Natalia I. Gokina

Pressure-induced actin polymerization in vascular smooth muscle as a mechanism underlying myogenic behavior

We hypothesize that actin polymerization within vascular smooth muscle (VSM) in response to increased intravascular pressure is a novel and previously unrecognized mechanism underlying arterial myogenic behavior. This hypothesis is based on the following observations. 1) Unlike skeletal or cardiac muscle, VSM contains a substantial pool of unpolymer‐ized globular (G) actin whose function is not known. 2) The cytosolic concentration of G‐actin is significantly reduced by an elevation in intravascular pressure, demonstrating the dynamic nature of actin within VSM and implying a shift in the F:G equilibrium in favor of F‐actin. 3) Agents that inhibit actin polymerization and stabilize the cytoskeleton (cytochalasins and latruncu‐lin) inhibit the development of myogenic tone and decrease the effectiveness of myogenic reactivity. 4) Depolymerization of F‐actin with cytochalasin D causes VSM relaxation and increased G‐actin content, whereas polymerization of F‐actin with jasplakinolide causes VSM contraction and decreased G‐actin content. These results are consistent with observations in other cell types in which actin dynamics have been implicated in contractility and/or motility. Actin filament formation in VSM may therefore underlie mechanotransduction and, by providing additional sites for interaction with myosin, enhance force production in response to pressure. Although the mechanism by which actin polymerization is stimulated by pressure is not known, it likely occurs via integrin‐mediated activation of signal trans‐duction pathways previously associated with VSM contraction (e.g., PKC activation, Rho A, and tyrosine phosphorylation).—Cipolla, M. J., Gokina, N. I., Osol, G. Pressure‐induced actin polymerization in vascular smooth muscle as a mechanism underlying myogenic behavior. FASEB J. 16, 72–76 (2002)

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.

Placental growth factor is a potent vasodilator of rat and human resistance arteries

The objectives of this study were to determine whether placental growth factor (PlGF) exerts a vasodilatory effect on rat uterine vessels (arcuate arteries and veins) and to examine regional differences in reactivity by comparing these responses to those of comparably sized mesenteric vessels. We also sought to examine and compare its effects on human uterine and subcutaneous vessels. All vessels were studied in vitro, under pressurized (rat) or isometric wire-mounted (human) conditions, and exposed to a range of PlGF concentrations. Inhibitors of nitric oxide and prostaglandin synthesis were included in an effort to understand the causal mechanism(s). In rat uterine arteries, the effects of receptor inhibition and activation using selective ligands for VEGFR-1 (PlGF) vs. VEGFR-2 (VEGF-E) were determined, and real-time RT-PCR was performed to evaluate the effect of pregnancy on relative abundance of VEGFR-1 and VEGFR-2 message in the vascular wall. PlGF was a potent vasodilator of all vessels studied, with greatest sensitivity observed in rat uterine arteries. Pregnancy significantly augmented dilator sensitivity to PlGF, and this effect was associated with selective upregulation of VEGFR-1 message in the pregnant state. The contribution of nitric oxide was appreciable in rat and human uterine arteries, with lesser effects in rat uterine veins and mesenteric arteries, and with no observable effect in human subcutaneous vessels. Based on these results, we conclude that PlGF is a potent vasodilator of several vessel types in both humans and rats. Its potency and mechanism vary with physiological state and vessel location and are mediated solely by the VEGFR-1 receptor subtype. Gestational changes in the uterine circulation suggest that this factor may play a role in modulating uterine vascular remodeling and blood flow during the pregnant state.

Role of Ca2+-Activated K+ Channels in the Regulation of Membrane Potential and Tone of Smooth Muscle in Human Pial Arteries

Smooth muscle cells (SMCs) in 58% of human pial arteries obtained during surgery showed no spontaneous contractions and displayed a stable resting membrane potential (MP) of -54.7 +/- 1.5 mV. Those that exhibited periodic spontaneous contractions associated with periodic depolarization and generation of spontaneous action potentials (APs) had a less negative MP of -43.1 +/- 0.5 mV (42%). Inhibition of calcium-activated potassium (KCa) channels in the silent arteries by charybdotoxin (CTX) and tetraethylammonium ions (TEA) induced dose-dependent depolarization, AP generation, and contraction. TEA and CTX enhanced the spontaneous depolarization and force in arteries that exhibited spontaneous activity. They also prolonged the spontaneous APs up to several times and increased their upstroke amplitude. Both TEA and CTX failed to produce significant depolarization in arteries treated with nifedipine. It is concluded that KCa channels are important regulators of human pial artery SMC resting MP and tone. They are also involved in the control of AP amplitude and duration and the associated contractions. These data suggest that alterations in the activity of SMC KCa channels could be responsible for the appearance of spontaneous activity in human pial arteries in vitro and that impaired function of these channels might be related to vasospastic phenomena in human cerebral circulation.

Augmented EDHF signaling in rat uteroplacental vasculature during late pregnancy

A successful pregnancy outcome relies on extensive maternal cardiovascular adaptation, including enhanced uteroplacental vasodilator mechanisms. The objective of the present study was to determine the contribution of the endothelium-derived hyperpolarizing factor (EDHF) signaling in pregnancy-enhanced uterine vasodilation, to define the role of Ca(2+)-activated K(+) channels in mediating EDHF effects, and to explore the impact of endothelial Ca(2+) signaling in pregnancy-specific upregulation of EDHF. Fura 2-based measurements of smooth muscle cell (SMC) and endothelial cell cytosolic Ca(2+) concentration ([Ca(2+)](i)) were performed simultaneously with measurements of the diameter of uterine radial arteries from nonpregnant (NP) and late pregnant (LP) rats. Changes in SMC membrane potential of pressurized arteries from LP rats were assessed using glass microelectrodes. After blockade of nitric oxide and prostacyclin production, a cumulative application of ACh induced rapid and effective dilatation of uterine vessels from both NP and LP rats. This vasodilation was associated with SMC hyperpolarization and SMC [Ca(2+)](i) reduction and was abolished by a high-K(+) solution, demonstrating that N(G)-nitro-L-arginine (L-NNA)- and indomethacin-resistant responses are attributable to EDHF. Pregnancy significantly potentiates EDHF-mediated vasodilation in part due to enhanced endothelial Ca(2+) signaling. L-NNA- and indomethacin-resistant responses were insensitive to iberiotoxin but abolished by a combined treatment with apamin and charybdotoxin, supporting the key role of small- and intermediate-conductance K(+) channels in mediating EDHF signaling in the maternal uterine resistance vasculature.

Induction of localized differences in rat uterine radial artery behavior and structure during gestation

OBJECTIVES The objectives of this study were to investigate differences in diameter and vasoconstriction of premyometrial versus uteroplacental radial arteries and to evaluate the contribution of nitric oxide (NO) to myogenic tone as a function of vessel location. STUDY DESIGN Radial arteries supplying either the myometrium or placenta were dissected from the uterus of pregnant rats. Constrictor responses to pressure elevation were studied before and after inhibition of endothelial NO synthase (eNOS). RESULTS Passive lumen diameters of premyometrial and proximal uteroplacental arteries were comparable and significantly smaller than those of distal uteroplacental vessels. High potassium- and pressure-induced responses were also similar in premyometrial and proximal but were virtually absent in distal uteroplacental segments. L-NNA enhanced pressure-induced tone, but was without effect in distal uteroplacental segments. CONCLUSION Gestational remodeling alters arterial structure and reactivity in a highly localized manner. During pregnancy, enhanced basal production of NO may be an important local mechanism for uterine blood flow regulation.

Postischemic Reperfusion Causes Smooth Muscle Calcium Sensitization and Vasoconstriction of Parenchymal Arterioles

Background and Purpose— Parenchymal arterioles (PAs) are high-resistance vessels in the brain that connect pial vessels to the microcirculation. We previously showed that PAs have increased vasoconstriction after ischemia and reperfusion that could increase perfusion deficit. Here, we investigated underlying mechanisms by which early postischemic reperfusion causes increased vasoconstriction of PAs. Methods— Isolated and pressurized PAs from within the middle cerebral artery territory were studied in male Wistar rats that were either nonischemic control (n=34) or after exposure to transient middle cerebral artery occlusion (MCAO) by filament occlusion for 2 hours with 30 minutes of reperfusion (MCAO; n=38). The relationships among pressure-induced tone, smooth muscle calcium (using Fura 2), and membrane potential were determined. Sensitivity of the contractile apparatus to calcium was measured in permeabilized arterioles using Staphylococcus aureus &agr;-toxin. Reactivity to inhibition of transient receptor potential melastanin receptor type 4 (9-phenanthrol), Rho kinase (Y27632), and protein kinase C (Gö6976) was also measured. Results— After MCAO, PAs had increased myogenic tone compared with controls (47±2% versus 35±2% at 40 mm Hg; P<0.01), without an increase in smooth muscle calcium (177±21 versus 201±16 nmol/L; P>0.05) or membrane depolarization (−38±4 versus −36±1 mV; P>0.05). In &agr;-toxin–permeabilized vessels, MCAO caused increased sensitivity of the contractile apparatus to calcium. MCAO did not affect dilation to transient receptor potential melastanin receptor type 4 or protein kinase C inhibition but diminished dilation to Rho kinase inhibition. Conclusions— The increased vasoconstriction of PAs during early postischemic reperfusion seems to be due to calcium sensitization of smooth muscle and could contribute to infarct expansion and limit neuroprotective agents from reaching their target tissue.

Temperature and protein kinase C modulate myofilament Ca2+ sensitivity in pressurized rat cerebral arteries

The effects of pharmacological activation and inhibition of protein kinase C (PKC) and temperature on the relationship between cytoplasmic Ca2+ and lumen diameter were studied in pressurized (50 mmHg) rat posterior cerebral arteries permeabilized with α-toxin. Increasing Ca2+ concentrations (30 nM-10 μM, 22°C) induced stable, concentration-dependent constrictions with a half-maximal effective concentration (EC50) of 112 nM. The maximal constriction was 80% of baseline diameter and 157% of that during depolarization of nonpermeabilized vessels with 124 mM KCl. Elevation of temperature to 37°C increased the EC50 to 246 nM and enhanced the steepness of concentration-response curves. Exposure of permeabilized arteries to indolactam V, an activator of PKC, resulted in a significant myofilament Ca2+sensitization (e.g., EC50 at 5 μM = 126 nM) without changing efficacy. The effects of calphostin C, a PKC inhibitor, on Ca2+sensitivity were minimal; however, the amplitude of Ca2+-induced constrictions in both control and indolactam-treated arteries was suppressed in a concentration-dependent manner. Thus 1) temperature is an important variable in studies of arterial Ca2+ sensitivity, and 2) changes in PKC activity can significantly alter both myofilament sensitivity to and constrictor efficacy of cytosolic Ca2+.The effects of pharmacological activation and inhibition of protein kinase C (PKC) and temperature on the relationship between cytoplasmic Ca2+ and lumen diameter were studied in pressurized (50 mmHg) rat posterior cerebral arteries permeabilized with alpha-toxin. Increasing Ca2+ concentrations (30 nM-10 microM, 22 degrees C) induced stable, concentration-dependent constrictions with a half-maximal effective concentration (EC50) of 112 nM. The maximal constriction was 80% of baseline diameter and 157% of that during depolarization of nonpermeabilized vessels with 124 mM KCl. Elevation of temperature to 37 degrees C increased the EC50 to 246 nM and enhanced the steepness of concentration-response curves. Exposure of permeabilized arteries to indolactam V, an activator of PKC, resulted in a significant myofilament Ca2+ sensitization (e.g., EC50 at 5 microM = 126 nM) without changing efficacy. The effects of calphostin C, a PKC inhibitor, on Ca2+ sensitivity were minimal; however, the amplitude of Ca2+-induced constrictions in both control and indolactam-treated arteries was suppressed in a concentration-dependent manner. Thus 1) temperature is an important variable in studies of arterial Ca2+ sensitivity, and 2) changes in PKC activity can significantly alter both myofilament sensitivity to and constrictor efficacy of cytosolic Ca2+.

Mechanisms of Enhanced Basal Tone of Brain Parenchymal Arterioles During Early Postischemic Reperfusion: Role of ET-1-Induced Peroxynitrite Generation

The contributions of vasoconstrictors (endothelin-1 (ET-1), peroxynitrite) and endothelium-dependent vasodilatory mechanisms to basal tone were investigated in parenchymal arterioles (PAs) after early postischemic reperfusion. Transient middle cerebral artery occlusion (tMCAO) was induced for 2 hours with 30 minutes reperfusion in male Wistar rats and compared with ischemia alone (permanent MCAO (pMCAO);2.5 hours) or sham controls. Changes in lumen diameter of isolated and pressurized PAs were compared. Quantitative PCR was used to measure endothelin type B (ETB) receptors. Constriction to intravascular pressure (‘basal tone’) was not affected by tMCAO or pMCAO. However, constriction to inhibitors of endothelial cell, small-(SK) and intermediate-(IK) conductance, Ca2+-sensitive K+ channels (apamin and TRAM-34, respectively) were significantly enhanced in PAs from tMCAO compared with pMCAO or sham. Addition of the ETB agonist sarafotoxin caused constriction in PAs from tMCAO but not from sham animals (21±4% versus 3±3% at 1 nmol/L;P<0.01) that was inhibited by the peroxynitrite scavenger FeTMPyP (5,10,15,20-tetrakis (N-methyl-4′-pyridyl) porphinato iron (III) chloride) (100 μmol/L). Expression of ETB receptors was not found on PA smooth muscle, suggesting that constriction to sarafotoxin after tMCAO was due to peroxynitrite and not ETB receptor expression. The maintenance of basal tone in PAs after tMCAO may restrict flow to the ischemic region and contribute to infarct expansion.

Inhibition of Nitric Oxide Synthases Abrogates Pregnancy-Induced Uterine Vascular Expansive Remodeling

Background/Aims: It was the aim of this study to test the hypothesis that hypertension and/or inhibition of nitric oxide (NO) synthases alters uterine vascular remodeling during pregnancy. Methods: Using a model of hypertension (NO synthase inhibition with L-NAME) in nonpregnant and pregnant rats, comparisons were made with age-matched controls, as well as with animals receiving hydralazine along with L-NAME to maintain normotension in the presence of NO synthase inhibition. Circumferential and axial remodeling of large (main uterine, MUA) and small (premyometrial radial) arteries were quantified and compared. Results:L-NAME treatment prevented expansive circumferential remodeling of the MUA; cotreatment with hydralazine was without effect. Circumferential remodeling of smaller premyometrial radial arteries was also significantly attenuated in hypertensive pregnant animals, while premyometrial radial arteries from rats receiving hydralazine with L-NAME were of intermediate diameter. Neither hypertension nor NO synthase inhibition had any effect on the substantial (200–300%) axial growth of MUA or premyometrial radial arteries. Conclusion: NO plays a major role in facilitating pregnancy-induced expansive remodeling in the uterine circulation, particularly in larger arteries. Some beneficial effects of hydralazine on expansive circumferential remodeling were noted in smaller radial vessels, and these may be linked to its prevention of systemic hypertension and/or to local effects on the arterial wall. Neither NO synthase inhibition nor hypertension had any effect on arterial longitudinal growth.