Myogenic and vasoconstrictor responsiveness of skeletal muscle arterioles is diminished by hindlimb unloading.

The purpose of the present study was to determine whether hindlimb unloading of rats alters vasoconstrictor and myogenic responsiveness of skeletal muscle arterioles. After either 2 wk of hindlimb unloading (HU) or cage control (C), second-order arterioles were isolated from the white portion of gastrocnemius (WG; C: n = 9, HU: n = 10) or soleus (Sol; C: n = 9, HU: n = 10) muscles and cannulated with two micropipettes connected to reservoir systems for in vitro study. Intraluminal pressure was set at 60 cmH2O. The arterioles were exposed to step changes in intraluminal pressure ranging from 20 to 140 cmH2O to determine myogenic responsiveness and to KCl (10-100 mM) and norepinephrine (10(-9)-10(-4) M) to determine vasoconstrictor responsiveness. Although maximal diameter of WG arterioles was not different between C (185 +/- 12 microm) and HU (191 +/- 14 microm) rats, WG arterioles from HU rats developed less spontaneous tone (C: 33 +/- 5%, HU 20 +/-3%), were unable to maintain myogenic tone at pressures from 140 to 100 cmH2O, and were less sensitive to the vasoconstrictor effects of KCl and norepinephrine (as indicated by a higher agonist concentration that produced 50% of maximal vasoconstrictor response). In contrast, maximal diameter of Sol arterioles from HU rats (117 +/- 12 microm) was smaller than that in C rats (148 +/- 14 microm). However, the development of spontaneous tone (C: 30 +/- 4%, HU: 36 +/- 5%), myogenic activity, and the responsiveness to vasoconstrictor agonists were not different between Sol arterioles from C and HU rats. These results indicate that hindlimb unloading diminishes the myogenic autoregulatory and contractile responsiveness of arterioles from muscle composed of type IIB fibers and suggest that the compromised ability to elevate vascular resistance after exposure to microgravity may be related to these vascular alterations. In addition, hindlimb unloading appears to induce vascular remodeling of arterioles from muscle composed of type I fibers, as indicated by the decrease in maximal diameter of arterioles from Sol muscle.     

Development of vasomotor responses in fetal mesenteric arteries

Changes in mesenteric arterial diameters were studied using intravital microscopy in chick fetuses at days 13 and 17 of incubation, corresponding to 0.6 and 0.8 fetal incubation time, both during 5 min of hypoxia followed by 5 min of reoxygenation and after topical administration of increasing concentrations (10(-6)-10(-2) M) of norepinephrine (NE) and acetylcholine (ACh). Baseline diameters of second-order mesenteric arteries increased from 56 microm at 0.6 incubation to 75 microm at 0.8 incubation. Acute hypoxia induced a reduction in arterial diameter to 87 +/- 4.4% of baseline at 0.6 incubation and to 44 +/- 6.7% at 0.8 incubation (P < 0.01). During reoxygenation, mesenteric arteries dilated to 118 +/- 6.5% and 121 +/- 7.5% of baseline at 0.6 and 0.8 fetal incubation time, respectively. Phentolamine did not affect the vasoconstriction during hypoxia at 0.6 incubation, whereas this alpha-adrenergic antagonist significantly attenuated the vasoconstrictor response at 0.8 incubation (to 93 +/- 2.7% of baseline, P < 0.01). Topical NE induced maximal vasoconstriction to 71 +/- 3% of baseline at 0.6 incubation and to 35 +/- 3.8% at 0.8 incubation (P < 0.01). Maximal vasodilation to topical ACh was 113 +/- 4.4% and 122 +/- 4.8% of baseline at 0.6 and 0.8 incubation, respectively. These in vivo findings show that fetal mesenteric arteries constrict in response to acute hypoxia and that the increase in magnitude of this vasoconstrictor response from 0.6 to 0.8 of fetal development results from an increase in adrenergic constrictor capacity.     

Effects of aging on vasoconstrictor and mechanical properties of rat skeletal muscle arterioles

Exercise capacity and skeletal muscle blood flow during exercise are reduced with advancing age. This reduction in blood flow capacity may be related to increased reactivity of skeletal muscle resistance vessels to vasoconstrictor stimuli. The purpose of this study was to test the hypothesis that aging results in increased vasoconstrictor responses of skeletal muscle resistance arterioles. First-order (1A) arterioles (90-220 microm) from the gastrocnemius and soleus muscles of young (4 mo) and aged (24 mo) Fischer-344 rats were isolated, cannulated, and pressurized via hydrostatic reservoirs. Vasoconstriction in response to increases in norepinephrine (NE; 1 x 10(-9)-1 x 10(-4) M) and KCl (20-100 mM) concentrations and increases in intraluminal pressure (10-130 cmH(2)O) were evaluated in the absence of flow. Responses to NE and KCl were similar in both soleus and gastrocnemius muscle arterioles from young and aged rats. In contrast, active myogenic responses to changes in intraluminal pressure were diminished in soleus and gastrocnemius arterioles from aged rats. To assess whether alterations in the mechanical properties of resistance arterioles underlie altered myogenic responsiveness, passive diameter responses to pressure and mechanical stiffness were evaluated. There was no effect of age on the structural behavior (passive pressure-diameter relationship) or stiffness of arterioles from either the soleus or gastrocnemius muscles. These results suggest that aging does not result in a nonspecific decrease in vasoconstrictor responsiveness of skeletal muscle arterioles. Rather, aging-induced adaptations of vasoreactivity of resistance arterioles appear to be limited to mechanisms that are uniquely involved in the signaling of the myogenic response.     

Conducted vasoconstriction in rat mesenteric arterioles: role for dihydropyridine-insensitive Ca(2+) channels

The aim of this study was to evaluate the role of voltage-operated Ca(2+) channels in the initiation and conduction of vasoconstrictor responses to local micropipette electrical stimulation of rat mesenteric arterioles (28 +/- 1 microm, n = 79) in vivo. Local and conducted (600 microm upstream from the pipette) vasoconstriction was not blocked by TTX (1 micromol/l, n = 5), nifedipine, or nimodipine (10 micromol/l, n = 9). Increasing the K(+) concentration of the superfusate to 75 mmol/l did not evoke vasoconstriction, but this depolarizing stimulus reversibly abolished vasoconstrictor responses to current stimulation (n = 7). Addition of the T-type Ca(2+) antagonist mibefradil (10 micromol/l, n = 6) to the superfusate reversibly blocked local and conducted vasoconstriction to current stimulation. With the use of RT-PCR techniques, it was demonstrated that rat mesenteric arterioles <40 microm do not express mRNA for L-type Ca(2+) channels (alpha(1C)-subunit), whereas mRNA coding for T-type subunits was found (alpha(1G)- and alpha(1H)-subunits). The data indicate that L-type Ca(2+) channels are absent from rat mesenteric arterioles (<40 microm). Rather, the vasoconstrictor responses appear to rely on other types of voltage-gated, dihydropyridine-insensitive Ca(2+) channels, possibly of the T-type.     

Vasoconstrictor responsiveness of hind body vascular beds is diminished in tail-suspended rats

It is well known that the mechanisms of occurrence of orthostatic intolerance induced by exposure to microgravity deal with multiple factors including alterations of arteries. In the previous works, the diminished contractile responsiveness of abdominal aorta and hind body medium-sized conduit arteries, mesenteric artery and femoral artery, were observed in tail-suspended rats, and the data showed that the femoral artery have subjected to the greatest changes. These results suggested that the vasoreactivity of resistance vessels might be affected by the real or simulated microgravity. Since the arterioles are the main site of peripheral resistance and of its regulation. Therefore, changes in responsiveness of arteriolar network, especially in the lower/hind body region, would be of primary importance in the genesis of postflight orthostatic intolerance. The aim of the present work was to examine whether simulated weightlessness may lead to an impairment in vasoconstrictor responsiveness in hind body vascular beds.     

Effects of aging on adipose resistance artery vasoconstriction: possible implications for orthostatic blood pressure regulation.

The purpose of this investigation was to determine mean arterial pressure (MAP) and regional vascular conductance responses in young and aged Fisher-344 rats during orthostatic stress, i.e., 70 degrees head-up tilt (HUT). Both groups demonstrated directionally different changes in MAP during HUT (young, 7% increase; aged, 7% decrease). Vascular conductance during HUT in young rats decreased in most tissues but largely remained unchanged in the aged animals. Based on the higher vascular conductance of white adipose tissue from aged rats during HUT, resistance arteries from white visceral fat were isolated and studied in vitro. There was diminished maximal vasoconstriction to phenylephrine and norepinephrine (NE: young, 42 +/- 5%; old, 18 +/- 6%) in adipose resistance arteries from aged rats. These results demonstrate that aging reduces the ability to maintain MAP during orthostatic stress, and this is associated with a diminished vasoconstriction of adipose resistance arteries.     

Diminished mesenteric vaso- and venoconstriction and elevated plasma ANP and BNP with simulated microgravity.

Diminished constriction of arteries and veins following exposure to microgravity or bed rest is associated with a reduced ability to augment peripheral vascular resistance (PVR) and stroke volume during orthostasis. We tested the hypothesis that small mesenteric arteries and veins, which are not exposed to large pressure shifts during simulated microgravity via head-down tail suspension (HDT), will exhibit decrements in adrenergic constriction after HDT in rats. Small mesenteric arteries and veins from control (Con; n = 41) and HDT (n = 35) male Sprague-Dawley rats were studied in vitro. Vasoactive responsiveness to norepinephrine (NE) in arteries (10(-9) to 10(-4) M) and veins (pressure-diameter responses from 2 to 12 cmH(2)O after incubation in 10(-6) or 10(-4) M NE) were evaluated. Plasma concentrations of atrial (ANP) and NH(2)-terminal prohormone brain (NT-proBNP) natriuretic peptides were also measured. In mesenteric arteries, sensitivity and maximal responsiveness to NE were reduced with HDT. In mesenteric veins there was a diminished venoconstriction to NE at any given pressure in HDT. Plasma concentrations of both ANP and NT-proBNP were increased with HDT, and maximal arterial and venous constrictor responses to NE after incubation with 10(-7) M ANP or brain natriuretic peptide (BNP) were diminished. These data demonstrate that, in a vascular bed not subjected to large hydrodynamic differences with HDT, both small arteries and veins have a reduced responsiveness to adrenergic stimulation. Elevated levels of circulating ANP or NT-proBNP could adversely affect the ability of these vascular beds to constrict in vivo and conceivably could alter the intrinsic constrictor properties of these vessels with long-term exposure.     

Increased nitric oxide production following chronic hypoxia contributes to attenuated systemic vasoconstriction

Attenuated vasoconstrictor reactivity following chronic hypoxia (CH) is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and diminished intracellular [Ca(2+)]. We tested the hypothesis that increased production of nitric oxide (NO) after CH contributes to blunted vasoconstrictor responsiveness. We found that basal NO production of mesenteric arteries from CH rats (barometric pressure = 380 Torr; 48 h) was greater than that of controls (barometric pressure = 630 Torr). In addition, studies employing pressurized mesenteric arteries (100-200 microM ID) abluminally loaded with the Ca(2+) indicator fura 2-AM demonstrated that although NO synthase (NOS) inhibition normalized agonist-induced vasoconstrictor responses between groups, VSM cell [Ca(2+)] in vessels from CH rats remained diminished compared with controls. To determine whether elevated NO production following CH results from increased NOS protein levels, we performed Western blots for NOS isoforms by using mesenteric arteries from control and CH rats. Endothelial NOS levels did not differ between groups, and other NOS isoforms were not detected in these samples. Selective endothelial loading of fura 2-AM was employed to test the hypothesis that elevated endothelial cell [Ca(2+)] following CH accounts for enhanced NOS activity. These experiments demonstrated greater endothelial cell [Ca(2+)] in mesenteric arteries isolated from CH rats compared with controls. We conclude that enhanced production of NO resulting from elevated endothelial cell [Ca(2+)] contributes to attenuated reactivity following CH by decreasing VSM cell Ca(2+) sensitivity.     

Alkaline pH shifts Ca2+ sparks to Ca2+ waves in smooth muscle cells of pressurized cerebral arteries

The effects of external pH (7.0-8.0) on intracellular Ca(2+) signals (Ca(2+) sparks and Ca(2+) waves) were examined in smooth muscle cells from intact pressurized arteries from rats. Elevating the external pH from 7.4 to 7.5 increased the frequency of local, Ca(2+) transients, or "Ca(2+) sparks," and, at pH 7.6, significantly increased the frequency of Ca(2+) waves. Alkaline pH-induced Ca(2+) waves were inhibited by blocking Ca(2+) release from ryanodine receptors but were not prevented by inhibitors of voltage-dependent Ca(2+) channels, phospholipase C, or inositol 1,4,5-trisphosphate receptors. Activating ryanodine receptors with caffeine (5 mM) at pH 7.4 also induced repetitive Ca(2+) waves. Alkalization from pH 7.4 to pH 7.8-8.0 induced a rapid and large vasoconstriction. Approximately 82% of the alkaline pH-induced vasoconstriction was reversed by inhibitors of voltage-dependent Ca(2+) channels. The remaining constriction was reversed by inhibition of ryanodine receptors. These findings indicate that alkaline pH-induced Ca(2+) waves originate from ryanodine receptors and make a minor, direct contribution to alkaline pH-induced vasoconstriction.     

Effects of hindlimb unloading on rat cerebral, splenic, and mesenteric resistance artery morphology.

Hindlimb unloading (HU) of rats induces a cephalic shift in body fluids. We hypothesized that the putative increase in cranial fluid pressure and decrease in peripheral fluid pressure would alter the morphology of resistance arteries from 2-wk HU male Sprague-Dawley rats. To test this hypothesis, the cerebral basilar, mesenteric, and splenic arteries were removed from control (C) and HU animals. The vessels were cannulated, and luminal pressure was set to 60 cmH(2)O. The resistance arteries were then relaxed with 10(-4) M nitroprusside, fixed, and cut into transverse cross sections (5 microm thick). Media cross-sectional area (CSA), intraluminal CSA, media layer thickness, vessel outer perimeter, and media nuclei number were determined. In the basilar artery, both media CSA (HU 17, 893 +/- 2,539 microm(2); C 12,904 +/- 1,433 microm(2)) and thickness (HU 33.9 +/- 4.1 microm; C 22.3 +/- 3.2 microm) were increased with hindlimb unloading (P < 0.05), intraluminal CSA decreased (HU 7,816 +/- 3,045 microm(2); C 13,469 +/- 5,500 microm(2)) (P < 0.05), and vessel outer perimeter and media nuclei number were unaltered. There were no differences in mesenteric or splenic resistance artery morphology between HU and C rats. These findings suggest that hindlimb unloading-induced increases in cephalic arterial pressure and, correspondingly, increases in circumferential wall stress result in the hypertrophy of basilar artery smooth muscle cells.     

Chronic intermittent hypoxia alters NE reactivity and mechanics of skeletal muscle resistance arteries.

Although arterial dilator reactivity is severely impaired during exposure of animals to chronic intermittent hypoxia (CIH), few studies have characterized vasoconstrictor responsiveness in resistance arteries of this model of sleep-disordered breathing. Sprague-Dawley rats were exposed to CIH (10% inspired O2 fraction for 1 min at 4-min intervals; 12 h/day) for 14 days. Control rats were housed under normoxic conditions. Diameters of isolated gracilis muscle resistance arteries (GA; 120-150 microm) were measured by television microscopy before and during exposure to norepinephrine (NE) and angiotensin II (ANG II) and at various intraluminal pressures between 20 and 140 mmHg in normal and Ca2+-free physiological salt solution. There was no difference in the ability of GA to constrict in response to ANG II (P = 0.42; not significant; 10(-10)-10(-7) M). However, resting tone, myogenic activation, and vasoconstrictor responses to NE (P < 0.001; 10(-9)-10(-6) M) were reduced in CIH vs. controls. Treatment of rats with the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol; 1 mM) in the drinking water restored myogenic responses and NE-induced constrictions of CIH rats, suggesting that elevated superoxide production during exposure to CIH attenuates vasoconstrictor responsiveness to NE and myogenic activation in skeletal muscle resistance arteries. CIH also leads to an increased stiffness and reduced vessel wall distensibility that were not correctable with oral tempol treatment.     

Effects of high-salt diet on CYP450-4A omega-hydroxylase expression and active tone in mesenteric resistance arteries

This study investigated the role of changes in the expression of the cytochrome P-450 4A (CYP450-4A) enzymes that produce 20-hydroxyeicosatetraenoic acid (20-HETE) in modulating the responses of rat mesenteric resistance arteries to norepinephrine (NE) and reduced Po(2) after short-term (3-day) changes in dietary salt intake. The CYP450-4A2, -4A3, and -4A8 isoforms were all detected by RT-PCR in arteries obtained from rats fed a high-salt (HS, 4% NaCl) diet, whereas only the CYP450-4A3 isoform was detected in vessels from rats fed a low-salt (LS, 0.4% NaCl) diet. Expression of the 51-kDa CYP450-4A protein was significantly increased by a HS diet. Inhibiting 20-HETE synthesis with 30 muM N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) reduced the vasoconstrictor response to NE in arteries obtained from rats fed either a LS or HS diet, but NE sensitivity after DDMS treatment was significantly lower in vessels from rats on a HS diet. DDMS treatment also restored the vasodilator response to reduced Po(2) that was impaired in arteries from rats on a HS diet. These findings suggest that 1) a HS diet increases the expression of CYP450-4A enzymes in the mesenteric vasculature, 2) 20-HETE contributes to the vasoconstrictor response to NE in mesenteric resistance arteries, 3) the contribution of 20-HETE to the vasoconstrictor response to NE is greater in rats fed a HS diet than in rats fed a LS diet, and 4) upregulation of the production of 20-HETE contributes to the impaired dilation of mesenteric resistance arteries in response to hypoxia in rats fed a HS diet.     

Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries

Increases in global Ca(2+) in the endothelium are a crucial step in releasing relaxing factors to modulate arterial tone. In the present study we investigated spontaneous Ca(2+) events in endothelial cells, and the contribution of smooth muscle cells to these Ca(2+) events, in pressurized rat mesenteric resistance arteries. Spontaneous Ca(2+) events were observed under resting conditions in 34% of cells. These Ca(2+) events were absent in arteries preincubated with either cyclopiazonic acid or U-73122, but were unaffected by ryanodine or nicotinamide. Stimulation of smooth muscle cell depolarization and contraction with either phenylephrine or high concentrations of KCl significantly increased the frequency of endothelial cell Ca(2+) events. The putative gap junction uncouplers carbenoxolone and 18alpha-glycyrrhetinic acid each inhibited spontaneous and evoked Ca(2+) events, and the movement of calcein from endothelial to smooth muscle cells. In addition, spontaneous Ca(2+) events were diminished by nifedipine, lowering extracellular Ca(2+) levels, or by blockers of non-selective Ca(2+) influx pathways. These findings suggest that in pressurized rat mesenteric arteries, spontaneous Ca(2+) events in the endothelial cells appear to originate from endoplasmic reticulum IP(3) receptors, and are subject to regulation by surrounding smooth muscle cells via myoendothelial gap junctions, even under basal conditions.     

Early gestation dexamethasone programs enhanced postnatal ovine coronary artery vascular reactivity

Excessive exposure of the fetus to maternally derived corticosteroids has been linked to the development of adult-onset diseases. To determine if early gestation corticosteroid exposure alters subsequent coronary artery reactivity, we administered dexamethasone (0.28 mg.kg(-1).day(-1)) to pregnant ewes at 27-28 days gestation (term being 145 days). Vascular responsiveness was assessed in endothelium-intact coronary and mesenteric arteries isolated from steroid-exposed and age-matched control fetal sheep at 123-126 days gestation and lambs at 4 mo of age. Lambs exposed to maternal dexamethasone had higher mean arterial blood pressures than the age-matched controls (93 +/- 3 vs. 83 +/- 5 mmHg, P < 0.05). Mesenteric arteries from the steroid-exposed fetuses displayed diminished responses to ANG II, relative to controls. In 4-mo-old lambs, prenatal dexamethasone exposure significantly increased coronary artery vasoconstriction to ANG II, ACh, and U-46619, but not KCl. In contrast, postnatal mesenteric artery reactivity was unaltered by steroid exposure. Compared with fetal mesenteric reactivity, postnatal mesenteric reactivity to ANG II, phenylephrine, and U-46619 was diminished, whereas the response to 120 mmol/l KCl was heightened. Coronary artery ANG II receptor protein expression was not significantly altered by steroid exposure in either age group. These findings demonstrate that early-gestation glucocorticoid exposure programs postnatal elevations in blood pressure and selectively enhances coronary artery responsiveness to second messenger-dependent vasoconstrictors. Glucocorticoid-induced alterations in coronary vascular smooth muscle structure or function may provide a mechanistic link between an adverse intrauterine environment and later cardiovascular disease.     

Vasopressin-induced vasoconstriction: two concentration-dependent signaling pathways.

Current scientific literature generally attributes the vasoconstrictor effects of [Arg(8)]vasopressin (AVP) to the activation of phospholipase C (PLC) and consequent release of Ca(2+) from the sarcoplasmic reticulum. However, half-maximal activation of PLC requires nanomolar concentrations of AVP, whereas vasoconstriction occurs when circulating concentrations of AVP are orders of magnitude lower. Using cultured vascular smooth muscle cells, we previously identified a novel Ca(2+) signaling pathway activated by 10-100 pM AVP. This pathway is distinguished from the PLC pathway by its dependence on protein kinase C (PKC) and L-type voltage-sensitive Ca(2+) channels (VSCC). In the present study, we used isolated, pressurized rat mesenteric arteries to examine the contributions of these different Ca(2+) signaling mechanisms to AVP-induced vasoconstriction. AVP (10(-14)-10(-6) M) induced a concentration-dependent constriction of arteries that was reversible with a V(1a) vasopressin receptor antagonist. Half-maximal vasoconstriction at 30 pM AVP was prevented by blockade of VSCC with verapamil (10 microM) or by PKC inhibition with calphostin-C (250 nM) or Ro-31-8220 (1 microM). In contrast, acute vasoconstriction induced by 10 nM AVP (maximal) was insensitive to blockade of VSCC or PKC inhibition. However, after 30 min, the remaining vasoconstriction induced by 10 nM AVP was partially dependent on PKC activation and almost fully dependent on VSCC. These results suggest that different Ca(2+) signaling mechanisms contribute to AVP-induced vasoconstriction over different ranges of AVP concentration. Vasoconstrictor actions of AVP, at concentrations of AVP found within the systemic circulation, utilize a Ca(2+) signaling pathway that is dependent on PKC activation and can be inhibited by Ca(2+) channel blockers.     

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.     

Analysis of tert-butyl hydroperoxide induced constrictions of perfused vascular beds in vitro

tert-Butyl hydroperoxide (t-BOOH), an inducer of oxidative stress in vitro, elicits constrictor responses of the isolated, rat kidney and mesenteric arteries perfused (5 ml/min) with physiological salt solutions (PSS) at 37 degrees C gassed with carbogen. We hypothesized that generation of superoxide anions (O(2)(-)) accounts for these responses. We assessed responses to t-BOOH in preparations with/without endothelium, and in the absence/presence of antioxidant compounds, catalase and tempol, scavengers of hydroxyl (OH(-)) radical and O(2)(-), respectively. t-BOOH (0.01-50 micromol) induced (expressed as % of 50 micromol KCl vasoconstriction) were abolished by endothelium denudation, perfusion with Ca(2+)-free PSS and by nifedipine, (1 nM). Infusion of t-BOOH (0.1 microM) did not significantly (P > 0.05) affect phenylepherine E(max) in the mesenteric arteries, however it reduced phenylepherine E(max) responses in the kidney from 94.9 +/- 3.9 % to 64.7 +/- 4.7 %. Nitroblue tetrazolium, as well as alpha-phenyl N-tert-butyl nitrone, at 100 microM, but not catalase (500 IU) significantly attenuated t-BOOH (10 micromol) vasoconstrictor responses. Tempol (100 microM), a membrane permeable antioxidant, also significantly reduced t-BOOH (10 micromol) responses from 17.0 +/- 1.9 % (control) to 9.6 +/- 0.5 % (+tempol) in the mesenteric arteries and from 40.4 +/- 4.2 % (control) to 20.7 +/- 1.5 % (+tempol) in the kidney. Our data suggest that t-BOOH elicits vasoconstriction via two distinct mechanisms: (i) increased influx of extracellular Ca(2+), and (ii) generation of free radicals including O(2)(-) anions.     

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ～70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.     

Differential regulation of L-type Ca2+ channels in cerebral and mesenteric arteries after simulated microgravity in rats and its intervention by standing

This study was designed to clarify whether simulated microgravity can induce differential changes in the current and protein expression of the L-type Ca(2+) channel (Ca(L)) in cerebral and mesenteric arteries and whether these changes can be prevented by daily short-duration -G(x) exposure. Tail suspension [hindlimb unloading (HU)] for 3 and 28 days was used to simulate short- and medium-term microgravity-induced deconditioning effects. Standing (STD) for 1 h/day was used to provide -G(x) as a countermeasure. Whole cell patch-clamp experiments revealed an increase in current density of Ca(L) of vascular smooth muscle cells (VSMCs) isolated from cerebral arteries of rats subjected to HU and a decrease in VSMCs from mesenteric arteries. Western blot analysis revealed a significant increase and decrease of Ca(L) channel protein expression in cerebral and small mesenteric arterial VSMCs, respectively, only after 28 days of HU. STD for 1 h/day did not prevent the increase of Ca(L) current density in cerebral arterial VSMCs, but it prevented completely (within 3 days) and partially (28 days) the decrease of Ca(L) current density in small mesenteric arterial VSMCs. Consistent with the changes in Ca(L) current, STD for 1 h/day did not prevent the increase of Ca(L) expression in cerebrovascular myocytes but did prevent the reduction of Ca(L) expression in mesenteric arterial VSMCs subjected to 28 days of HU. These data indicate that simulated microgravity up- and downregulates the current and expression of Ca(L) in cerebral and hindquarter VSMCs, respectively. STD for 1 h/day differentially counteracted the changes of Ca(L) function and expression in cerebral and hindquarter arterial VSMCs of HU rats, suggesting the complexity of the underlying mechanisms in the effectiveness of intermittent artificial gravity for prevention of postflight cardiovascular deconditioning, which needs further clarification.     

Elevation of mitochondrial calcium by ryanodine-sensitive calcium-induced calcium release

Calcium is an important regulator of mitochondrial function. Since there can be tight coupling between inositol 1,4, 5-trisphosphate-sensitive Ca(2+) release and elevation of mitochondrial calcium concentration, we have investigated whether a similar relationship exists between the release of Ca(2+) from the ryanodine receptor and the elevation of mitochondrial Ca(2+). Perfusion of permeabilized A10 cells with inositol 1,4, 5-trisphosphate resulted in a large transient elevation of mitochondrial Ca(2+) to about 8 microm. The response was inhibited by heparin but not ryanodine. Perfusion of the cells with Ca(2+) buffers in excess of 1 microm leads to large increases in mitochondrial Ca(2+) that are much greater than the perfused Ca(2+). These increases, which average around 10 microm, are enhanced by caffeine and inhibited by ryanodine and depletion of the intracellular stores with either orthovanadate or thapsigargin. We conclude that Ca(2+)-induced Ca(2+) release at the ryanodine receptor generates microdomains of elevated Ca(2+) that are sensed by adjacent mitochondria. In addition to ryanodine-sensitive stores acting as a source of Ca(2+), Ca(2+)-induced Ca(2+) release is required to generate efficient elevation of mitochondrial Ca(2+).