(-6) M) stimulation of intact aorta produced a sustained contraction, while the increase in [Ca(2+)](i) was transient. In irradiated tissues, PDBu-induced contractions were greater than those seen in control tissues but there was no elevation in [Ca(2+)](i). Taken together, these data strongly support the hypothesis that irradiation increases the sensitivity of vascular smooth muscle myofilaments to Ca(2+) and this effect is dependent on activation of protein kinase C.     

Endothelin-induced contraction of bronchiole and pulmonary arteriole smooth muscle cells is regulated by intracellular Ca2+ oscillations and Ca2+ sensitization

Endothelin-1 (ET) induces increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) sensitization, and contraction of both bronchiole and pulmonary arteriole smooth muscle cells (SMCs) and may play an important role in the pathophysiology of asthma and pulmonary hypertension. However, because it remains unclear how changes in [Ca(2+)](i) and the Ca(2+) sensitivity regulate SMC contraction, we have studied mouse lung slices with phase-contrast and confocal microscopy to correlate the ET-induced contraction with the changes in [Ca(2+)](i) and Ca(2+) sensitivity of bronchiole and arteriole SMCs. In comparison with acetylcholine (ACh) or serotonin (5-HT), ET induced a stronger and long-lasting contraction of both bronchioles and arterioles. This ET-induced contraction was associated with prominent asynchronous Ca(2+) oscillations that were propagated as Ca(2+) waves along the SMCs. These Ca(2+) oscillations were mediated by cyclic intracellular Ca(2+) release and required external Ca(2+) for their maintenance. Importantly, as the frequency of the Ca(2+) oscillations increased, the extent of contraction increased. ET-induced contraction was also associated with an increase in Ca(2+) sensitivity. In "model" slices in which the [Ca(2+)](i) was constantly maintained at an elevated level by pretreatment of slices with caffeine and ryanodine, the addition of ET increased bronchiole and arteriole contraction. These results indicate that ET-induced contraction of bronchiole and arteriole SMCs is regulated by the frequency of Ca(2+) oscillations and by increasing the sensitivity of the contractile machinery to Ca(2+).     

Contribution of both Ca2+ entry and Ca2+ sensitization to the alpha1-adrenergic vasoconstriction of rat penile small arteries

Sympathetic adrenergic nerves maintain the flaccid state of the penis through the tonic release of norepinephrine that contracts trabecular and arterial smooth muscle. Simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension and experiments with alpha-toxin-permeabilized arteries were performed in branches of the rat dorsal penile artery to investigate the intracellular Ca(2+) signaling pathways underlying alpha(1)-adrenergic vasoconstriction. Phenylephrine increased both [Ca(2+)](i) and tension, these increases being abolished by extracellular Ca(2+) removal and reduced by about 50% by the L-type Ca(2+) channel blocker nifedipine (0.3 microM). Non-L-type Ca(2+) entry through store-operated channels was studied by inhibiting the sarcoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA (30 microM) induced variable phasic contractions that were abolished by extracellular Ca(2+) removal and by the store-operated channels antagonist 2-aminoethoxydiphenyl borate (2-APB, 50 microM) and largely inhibited by nifedipine (0.3 microM). CPA induced a sustained increase in [Ca(2+)](i) that was reduced in a Ca(2+)-free medium. Under conditions of L-type channels blockade, Ca(2+) readmission after store depletion with CPA evoked a sustained and marked elevation in [Ca(2+)](i) not coupled to contraction. 2-APB (50 microM) inhibited the rise in [Ca(2+)](i) evoked by CPA and the nifedipine-insensitive increases in both [Ca(2+)](i) and contraction elicited by phenylephrine. In alpha-toxin-permeabilized penile arteries, activation of G proteins with guanosine 5'-O-(3-thiotriphosphate) and of the alpha(1)-adrenoceptor with phenylephrine both enhanced the myofilament sensitivity to Ca(2+). This Ca(2+) sensitization was reduced by selective inhibitors of PKC, tyrosine kinase (TK), and Rho kinase (RhoK) by 43%, 67%, and 82%, respectively. As a whole, the present data suggest the alpha(1)-adrenergic vasoconstriction in penile small arteries involves Ca(2+) entry through both L-type and 2-APB-sensitive receptor-operated channels, as well as Ca(2+) sensitization mechanisms mediated by PKC, TK, and RhoK. A capacitative Ca(2+) entry coupled to noncontractile functions of the smooth muscle cell is also demonstrated.     

Cell type-dependent regulation of human DNA topoisomerase III alpha gene expression by upstream stimulatory factor 2

Cyclosporin A (CsA) is a widely used immunosuppressive agent with severe side effects including hypertension. Here, we investigated the effects of CsA on intracellular free calcium ([Ca(2+)](i)) and the mechanisms involved in vasoconstriction in cultured human coronary myocytes. We used the Fura-2 technique for Ca(2+) imaging. Acute application of CsA at therapeutic concentrations (0.1-10 micromol/l) had no effect. Chronic exposure to CsA (1 micromol/l) for 24 h induced a small (20 nmol/l) but highly significant increase of basal [Ca(2+)](i) and enhanced the occurrence of spontaneous Ca(2+) oscillations. Endothelin- and vasopressin-induced rises of [Ca(2+)](i) were also enhanced. The demonstration that CsA increases basal [Ca(2+)](i) in addition to its impact on agonist receptor stimulation is of major importance for new therapeutic approaches.     

Enhancement of myofilament calcium sensitivity by acute hypoxia in rat distal pulmonary arteries

Hypoxic contraction of pulmonary arterial smooth muscle is thought to require increases in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity, which may or may not be endothelium-dependent. To examine the effects of hypoxia and endothelium on Ca(2+) sensitivity in pulmonary arterial smooth muscle, we measured the relation between [Ca(2+)](i) and isometric force at 37°C during normoxia (21% O(2)-5% CO(2)) and after 30 min of hypoxia (1% O(2)-5% CO(2)) in endothelium-intact (E+) and -denuded (E-) rat distal intrapulmonary arteries (IPA) permeabilized with staphylococcal α-toxin. Endothelial denudation enhanced Ca(2+) sensitivity during normoxia but did not alter the effects of hypoxia, which shifted the [Ca(2+)](i)-force relation to higher force in E+ and E- IPA. Neither hypoxia nor endothelial denudation altered Ca(2+) sensitivity in mesenteric arteries. In E+ and E- IPA, hypoxic enhancement of Ca(2+) sensitivity was abolished by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (30 μM), which shifted normoxic [Ca(2+)](i)-force relations to higher force. In E- IPA, the Rho kinase antagonist Y-27632 (10 μM) shifted the normoxic [Ca(2+)](i)-force relation to lower force but did not alter the effects of hypoxia. These results suggest that acute hypoxia enhanced myofilament Ca(2+) sensitivity in rat IPA by decreasing nitric oxide production and/or activity in smooth muscle, thereby revealing a high basal level of Ca(2+) sensitivity, due in part to Rho kinase, which otherwise did not contribute to Ca(2+) sensitization by hypoxia.