Vascular smooth muscle cell stress as a determinant of cerebral artery myogenic tone

Although the level of myogenic tone (MT) varies considerably from vessel to vessel, the regulatory mechanisms through which the actual diameter set point is determined are not known. We hypothesized that a unifying principle may be the equalization of active force at the contractile filament level, which would be reflected in a normalization of wall stress or, more specifically, media stress. Branched segments of rat cerebral arteries ranging from <50 microm to >200 microm in diameter were cannulated and held at 60 mmHg with the objectives of: 1) evaluating the relationship between arterial diameter and the extent of myogenic tone, 2) determining whether differences in MT correlate with changes in cytosolic calcium ([Ca(2+)](i)), and 3) testing the hypothesis that a normalization of wall or media stress occurs during the process of tone development. The level of MT increased significantly as vessel size decreased. At 60 mmHg, vascular smooth muscle [Ca(2+)](i) concentrations were similar in all vessels studied (averaging 230 +/- 9.2 nM) and not correlated with vessel size or the extent of tone. Wall tension increased with increasing arterial size, but wall stress and media stress were similar in large versus small arteries. Media stress, in particular, was quite uniform in all vessels studied. Both morphological and calcium data support the concept of equalization of media stress (and, hence, vascular smooth muscle cell stress and force) as an underlying mechanism in determining the level of tone present in any particular vessel. The equalization of active (vascular smooth muscle cell) stress may thus explain differences in MT observed in the different-sized vessels constituting the arterial network and provide a link between arterial structure and function, in both short- and long-term (hypertension) pressure adaptation.     

Myogenic response of rat femoral small arteries in relation to wall structure and [Ca(2+)](i)

The present study investigated the influence of media thickness on myogenic tone and intracellular calcium concentration ([Ca(2+)](i)) in rat skeletal muscle small arteries. A ligature was loosely tied around one external iliac artery of 5-wk-old spontaneously hypertensive rats. At 18 wk of age, femoral artery blood pressure was 102 +/- 11 mmHg (n = 15) on the ligated side and 164 +/- 6 mmHg (n = 15) on the contralateral side. Small arteries feeding the gracilis muscle had a reduced media cross-sectional area and a reduced media-to-lumen ratio on the ligated side, where also the range of myogenic constriction was shifted to lower pressures. However, when expressed as a function of wall stress, diameter responses were nearly identical. [Ca(2+)](i) was higher in vessels from the ligated hindlimb at pressures above 10 mmHg, but vasoconstriction was not accompanied by changes in [Ca(2+)](i). Thus the myogenic constriction here seems due primarily to changes in intracellular calcium sensitivity, which are determined mainly by the force per cross-sectional area of the wall and therefore altered by changes in vascular structure.     

Ionic mechanisms mediating the myogenic response in newborn porcine cerebral arteries

Mechanisms that underlie autoregulation in the newborn vasculature are unclear. Here we tested the hypothesis that in newborn porcine cerebral arteries intravascular pressure elevates wall tension, leading to an increase in intracellular calcium concentration ([Ca2+]i) and a constriction that is opposed by pressure-induced K+ channel activation. Incremental step (20 mmHg) elevations in intravascular pressure between 10 and 90 mmHg induced an immediate transient elevation in arterial wall [Ca2+]i and a short-lived constriction that was followed by a smaller steady-state [Ca2+]i elevation and sustained constriction. Pressures between 10 and 90 mmHg increased steady-state arterial wall [Ca2+]i between approximately 142 and 299 nM and myogenic (defined as passive-active) tension between 25 and 437 dyn/cm. The relationship between pressure and myogenic tension was strongly Ca2+ dependent until forced dilation. At low pressure, 60 mM K+ induced a steady-state elevation in arterial wall [Ca2+]i and a constriction. Nimodipine, a voltage-dependent Ca2+ channel blocker, and removal of extracellular Ca2+ similarly dilated arteries at low or high pressures. 4-Aminopyridine, a voltage-dependent K+ (Kv) channel blocker, induced significantly larger constrictions at high pressure, when compared with those at low pressure. Although selective Ca2+-activated K+ (KCa) channel blockers and intracellular Ca2+ release inhibitors induced only small constrictions at low and high pressures, a low concentration of caffeine (1 microM), a ryanodine-sensitive Ca2+ release (RyR) channel activator, increased KCa channel activity and induced dilation. These data suggest that in newborn cerebral arteries, intravascular pressure elevates wall tension, leading to voltage-dependent Ca2+ channel activation, an increase in wall [Ca2+]i and Ca2+-dependent constriction. In addition, pressure strongly activates Kv channels that opposes constriction but only weakly activates KCa channels.     

Caveolin-1 abolishment attenuates the myogenic response in murine cerebral arteries

Intravascular pressure-induced vasoconstriction (the "myogenic response") is intrinsic to smooth muscle cells, but mechanisms that underlie this response are unresolved. Here we investigated the physiological function of arterial smooth muscle cell caveolae in mediating the myogenic response. Since caveolin-1 (cav-1) ablation abolishes caveolae formation in arterial smooth muscle cells, myogenic mechanisms were compared in cerebral arteries from control (cav-1(+/+)) and cav-1-deficient (cav-1(-/-)) mice. At low intravascular pressure (10 mmHg), wall membrane potential, intracellular calcium concentration ([Ca(2+)](i)), and myogenic tone were similar in cav-1(+/+) and cav-1(-/-) arteries. In contrast, pressure elevations to between 30 and 70 mmHg induced a smaller depolarization, [Ca(2+)](i) elevation, and myogenic response in cav-1(-/-) arteries. Depolarization induced by 60 mM K(+) also produced an attenuated [Ca(2+)](i) elevation and constriction in cav-1(-/-) arteries, whereas extracellular Ca(2+) removal and diltiazem, an L-type Ca(2+) channel blocker, similarly dilated cav-1(+/+) and cav-1(-/-) arteries. N(omega)-nitro-l-arginine, an nitric oxide synthase inhibitor, did not restore myogenic tone in cav-1(-/-) arteries. Iberiotoxin, a selective Ca(2+)-activated K(+) (K(Ca)) channel blocker, induced a similar depolarization and constriction in pressurized cav-1(+/+) and cav-1(-/-) arteries. Since pressurized cav-1(-/-) arteries are more hyperpolarized and this effect would reduce K(Ca) current, these data suggest that cav-1 ablation leads to functional K(Ca) channel activation, an effect that should contribute to the attenuated myogenic constriction. In summary, data indicate that cav-1 ablation reduces pressure-induced depolarization and depolarization-induced Ca(2+) influx, and these effects combine to produce a diminished arterial wall [Ca(2+)](i) elevation and constriction.     

Effects of Rho kinase inhibition on cerebral artery myogenic tone and reactivity.

Several recent studies have implicated the RhoA-Rho kinase pathway in arterial myogenic behavior. The goal of this study was to determine the effects of Rho kinase inhibition (Y-27632) on cerebral artery calcium and diameter responses as a function of transmural pressure. Excised segments of rat posterior cerebral arteries (100-200 microm) were cannulated and pressurized in an arteriograph at 37 degrees C. Increasing pressure from 10 to 60 mmHg triggered an elevation of cytosolic calcium concentration ([Ca(2+)](i)) from 113 +/- 9 to 199 +/- 12 nM and development of myogenic tone. Further elevation of pressure to 120 mmHg induced only a minor additional increase in [Ca(2+)](i) and constriction. Y-27632 (0.3-10 microM) inhibited myogenic tone in a concentration-dependent manner at 60 and 120 mmHg with comparable efficacy; conversely, sensitivity was decreased at 120 vs. 60 mmHg (50% inhibitory concentration: 2.5 +/- 0.3 vs. 1.4 +/- 0.1 microM; P < 0.05). Dilation was accompanied by further increases in [Ca(2+)](i) and an enhancement of Ca(2+) oscillatory activity. Y-27632 also effectively dilated the vessels permeabilized with alpha-toxin in a concentration-dependent manner. However, dilator effects of Y-27632 at low concentrations were larger at 60 vs. 100 mmHg. In summary, the results support a significant role for RhoA-Rho kinase pathway in cerebral artery mechanotransduction of pressure into sustained vasoconstriction (myogenic tone and reactivity) via mechanisms that augment smooth muscle calcium sensitivity. Potential downstream events may involve inhibition of myosin phosphatase and/or stimulation of actin polymerization, both of which are associated with increased smooth muscle force production.     

beta(1)-Subunit of BK channels regulates arterial wall[Ca(2+)] and diameter in mouse cerebral arteries.

Mice with a disrupted beta(1) (BK beta(1))-subunit of the large-conductance Ca(2+)-activated K(+) (BK) channel gene develop systemic hypertension and cardiac hypertrophy, which is likely caused by uncoupling of Ca(2+) sparks to BK channels in arterial smooth muscle cells. However, little is known about the physiological levels of global intracellular Ca(2+) concentration ([Ca(2+)](i)) and its regulation by Ca(2+) sparks and BK channel subunits. We utilized a BK beta(1) knockout C57BL/6 mouse model and studied the effects of inhibitors of ryanodine receptor and BK channels on the global [Ca(2+)](i) and diameter of small cerebral arteries pressurized to 60 mmHg. Ryanodine (10 microM) or iberiotoxin (100 nM) increased [Ca(2+)](i) by approximately 75 nM and constricted +/+ BK beta(1) wild-type arteries (pressurized to 60 mmHg) with myogenic tone by approximately 10 microm. In contrast, ryanodine (10 microM) or iberiotoxin (100 nM) had no significant effect on [Ca(2+)](i) and diameter of -/- BK beta(1)-pressurized (60 mmHg) arteries. These results are consistent with the idea that Ca(2+) sparks in arterial smooth muscle cells limit myogenic tone through activation of BK channels. The activation of BK channels by Ca(2+) sparks reduces the voltage-dependent Ca(2+) influx and [Ca(2+)](i) through tonic hyperpolarization. Deletion of BK beta(1) disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in global [Ca(2+)](i).     

Pregnancy attenuates uterine artery pressure-dependent vascular tone: role of PKC/ERK pathway

The mechanisms of adaptation of uterine artery vascular tone to pregnancy are not fully understood. The present study tested the hypothesis that pregnancy decreases the PKC-mediated Ca(2+) sensitivity of the contractile process and attenuates myogenic tone in resistance-sized uterine arteries. In pressurized uterine arteries from nonpregnant (NPUA) and near-term pregnant (PUA) sheep, we measured, simultaneously in the same tissue, vascular diameter and vessel wall intracellular Ca(2+) concentration ([Ca(2+)](i)) as a function of intraluminal pressure. In both NPUA and PUA, membrane depolarization with KCl caused a rapid increase in [Ca(2+)](i) and a decrease in diameter. A pressure increase from 20 to 100 mmHg resulted in a transient increase in diameter that was associated with an increase in [Ca(2+)](i), followed by myogenic contractions in the absence of further changes in [Ca(2+)](i). In addition, activation of PKC by phorbol 12,13-dibutyrate induced a decrease in diameter in the absence of changes in [Ca(2+)](i). Pressure-dependent myogenic responses were significantly decreased in PUA compared with NPUA. However, pressure-induced increases in [Ca(2+)](i) were not significantly different between PUA and NPUA. The ratio of changes in diameter to changes in [Ca(2+)](i) was significantly greater for pressure-induced contraction of NPUA than that of PUA. Inhibition of PKC by calphostin C significantly attenuated the pressure-induced vascular tone and eliminated the difference of myogenic responses between NPUA and PUA. In contrast, the MAPKK (MEK) inhibitor PD-098059 had no effect on NPUA but significantly enhanced myogenic responses of PUA. In the presence of PD-098059, there was no difference in pressure-induced myogenic responses between NPUA and PUA. The results suggest that pregnancy downregulates pressure-dependent myogenic tone of the uterine artery, which is partly due to increased MEK/ERK activity and decreased PKC signal pathway leading to a decrease in Ca(2+) sensitivity of myogenic mechanism in the uterine artery during pregnancy.     

Transient increases in diameter and [Ca(2+)](i) are not obligatory for myogenic constriction

Studies were performed to determine the significance of temporal variation in vascular smooth muscle Ca(2+) signaling during acute arteriolar myogenic constriction and, in particular, the importance of the stretch-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) transient in attaining a steady-state mechanical response. Rat cremaster arterioles (diameter approximately 100 microm) were dissected from surrounding tissues, and vessel segments were pressurized in the absence of intraluminal flow. For [Ca(2+)](i) measurements, vessels were loaded with fura 2 and fluorescence emitted by excitation at 340 and 380 nm was measured using video-based image analysis. Ca(2+) and diameter responses were examined after increases in intravascular pressure were applied as an acute step increase or a ramp function. Additional studies examined the effect of longitudinal vessel stretch on [Ca(2+)](i) and arteriolar diameter. Step increase in intraluminal pressure (from 50 to 120 mmHg) caused biphasic change in [Ca(2+)](i) and diameter. [Ca(2+)](i) transiently increased to 114.0 +/- 2.0% of basal levels and subsequently declined to 106.7 +/- 4.4% at steady state. Diameter initially distended to 125.4 +/- 2.1% of basal levels before constricting to 71.1 +/- 1.2%. In contrast, when the same pressure increase was applied as a ramp function (over 5 min) transient vessel distension and transient increase in [Ca(2+)](i) were prevented, yet at steady state vessels constricted to 71.3 +/- 2.5%. Longitudinal stretch resulted in a large [Ca(2+)](i) transient (158 +/- 19% of basal) that returned to baseline despite maintenance of the stretch stimulus. The data demonstrate that the initial vessel distension (reflecting myocyte stretch) and associated global [Ca(2+)](i) transient are not obligatory for myogenic contraction. Thus, although arteriolar smooth muscle cells are responsive to acute stretch, the resulting changes in myogenic tone may be more closely related to other mechanical variables such as wall tension.     

Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries

Myogenic tone in the pulmonary vasculature of normoxic adult animals is minimal or nonexistent. Whereas chronic hypoxia (CH) increases basal tone in pulmonary arteries, it is unclear if a portion of this elevated tone is due to development of myogenicity. Since basal arterial RhoA activity and Rho kinase (ROK) expression are augmented by CH, we hypothesized that CH elicits myogenic reactivity in pulmonary arteries through ROK-dependent vascular smooth muscle (VSM) Ca(2+) sensitization. To test this hypothesis, we assessed the contribution of ROK to basal tone and pressure-induced vasoconstriction in endothelium-disrupted pulmonary arteries [50-300 microm inner diameter (ID)] from control and CH [4 wk at 0.5 atmosphere (atm)] rats. Arteries were loaded with fura-2 AM to continuously monitor VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). Basal VSM [Ca(2+)](i) was not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 microM), caused a concentration-dependent reduction of basal tone in CH arteries but had no effect in control vessels. In contrast, PKC inhibition with GF109203X (1 microM) did not alter basal tone. Furthermore, significant vasoconstriction in response to stepwise increases in intraluminal pressure (5-45 mmHg) was observed at 12, 15, 25, and 35 mmHg in arteries (50-200 microm ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 microM) but not by GF109203X. VSM [Ca(2+)](i) was unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200-300 microm ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament Ca(2+) sensitization.     

Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases in Ca2+ in rat cerebral arteries

Although postnatal maturation potently modulates agonist-induced cerebrovascular contractility, its effects on the mechanisms mediating cerebrovascular myogenic tone remain poorly understood. Because the regulation of calcium influx and myofilament calcium sensitivity change markedly during early postnatal life, the present study tested the general hypothesis that early postnatal maturation increases the pressure sensitivity of cerebrovascular myogenic tone via age-dependent enhancement of pressure-induced calcium mobilization and myofilament calcium sensitivity. Pressure-induced myogenic tone and changes in artery wall intracellular calcium concentrations ([Ca(2+)](i)) were measured simultaneously in endothelium-denuded, fura-2-loaded middle cerebral arteries (MCA) from pup [postnatal day 14 (P14)] and adult (6-mo-old) Sprague-Dawley rats. Increases in pressure from 20 to 80 mmHg enhanced myogenic tone in MCA from both pups and adults although the normalized magnitudes of these increases were significantly greater in pup than adult MCA. At each pressure step, vascular wall [Ca(2+)](i) was also significantly greater in pup than in adult MCA. Nifedipine significantly attenuated pressure-evoked constrictions in pup MCA and essentially eliminated all responses to pressure in the adult MCA. Both pup and adult MCA exhibited pressure-dependent increases in calcium sensitivity, as estimated by changes in the ratio of pressure-induced myogenic tone to wall [Ca(2+)](i). However, there were no differences in the magnitudes of these increases between pup and adult MCA. The results support the view that regardless of postnatal age, changes in both calcium influx and myofilament calcium sensitivity contribute to the regulation of cerebral artery myogenic tone. The greater cerebral myogenic response in P14 compared with adult MCA appears to be due to greater pressure-induced increases in [Ca(2+)](i), rather than enhanced augmentation of myofilament calcium sensitivity.     

Pressure-dependent myogenic constriction of cerebral arteries occurs independently of voltage-dependent activation

Pressure-induced decreases in arterial diameter are accompanied by membrane depolarization and Ca(2+) entry via voltage-gated Ca(2+) channels. Recent evidence also suggests the involvement of Ca(2+) sensitization of the contractile proteins. Both PKC and Rho kinase are candidate second messengers for the mediation of the sensitization process. We investigated the signaling pathways of pressure-induced decreases in rat cerebral artery diameter in vessels that were depolarized with a 60 mM potassium-physiological salt solution (KPSS). Arteries were mounted on a pressure myograph, and pressure-induced constrictions were recorded. In some experiments simultaneous changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) were recorded by using fura 2 fluorescence photometry. Pressure increases induced constriction with significant changes in [Ca(2+)](i) at high pressures (60-100 mmHg). The ratio of the change in diameter to change in [Ca(2+)](i) was greater for pressure-induced constriction compared with constriction produced by depolarization with 60 mM KPSS, suggesting that in addition to increases in [Ca(2+)](i), enhanced myofilament Ca(2+) sensitivity occurs during pressure-induced decreases in arterial diameter. Depolarizing the membrane with 60 mM KPSS increased [Ca(2+)](i) via a Ca(2+) influx pathway insensitive to PKC inhibition. Cerebral arteries were able to maintain their diameters in the continued presence of 60 mM KPSS. Pressure-induced constriction under these conditions was not associated with further increases in Ca(2+) but was abolished by selective inhibitors of PLC, PKC, and Rho kinase. We report for the first time that in rat cerebral arteries, pressure-induced decreases in arterial diameter are not only due to increases in voltage-gated Ca(2+) influx but also to accompanying increases in myofilament sensitivity to Ca(2+) mediated by PKC/Rho kinase activation.     

Actin cytoskeletal modulation of pressure-induced depolarization and Ca(2+) influx in cerebral arteries

The objective of this study was to examine the role of the actin cytoskeleton in the development of pressure-induced membrane depolarization and Ca(2+) influx underlying myogenic constriction in cerebral arteries. Elevating intraluminal pressure from 10 to 60 mmHg induced membrane depolarization, increased intracellular cytosolic Ca(2+) concentration ([Ca(2+)](i)) and elicited myogenic constriction in both intact and denuded rat posterior cerebral arteries. Pretreatment with cytochalasin D (5 microM) or latrunculin A (3 microM) abolished constriction but enhanced the [Ca(2+)](i) response; similarly, acute application of cytochalasin D to vessels with tone, or in the presence of 60 mM K(+), elicited relaxation accompanied by an increase in [Ca(2+)](i). The effects of cytochalasin D were inhibited by nifedipine (3 microM), demonstrating that actin cytoskeletal disruption augments Ca(2+) influx through voltage-sensitive L-type Ca(2+) channels. Finally, pressure-induced depolarization was enhanced in the presence of cytochalasin D, further substantiating a role for the actin cytoskeleton in the modulation of ion channel function. Together, these results implicate vascular smooth muscle actin cytoskeletal dynamics in the control of cerebral artery diameter through their influence on membrane potential as well as via a direct effect on L-type Ca(2+) channels.     

Selected contribution: NO released to flow reduces myogenic tone of skeletal muscle arterioles by decreasing smooth muscle Ca(2+) sensitivity.

To clarify the contribution of intracellular Ca(2+) concentration ([Ca(2+)](i))-dependent and -independent signaling mechanisms in arteriolar smooth muscle (aSM) to modulation of arteriolar myogenic tone by nitric oxide (NO), released in response to increases in intraluminal flow from the endothelium, changes in aSM [Ca(2+)](i) and diameter of isolated rat gracilis muscle arterioles (pretreated with indomethacin) were studied by fluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca(2+)](i) significantly increased and myogenic tone developed in response to elevations of extracellular Ca(2+) concentration. The Ca(2+) channel inhibitor nimodipine substantially decreased [Ca(2+)](i) and completely inhibited myogenic tone. Dilations to intraluminal flow (that were inhibited by N(omega)-nitro-L-arginine methyl ester) or dilations to the NO donor S-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were not accompanied by substantial decreases in aSM [Ca(2+)](i). 8-Bromoguanosine cGMP and the cGMP-specific phosphodiesterase inhibitor zaprinast significantly dilated arterioles yet elicited only minimal decreases in [Ca(2+)](i). Thus flow-induced endothelial release of NO elicits relaxation of arteriolar smooth muscle by a cGMP-dependent decrease of the Ca(2+) sensitivity of the contractile apparatus without substantial changes in the pressure-induced level of [Ca(2+)](i).     

Decreased function of voltage-gated potassium channels contributes to augmented myogenic tone of uterine arteries in late pregnancy

Increased pressure-induced (myogenic) tone in small uteroplacental arteries from late pregnant (LP) rats has been previously observed. In this study, we hypothesized that this response may result from a diminished activity of vascular smooth muscle cell (SMC) voltage-gated delayed-rectifier K(+) (K(v)) channels, leading to membrane depolarization, augmented Ca(2+) influx, and vasoconstriction (tone). Elevation of intraluminal pressure from 10 to 60 and 100 mmHg resulted in a marked, diltiazem-sensitive rise in SMC cytosolic Ca(2+) concentration ([Ca(2+)](i)) associated with a vasoconstriction of uteroplacental arteries of LP rats. In contrast, these changes were significantly diminished in uterine arteries from nonpregnant (NP) rats. Gestational augmentation of pressure-induced Ca(2+) influx through L-type Ca(2+) channels was associated with an enhanced SMC depolarization, the appearance of electrical and [Ca(2+)](i) oscillatory activities, and vasomotion. Exposure of vessels from NP animals to 4-aminopyridine, which inhibits the activity of K(v) channels, mimicked the effects of pregnancy by increasing pressure-induced depolarization, elevation of [Ca(2+)](i), and development of myogenic tone. Furthermore, currents through K(v) channels were significantly reduced in myocytes dissociated from arteries of LP rats compared with those of NP controls. Based on these results, we conclude that decreased K(v) channel activity contributes importantly to enhanced pressure-induced depolarization, Ca(2+) entry, and increase in myogenic tone present in uteroplacental arteries from LP rats.     

Characteristics of the myogenic behaviour of arteries of the common European frog (Rana temporaria)

Mammalian small arteries exhibit pressure-dependent myogenic behaviour characterised by an active constriction in response to an increased transmural pressure or an active dilatation in response to a decreased transmural pressure. This study aimed to determine whether pressure-dependent myogenic responses are a functional feature of amphibian arteries. Mesenteric and skeletal muscle arteries from the common European frog (Rana temporaria) were cannulated at either end with two fine glass micropipettes in the chamber of an arteriograph. Arterial pressure-diameter relationships (5-40 mmHg) were determined in the presence and absence of Ca2+. All arteries dilated passively with increasing pressure in the absence of Ca2+. In the presence of Ca2+ proximal mesenteric branches and tibial artery branches dilated with increasing transmural pressure but tone (p < 0.05) was evident in both arteries. A clear myogenic response to a step increase or decrease in pressure was observed in small distal arteries (6 of 13 mesenteric and 7 of 10 sciatic branches) resulting in significantly (p < 0.05) narrower diameters in Ca2+ in the range 10-40 mmHg in mesenteric and 20-40 mmHg in sciatic arteries, respectively. The results demonstrate that arteries of an amphibian can generate spontaneous pressure-dependent tone. This is the first study to demonstrate myogenic contractile behaviour in arteries of nonmammalian origin.     

Pressure-induced smooth muscle cell depolarization in pulmonary arteries from control and chronically hypoxic rats does not cause myogenic vasoconstriction.

Chronic obstructive pulmonary diseases, as well as prolonged residence at high altitude, can result in generalized airway hypoxia, eliciting an increase in pulmonary vascular resistance. We hypothesized that a portion of the elevated pulmonary vascular resistance following chronic hypoxia (CH) is due to the development of myogenic tone. Isolated, pressurized small pulmonary arteries from control (barometric pressure congruent with 630 Torr) and CH (4 wk, barometric pressure = 380 Torr) rats were loaded with fura 2-AM and perfused with warm (37 degrees C), aerated (21% O(2)-6% CO(2)-balance N(2)) physiological saline solution. Vascular smooth muscle (VSM) intracellular Ca(2+) concentration ([Ca(2+)](i)) and diameter responses to increasing intraluminal pressure were determined. Diameter and VSM cell [Ca(2+)](i) responses to KCl were also determined. In a separate set of experiments, VSM cell membrane potential responses to increasing luminal pressure were determined in arteries from control and CH rats. VSM cell membrane potential in arteries from CH animals was depolarized relative to control at each pressure step. VSM cells from both groups exhibited a further depolarization in response to step increases in intraluminal pressure. However, arteries from both control and CH rats distended passively to increasing intraluminal pressure, and VSM cell [Ca(2+)](i) was not affected. KCl elicited a dose-dependent vasoconstriction that was nearly identical between control and CH groups. Whereas KCl administration resulted in a dose-dependent increase in VSM cell [Ca(2+)](i) in arteries taken from control animals, this stimulus elicited only a slight increase in VSM cell [Ca(2+)](i) in arteries from CH animals. We conclude that the pulmonary circulation of the rat does not demonstrate pressure-induced vasoconstriction.     

Rho kinase inhibition partly weakens myogenic reactivity in rat small arteries by changing calcium sensitivity

The hypothesis that Rho kinase is involved in myogenic reactivity was investigated in pressurized rat tail small arteries using videomicroscopic diameter determination and calcium fluorimetry. The potent Rho kinase inhibitor Y-27632 reversibly increased vessel diameter at 80 mmHg without changing the intracellular calcium concentration ([Ca](i)) shifting the relationship between diameter change and [Ca](i) to higher calcium levels. Neither endothelium removal nor inhibition of neural transmission affected the Y-27632-induced effect. Y-27632 at 3 x 10(-6) mol/l attenuated the myogenic response in the pressure range from 10 to 120 mmHg, shifting the relationship between vessel tone and [Ca](i) to higher calcium levels. In addition, the Y-27632-induced shift of the relationship between vessel tone and [Ca](i) was larger at 80 than at 10 mmHg. These results suggest that smooth muscle cell Rho kinase in rat tail small arteries 1) is in an active state partly determining the level of the myogenic tone, and 2) alters the strength of the myogenic response by changing calcium sensitivity, probably caused by the pressure-induced activation of the kinase.     

Evidence for a functional calcium-sensing receptor that modulates myogenic tone in rat subcutaneous small arteries

Myogenic tone of small arteries is dependent on the presence of extracellular calcium (Ca(o)(2+)), and, recently, a receptor that senses changes in Ca(2+), the calcium-sensing receptor (CaR), has been detected in vascular tissue. We investigated whether the CaR is involved in the regulation of myogenic tone in rat subcutaneous small arteries. Immunoblot analysis using a monoclonal antibody against the CaR demonstrated its presence in rat subcutaneous arteries. To determine whether the CaR was functionally active, segments of artery (< 250 microm internal diameter) mounted in a pressure myograph with an intraluminal pressure of 70 mmHg were studied after the development of myogenic tone. Increasing Ca(o)(2+) concentration ([Ca(2+)](o)) cumulatively from 0.5 to 10 mM induced an initial constriction (0.5-2 mM) followed by dilation (42 +/- 5% loss of tone). The dose-dependent dilation was mimicked by other known CaR agonists including magnesium (1-10 mM) and the aminoglycosides neomycin (0.003-10 mM) and kanamycin (0.003-3 mM). PKC activation with the phorbol ester phorbol-12,13-dibutyrate (20nM) inhibited the dilation induced by high [Ca(2+)](o) or neomycin, whereas inhibition of PKC with GF109203X (10 microM) increased the responses to Ca(o)(2+) or neomycin, consistent with the role of PKC as a negative regulator of the CaR. We conclude that rat subcutaneous arteries express a functionally active CaR that may be involved in the modulation of myogenic tone and hence the regulation of peripheral vascular resistance.     

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 of myogenic mechanisms and hypoxic dilation in rat middle cerebral arteries

The goal of this study was to determine how myogenic responses and vascular responses to reduced Po(2) interact to determine vascular smooth muscle (VSM) transmembrane potential and active tone in isolated middle cerebral arteries from Sprague-Dawley rats. Stepwise elevation of transmural pressure led to depolarization of the VSM cells and myogenic constriction, and reduction of the O(2) concentration of the perfusion and superfusion reservoirs from 21% O(2) to 0% O(2) caused vasodilation and VSM hyperpolarization. Myogenic constriction and VSM depolarization in response to transmural pressure elevation still occurred at reduced Po(2). Arterial dilation in response to reduced Po(2) was not impaired by pressure elevation but was significantly reduced at the lowest transmural pressure (60 mmHg). However, the magnitude of VSM hyperpolarization was unaffected by transmural pressure elevation. This study demonstrates that myogenic activation in response to transmural pressure elevation does not override hypoxic relaxation of middle cerebral arteries and that myogenic responses and hypoxic relaxation can independently regulate vessel diameter despite substantial changes in the other variable.