Differential activation of alpha 1- and alpha 2-adrenoceptors on microvascular smooth muscle during sympathetic nerve stimulation

The relative contribution of postjunctional alpha 1- and alpha 2-adrenoceptors to constriction of microvessels was examined during sympathetic nerve stimulation and sympathetic escape (difference between peak and steady-state constriction). Large arterioles (120 +/- 4 microns control diameter) and venules (174 +/- 6 microns) and small arterioles (13 +/- 4 microns) were examined in rat cremaster skeletal muscle during stimulation of the cremaster efferent innervation (decentralized lumbar sympathetic chain, 0.5-16 Hz, 2-minute train). The muscle was suspended in a tissue bath, and diameter was measured with intravital microscopy. Frequency-response curves were obtained after vehicle (prazosin or rauwolscine) was added to the bath. In large arterioles, prazosin (10(-7) M) significantly attenuated constriction by 60-80%; a fivefold higher concentration had no additional effect. In contrast, rauwolscine (1 to 5 x 10(-7) M) had no effect. Venules evidenced minimal response to nerve stimulation. In small arterioles, rauwolscine (5 x 10(-7) M) significantly attenuated constriction by 50-60%, while prazosin (10(-7) M) had no effect. These data suggest that for large arterioles, which are known to possess both receptors, alpha 1-adrenoceptors are preferentially stimulated by nerve-released norepinephrine. In contrast, sympathetic constriction of small arterioles is mediated by alpha 2-adrenoceptors. Compared with large arterioles, small arterioles exhibited greater peak and steady-state constriction at all frequencies, with maximal responses achieved over the 0.5-4 Hz range. Large arterioles exhibit graded constriction over the entire frequency range. Sympathetic escape exhibited a small, negatively correlated frequency dependence for large arterioles, tended to be greater for small arterioles, and was more evident in large arterioles during alpha 2-adrenoceptor constriction at low-frequency stimulation. This distinct neural control of large resistance vessels by alpha 1-adrenoceptors and small terminal arterioles by alpha 2-adrenoceptors may allow neurogenic regulation of these vessel segments to be differentially susceptible to modulation by other extrinsic and intrinsic vasoactive controls that preferentially interact with alpha 1- and alpha 2-adrenergic contractile mechanisms.     

In situ analysis of alpha-adrenoceptors on arteriolar and venular smooth muscle in rat skeletal muscle microcirculation

The purpose of this study was to determine whether both alpha 1- and alpha 2-adrenergic receptors exist on vascular smooth muscle of microvessels and whether adrenergic constriction of anatomically distinct microvascular segments is differentially subserved by either receptor subtype. The cremaster skeletal muscle of anesthetized rats was acutely denervated and suspended in a Krebs bath containing cocaine, normetanephrine, and propranolol to block uptake1, uptake2, and beta-receptors, respectively. Intravital microscopy was used to study large distributing arterioles (mean diameter, 100 microns), small precapillary arterioles (25 microns), and capacitance venules (140 microns). Concentration-response (diameter change) curves were obtained for bath-added agonists norepinephrine (mixed alpha 1/alpha 2), phenylephrine (alpha 1), and B-HT 933 (alpha 2) in the absence or presence of antagonists prazosin (alpha 1) and yohimbine (alpha 2). Apparent pD2(-log ED50) values for large arterioles and venules were, respectively, as follows: norepinephrine (7.41 and 7.15), phenylephrine (5.95 and 5.41), and B-HT 933 (5.05 and 5.06). Low concentrations of prazosin (10(-8) M) and yohimbine (10(-7) M) produced receptor subtype-selective antagonism and parallel, dextral displacement of norepinephrine curves for large arterioles and venules. The large arteriole pKB (-log KB) was 7.83 +/- 0.65 for prazosin and 7.36 +/- 0.46 for yohimbine. Higher concentrations of prazosin (10(-7) and 3 X 10(-7) M) and yohimbine (10(-6) M) produced further dextral but nonparallel displacement of norepinephrine curves. In contrast, receptor subtype-selective concentrations of only yohimbine inhibited adrenergic constriction of small, precapillary arterioles; but prazosin had no effect at receptor subtype-selective concentrations. These data suggest that adrenergic regulation of large arterioles and venules in skeletal muscle uses both alpha 1- and alpha 2-adrenoceptors. Precapillary arterioles, however, may be subserved predominantly by alpha 2-receptors.     

Microvascular effects of atrial natriuretic factor: interaction with alpha 1- and alpha 2-adrenoceptors

The cremaster skeletal muscle of anesthetized rats was denervated and extended with intact circulation into a tissue bath. Intravital microscopy was used to measure microvessel diameter at three different anatomical levels within the microcirculation: large distributing arterioles (x control diameter = 100 +/- 7 micron), large capacitance venules (147 +/- 8 micron), and small terminal arterioles (17 +/- 1 micron). Norepinephrine (NE) was added to the cremaster bath to produce intermediate reductions in diameter of large arterioles and venules (55% and 38% of maximum constriction, respectively). In the presence of NE tone, bath-added atrial natriuretic factor (ANF) produced concentration-dependent dilation of both arterioles and venules. Arteriolar IC25 = 18 pmol and IC50 = 1.2 X 10(-10) M; venules exhibited similar sensitivity. However, the highest ANF concentration examined (10(-7) M) only reversed NE-induced tone by 70%. In a second large vessel group ANF completely reversed constriction induced by the alpha 1-adrenoceptor agonist, phenylephrine, in the presence of 5 X 10(-7) M yohimbine. However, vessels constricted with the alpha 2-receptor agonist UK-14,304 (in the presence of 10(-8) M prazosin) were insensitive to ANF. A third group of terminal arterioles, which possess considerable spontaneous "intrinsic" tone, were studied in the absence of alpha-receptor agonists. Significant dilation occurred at greater than 10(-7) M, and the maximal response was only 25% of complete dilation with adenosine. These data indicate that ANF exhibits a high potency and selectivity for reversal of alpha 1-adrenoceptor-mediated constriction of large arterioles and venules. Constriction produced by alpha 2-adrenoceptor occupation or by nonadrenergic "intrinsic" mechanisms appears to be insensitive to ANF. We propose that the ability of ANF to reduce microvascular resistance depends on the relative contribution of alpha 1-, alpha 2-, and intrinsic vasoconstrictor components to the prevailing level of smooth muscle tone. Differences in these components among regional circulations and between arterial and venous smooth muscle may contribute to the systemic hemodynamic pattern produced by ANF.     

Preservation of venular but not arteriolar smooth muscle alpha-adrenoceptor sensitivity during reduced blood flow.

To compare arteriolar versus venular smooth muscle sensitivity to myogenic and metabolic inhibition during reduced blood pressure and flow, we measured the diameter of first-order venules (diameter, 230 microns) and arterioles (diameter, 156 microns) of the denervated, blood-perfused rat cremaster skeletal muscle that was suspended in a tissue bath. Sensitivity was determined for bath-added norepinephrine in the presence of yohimbine or prazosin to produce alpha 1- and alpha 2-adrenoceptor constriction, respectively, and for KCl to examine non-receptor-mediated sensitivity. To reduce venular pressure and flow, vasopressin, which constricts cremaster arterioles but not venules, was applied locally at a maximally effective concentration. This arteriolar constriction had no effect on venular sensitivity to alpha 1-adrenoceptor and KCl-mediated constriction. Venular sensitivity (-log M EC50) to alpha 1 and to KCl activation was 6.20 +/- 0.10 and 1.20 +/- 0.04 in the absence and 6.34 +/- 0.09 and 1.30 +/- 0.03 in the presence of arteriolar constriction, respectively. Venular sensitivity to alpha 2 activation was actually greater during arteriolar constriction (6.25 +/- 0.11 in the absence of constriction versus 7.06 +/- 0.13 in the presence of constriction, p less than 0.001). In a second series, the effect of reduced cremaster perfusion pressure and flow on both arteriolar and venular sensitivity was examined by mechanically lowering cremaster inflow. Reduction of first-order arteriolar and venular flow by 82-85% attenuated arteriolar alpha 1 and abolished alpha 2 sensitivity but had no effect on venular adrenergic sensitivity; KCl sensitivity was increased. These data indicate that, in contrast to arteriolar smooth muscle, venular smooth muscle alpha-adrenoceptor sensitivity is preserved during reduced pressure and flow and, thus, is little affected by metabolic and myogenic regulation. The selective depressant effect on arteriolar adrenergic but not KCl constriction suggests that myogenic/metabolic inhibition of arterioles is receptor specific.     

Effect of acidosis on contraction of microvascular smooth muscle by alpha 1- and alpha 2-adrenoceptors. Implications for neural and metabolic regulation

Our previous studies have identified that adrenergic regulation of large arterioles and venules in skeletal muscle uses both postjunctional alpha 1- and alpha 2-adrenoceptors, whereas terminal arterioles appear to be subserved primarily by alpha 2-receptors. Adrenergic constriction of terminal arterioles is known to be particularly susceptible to inhibition by increased tissue metabolic rate. The purpose of this study was to examine the influence of tissue acidosis on alpha 1- and alpha 2-adrenoceptor constriction of skeletal muscle microvessels to determine if this differential receptor distribution might have significance in neural-metabolic interactions. Intravital microscopy of rat cremaster skeletal muscle was used to obtain concentration-response curves (diameter changes) of large distributing arterioles (mean diameter, 100 microns), small precapillary arterioles (20 microns), and capacitance venules (150 microns) for addition to the tissue bath of alpha-adrenergic agonists during normal pH (7.4) and during tissue bath acidosis (pH 7.1) produced by increasing bath PCO2. The following alpha-agonists were used: phenylephrine (alpha 1), B-HT 933 (alpha 2), and norepinephrine (mixed alpha 1/alpha 2). Acidosis had no effect on baseline diameter of the three vessel types, indicating a lack of effect on "intrinsic tone." Acidosis also had no effect on large microvessel sensitivity to phenylephrine but markedly reduced responses to B-HT 933. Acidosis had no effect on large arteriolar and venular sensitivity to norepinephrine but markedly decreased (x300) small precapillary arteriolar sensitivity. These data suggest that 1) alpha 2- but not alpha 1-adrenoceptor-mediated constriction of microvessels may be selectively sensitive to modest reductions in tissue pH, and 2) the prevalence of alpha 2-receptors on terminal arterioles and the marked sensitivity of alpha 2 constriction to tissue acidosis may contribute to the particular susceptibility of neural constriction at this level of the microcirculation to metabolic inhibition.     

Endothelin mediates a component of the enhanced myogenic responsiveness of arterioles from hypertensive rats

OBJECTIVE: To determine if the enhanced pressure-induced constriction of arterioles isolated from hypertensive rats is mediated by the endothelium. METHODS: We utilized isolated, cannulated first-order arterioles (80 to 110 microns, i.d.) from the rat cremaster muscle of spontaneously hypertensive (SHR) and normotensive (WKY) rats. Arteriolar diameter was measured in response to changes in intraluminal pressures as well as various pharmacological agents in the presence and absence of an intact endothelial cell lining. RESULTS: All arterioles developed intrinsic tone (approximately 74% of passive). Pressure-diameter relationships over a pressure range of 30 to 170 cm H2O demonstrated that the myogenic response of arterioles derived from both WKY and SHR was not dependent upon an intact endothelium. However, at higher pressures (> 170 cm H2O) the ability of the denuded arteriole from the SHR to maintain a constricted diameter was completely abolished. Treatment of arterioles from the SHR having intact endothelium with diclofenac (10(-5) M; 30 min) to inhibit the effect of cyclooxygenase had no effect on the high pressure constriction. In contrast, application of BQ-123 (10(-7) M; 30 min), an ET-A receptor blocker used to inhibit vascular smooth muscle responses to endothelin, completely abolished the arteriolar constriction at higher pressures. CONCLUSIONS: Therefore, in the hypertensive, arteriolar vasomotor responses to changes in intraluminal pressure is due to at least two mechanisms; one that is intrinsic to vascular smooth muscle (i.e., myogenic) and a second that involves an endothelial cell release of endothelin.     

Vessel- and vasoconstrictor-dependent role of rho/rho-kinase in renal microvascular tone

We examined the role of Rho/Rho-kinase in renal afferent and efferent arteriolar tone induced by angiotensin (Ang) II, KCl and elevated renal arterial pressure (from 80 to 180 mm Hg), using isolated perfused rat hydronephrotic kidney. In the condition with no vasoconstrictor stimuli, Y-27632, a Rho-kinase inhibitor, dilated only afferent (from 11.6 +/- 0.4 to 14.1 +/- 0.5 microm) but not efferent arterioles (from 11.6 +/- 0.2 to 12.6 +/- 0.7 microm) at 10(-5) mol/l. During renal vasoconstriction by Ang II, Y-27632 restored the afferent arteriolar constriction (141 +/- 10% reversal at 10(-5) mol/l), whereas the ability of Y-27632 to inhibit the Ang II-induced efferent arteriolar constriction was diminished (73 +/- 7% reversal). A similar action was observed with fasudil, another Rho-kinase inhibitor. Furthermore, Y-27632 impaired myogenic afferent arteriolar constriction, with 117 +/- 17% inhibition at 10(-5) mol/l. The inhibition by Y-27632 of the myogenic vasoconstriction was almost the same as that of the Ang II-induced tone of this vessel type. However, Y-27632 had a modest effect on KCl-induced vasoconstriction of afferent arterioles. In conclusion, the present study demonstrates a predominant role of Rho/Rho-kinase in mediating the basal and Ang II-induced tone of afferent, but not efferent, arterioles. Furthermore, the role of Rho/Rho-kinase in afferent arteriolar constriction differs, with a substantial contribution to Ang II-induced and myogenic constriction but a minimal role in depolarization-induced constriction. Since Ang II-induced, KCl-induced and myogenic constriction of afferent arterioles require calcium entry through voltage-dependent calcium channels, the interaction between Rho/Rho-kinase and the calcium entry pathway may determine the afferent arteriolar tone induced by these stimuli.     

Microvascular responses to E. coli endotoxin with altered adrenergic activity

The cremaster muscle microcirculation of pentobarbital-anesthetized Wistar rats was studied using videomicroscopy. The left cremaster muscle was spread over an optical port in a bath filled with modified Krebs solution (pH 7.4, 34 degrees C). The right femoral artery was cannulated for determination of mean arterial pressure (Pm). Following control measurements of Pm and arteriolar and venular dimensions, dose-response curves of arteriolar and venular dimensions to topical norepinephrine (10(-10) M to 10(-3) M) was obtained. The rats were then administered E. coli endotoxin (6 mg/kg, iv, LD100) over a 1-hr period. The dose response curves were than repeated at intervals of 30 min. Before endotoxin the threshold dose for norepinephrine was consistently 10(-9) M or 0(-8) M. Pm decreased progressively with time postendotoxin. After endotoxin infusion, there was a gradual and progressive constriction of both arterioles and venules. The threshold dosage for norepinephrine to produce constriction of both arterioles and venules increased progressively with time. At 3 hr postendotoxin the threshold dose had increased to 10(-6) M to 10(-4) M. This is the dose that produces maximum constriction of arterioles in the preendotoxin control period. The study was terminated when the animal died or the field was obscured by petechiae. The microvessel sensitivity to norepinephrine is markedly reduced during endotoxin shock possibly due to increase in the active state of the vascular smooth muscle or to change in length of the muscle fibers or to changes in sympathetic alpha-adrenergic activity. The response was not prevented by H1 and H2 receptor blockade, but was prevented by alpha-adrenergic blockade with phentolamine.     

Stimulation of calcium flows induced by endothelin in cultured smooth muscle cells

Endothelin is a potent vasoconstrictor peptide isolated from the conditioned medium of porcine aortic endothelial cells. The action of endothelin is thought to be associated with calcium entry via calcium potential channels (Yanagisawa et. al. Nature 1988; 38:411-415). The present study was designed to determine the effect of endothelin on calcium fluxes (influx and efflux) on rat aortic smooth muscle cells in culture. The unidirectional influx of calcium was measured 15, 45, 75 and 105 seconds after the addition of trace amounts of 45Ca++ (5 microCi/ml) to the cells incubated with or without endothelin. Endothelin (50nM) stimulated calcium influx from a basal level of 312 +/- 17 to 537 +/- 12 pmol/mn/10(6) cells. This stimulation was dose-dependent with an EC50 value of about 10 nM. When cells were preincubated with calcium antagonists (nifedipine, dilttiazem, D600, nicardipine and flunarizine) at a final concentration of 1 microM, the endothelin-stimulated calcium influx was not modified. The unidirectional efflux of calcium was measured after an overload of cells with 45Ca++ (5 microCi/ml) for 18 hours, over 10 seconds intervals. In the first 30 seconds after the addition of endothelin (100 nM), the amount of 45Ca++ released was 3 times that in the absence of the peptide. The effect of endothelin was concentration dependent and similar to those observed with other vasoconstrictor peptides (vasopressin and angiotensin II). The results indicate that endothelin does not directly act on voltage-dependent calcium channels. The endothelin-stimulated calcium efflux suggests a mobilization of calcium from intracellular store sites followed by extrusion through an activation of a specific receptor-dependent calcium channel.     

Delineation of the distribution of beta-adrenergic receptor subtypes in canine myocardium

beta 2-Receptors constitute only 10-30% of the total beta-adrenergic receptors in mammalian ventricular myocardium, but their precise tissue location cannot be determined easily by measuring physiological variables. To delineate the distribution of beta-receptor subtypes in myocytic and vascular components of the heart, we incubated transmural sections of canine left ventricle with [125Iodo]cyanopindolol and selected concentrations of the beta 1-selective antagonist betaxolol or the beta 2-selective antagonist ICI 118,551. Detailed competition binding data were best accounted for by a two-site model in which approximately 75% of total sites were beta 1- and 25% were beta 2-receptors. The relative proportions of beta-receptor subtypes in myocytic and vascular components were assessed autoradiographically by analyzing the density of binding sites in transmural sections incubated with radioligand and subtype-selective displacers. Betaxolol (10(-7) M) reduced the density of radioligand binding sites by 44% in regions composed primarily of ventricular myocytes but by less than 5% in small coronary arterioles. ICI 118,551 (10(-7) M) reduced radioligand binding-site density by 18% in myocytic regions and by 55% in small arterioles. In myocytic regions, these data indicated a subtype composition of approximately 85% beta 1- and 15% beta 2-sites. In contrast, arterioles contained almost exclusively the beta 2-subtype. The diameters of coronary vessels in which beta 2-receptors were found to be selectively increased fell within a narrow range (mean +/- SD, 35 +/- 11 microns; range, 16-55 microns). Small mural arteries and venules did not contain a significantly higher proportion of beta 2-receptors than adjacent myocytic regions.     

Effects of endothelin, platelet activating factor and thromboxane A2 in ferret lungs.

We have compared the effects of three vasoactive agents, endothelin, platelet activating factor and thromboxane A2 analogue, U 46,619, in the pulmonary circulation of ferrets. Lungs of nine adult ferrets, body weight 1.06 +/- 0.27 kg, were isolated and perfused with sheep red blood cells suspended in Kreb's solution with 2 g% Dextran 70 (hematocrit 33 +/- 6%), under conditions of constant flow in zone 3. Endothelin-1 (ET: 0.1-1.35 micrograms/kg), platelet activating factor (PAF: 0.5-5.5 micrograms/kg) or thromboxane A2 analogue (U 46,619: 1 microgram/kg), was infused into the pulmonary artery and the pressure response determined. To locate the site of action of the agents, the pulmonary circulation was partitioned into arteries, microvessels and veins by measuring pressures in 20-50 microns diameter subpleural arterioles and venules by micropuncture, both during baseline and after the peak response to each vasoactive drug. We found that the ferret pulmonary circulation constricted in response to ET, PAF and U 46,619, but the magnitude of constriction varied. Ferret lungs were most sensitive to U 46,619 and least sensitive to PAF. The major site of action also differed among the agents; U 46,619 and ET predominantly constricted veins whereas PAF predominantly constricted arteries. We conclude that the ferret pulmonary circulation demonstrates differential responsiveness to vasoactive agents, that venous constriction is a common feature and that the predominant site of action varies with the specific agent.     

Presence of a specific binding site of endothelin on cultured smooth muscle cells

The 21 amino-acids endothelium-derived peptide, endothelin, recently isolated by Yanagisawa et al. (Nature 1988; 33, 411-5) possesses potent vasoconstrictive properties in vivo and in vitro. In the present study, we investigated the binding of endothelin on cultured rat aortic smooth muscle cells using 125I-iodotyrosyl-endothelin labelled by the chloramine T method. 125I-endothelin bound to a single class of hight affinity binding sites in vascular smooth muscle cells. After 2 hours incubation at 37 degrees C, dissociation constant (Kd) was 1.2 +/- 0.3 nM and binding capacity (Bmax) was 59 +/- 11 fmol/10(6) cells (n = 5). 125I-endothelin was displaced by unlabelled endothelin with a inhibition constant (Ki) of 0.2 nM, whereas an absence of competition was observed with 1 microM of vasoactive substances such as angiotensin II, arg-vasopressin, atrial natriuretic factor, histamine, epinephrine and norepinephrine, and with the calcium entry blocks nifedipine, diltiazem and D 600. 125I-endothelin binding was not reversible by addition of unlabelled endothelin (1 microM) and not dissociable by acetic acid (10 mM) or trypsin (0.1 p. 100) treatment of the cells. Furthermore, preincubation of vascular smooth muscle cells with endothelin (1 nM) at 37 degrees C induced a rapid down-regulation of endothelin binding capacity by about 50 p. 100. These data indicate that specific endothelin bindind sites are present in smooth muscle cells, and suggest a tight binding or a rapid captation of endothelin into the cell membrane leading to contractile events.     

In VIVO Arteriolar Reactivity to Norepinephrine and Calcium in One-Kidney,One-Clip Goldblatt Hypertensive Rats

The in vivo reactivity of small arterioles to norepinephrine and to changes in external calcium was investigated in normotensive (NT) and one-kidney, one-clip Goldblatt hypertensive rats (1K1C). Rats were anesthetized with sodium pentobarbital (50 mg/kg) and arterioles in the cremaster muscle were exposed to increasing concentrations of either norepinephrine (10^?10 to 10^?5 M) or of bath calcium (0 to 5.1 mM). Third-order arterioles of 1K1C showed almost a ten-fold increased reactivity to NE compared to arterioles of NT rats (pD₂ values of 7.88 ±.43 vs 6.92 ±.30). Arterioles of 1K1C rats showed an increased reactivity to re-exposure to calcium (0.65 to 5.10 mM). Following exposure to phentolamine this hyper-reactivity was abolished and arterioles of 1K1C and NT exhibited similar responses to changes in bath calcium concentrations, suggesting that the increased reactivity in the 1K1C was due to stimulation of endogenous norepinephrine release. When arterioles were exposed to increasing bath concentrations of the calcium entry blockers, verapamil and diltiazem, dilator responses were similar for 1K1C and NT groups. Collectively, these data suggest that during the development of renovascular hypertension, observed increases in arteriolar reactivity involve an increase in the receptor mediated entry of extracellular calcium into vascular smooth muscle and no change in non-receptor-mediated entry of calcium.     

Effect of reduced blood flow on alpha 1- and alpha 2-adrenoceptor constriction of rat skeletal muscle microvessels.

Adrenergic constriction of skeletal muscle arterioles, particularly small terminal arterioles, is opposed by decreased blood flow or increased metabolic rate. Our previous studies indicate that neural constriction of large arterioles, which have both postjunctional alpha 1- and alpha 2-adrenoceptors, is mediated by alpha 1-receptors; small arterioles depend on alpha 2-receptors. Also, alpha 2, but not alpha 1, constriction is reduced by acidosis. Differential sensitivity of alpha 1 versus alpha 2 constriction to metabolic signals such as H+ may underlie the sensitivity of arteriolar adrenergic constriction to metabolic inhibition. To examine this hypothesis, we studied the effect of reduced perfusion on alpha 1- versus alpha 2-mediated constriction of large arterioles and venules. Intravital microscopy of rat cremaster skeletal muscle was used to obtain concentration-response curves for phenylephrine (alpha 1-agonist) and UK-14,304 (alpha 2-agonist). Thirty percent reduction in cremasteric artery flow by venous outflow obstruction had no effect on baseline diameter, indicating no effect on "intrinsic tone." Reduced perfusion also had no effect on arteriolar or venular sensitivity to phenylephrine or venular sensitivity to UK-14,304 but significantly attenuated arteriolar response to UK-14,304. To examine a possible mechanism for the selective inhibition of alpha 2 constriction by acidosis, we determined the effect of acidosis on the partial alpha 1-agonist St587. Like alpha 2 constriction, St587-mediated constriction of arterioles was reduced during acidosis and was attenuated by nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of T-type selective calcium antagonist on renal microcirculation: studies in the isolated perfused hydronephrotic kidney

Although calcium antagonists exert preferential vasodilation of renal afferent arterioles, we have recently demonstrated that nilvadipine and efonidipine, possessing both L-type and T-type calcium channel blocking action, reverse the angiotensin (Ang) II-induced afferent and efferent arteriolar constriction. In the present study, we investigated the role of T-type calcium channels in mediating the Ang II-induced efferent arteriolar tone using the selective T-type calcium channel blocker mibefradil. Isolated perfused hydronephrotic rat kidneys were used for direct visualization of renal microcirculation. Administration of Ang II (0.3 nmol/L) caused marked constriction of afferent (from 13.5+/-0.6 to 9.2+/-0.6 microm, P<0.01, n=6) and efferent (from 11.5+/-1.0 to 7.4+/-0.7 microm, P<0.01, n=5) arterioles. Mibefradil (1 micromol/L) dilated both vessels, with 82+/-11% and 72+/-7% reversal of afferent and efferent arterioles, respectively. Similarly, nickel chloride (100 micromol/L) caused dilation of both arterioles, similar in magnitude in afferent (68+/-10%, n=7) and efferent (80+/-7%, n=7) arterioles. To eliminate the possibility that the mibefradil-induced dilation was mediated by L-type channel blockade, mibefradil was administered in the presence of nifedipine (1 micromol/L). Thus, nifedipine caused modest efferent arteriolar dilation (30+/-6% reversal, n=9), and subsequent addition of mibefradil elicited further dilation of this vessel (80+/-4%, P<0.01 versus nifedipine). Furthermore, mibefradil reversed the Ang II-induced efferent arteriolar constriction even in the presence of nifedipine and phentolamine. These findings demonstrate that T-type calcium antagonists markedly dilate the Ang II-induced efferent arteriolar constriction, but the action is not mediated by inhibition of catecholamine release. This potent activity would contribute to the efferent arteriolar response to nilvadipine and efonidipine and may offer benefit in light of glomerular hemodynamics.     

Mediators of CD18/P-selectin-dependent constriction of venule-paired arterioles in hypercholesterolemia

This study addresses the role of venule-derived mediators in the arteriolar constriction that accompanies hypercholesterolemia. Constriction was assessed by measuring the tone of small arterioles closely paired with venules in the mesentery of normal cholesterol rats (NC), high cholesterol rats (HC), HC rats injected with antibodies against CD18 and P-selectin (HC/mAbs), HC rats treated with the thromboxane synthase inhibitor, ozagrel (HC/ozagrel), and HC rats pretreated with anti-platelet serum (HC/APS). Venule-paired arterioles in the untreated HC group demonstrated enhanced tone compared with arterioles in the NC group, while no difference was found between unpaired arterioles of the two groups. Perivascular nitric oxide (NO) concentrations were found to be significantly decreased in venule-paired arterioles of HC rats (238+/-14 nM) compared with those of NC rats (426+/-42 nM). The injection of anti-adhesion antibodies successfully attenuated the enhanced arteriolar tone and venular leukocyte adherence in the HC group, and tended to increase levels of NO in venule-paired arterioles by 33% (to 326+/-19 nM; still lower than that of the NC group). Ozagrel and platelet depletion attenuated the enhanced arteriolar tone by 53% and 33%, respectively, without affecting NO concentrations. These findings indicate that the mechanism of blood cell-dependent arteriolar constriction during hypercholesterolemia may be dependent on thromboxane, a decrease in NO, and the proximity of the arterioles to postcapillary venules.     

Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules

At the onset of the inflammatory process, leukocytes roll along venular but not arteriolar walls before they firmly attach and emigrate. To test whether differences in hydrodynamic flow conditions are responsible for the preferential occurrence of leukocyte rolling in venules, we varied wall shear rate, gamma w, between 30 and 2,000 sec-1 by selective micro-occlusion of side branches in venules and arterioles (diameter, 20-37 microns) of the exposed mesentery of anesthetized rats. In venules, 39% (range, 6-77%) of all passing leukocytes were found interacting with the endothelium (rolling), whereas this fraction was only 0.6% in arterioles. The fraction of rolling leukocytes in venules decreased from 49 +/- 13% at gamma w less than 100 sec-1 (N = 12) to 24 +/- 13% at gamma w greater than 400 sec-1 (N = 12). Mean leukocyte rolling velocity in venules increased with gamma w, but the most frequent rolling velocity class was 20-40 microns/sec at all shear rates. In arterioles, even prolonged (up to 90 minutes) conditions of reduced flow (gamma w less than 150 sec-1) did not induce leukocyte rolling. Radial distribution of freely flowing leukocytes not different in arterioles and venules. The data indicate that hemodynamic factors are not responsible for the difference of leukocyte adhesion between arterioles and venules. The venular endothelium appears to be specialized to support leukocyte adhesion during inflammation. This finding correlates with reports on preferential expression of various endothelial-leukocyte adhesion molecules on venular endothelial cells.     

Vascular smooth muscle structure and juvenile growth in rat intestinal venules

The morphological structure of individual vascular smooth muscle cells from intestinal venules was evaluated with a combination of quantitative scanning (SEM) and transmission (TEM) electron microscopy techniques. In addition, growth of individual venular smooth muscle cells and of the overall vessel wall was compared from measurements of these variables during the rapid juvenile growth spurt from ages 4 to 6 and 10 to 12 weeks in Wistar-Kyoto rats. SEM revealed that smooth muscle cells of intestinal venules in weanling rats are very long (379 +/- 91 [SD] microns) and wide (6.0 +/- 1.3 microns) and very little further cell enlargement occurs during rapid juvenile growth. TEM studies indicated that passive inner vessel diameter and total muscle layer cross-sectional area of both the largest and intermediate diameter venules of young rats, as well as the percentage of the total wall area as muscle tissue in each venule type, did not significantly increase during body growth. These observations indicate that both the intestinal venules and their smooth muscle cells reach mature dimensions at a very early stage of life. Comparison of intestinal vascular smooth muscle cell dimensions indicates that venular smooth muscle cells are much larger in both cell length and volume than comparable arteriolar smooth muscle cells.     

Nonuniform vasomotor responses of the coronary microcirculation to serotonin and vasopressin

Large-conduit coronary arteries respond to vasoactive stimuli differently than smaller coronary arterioles, but the quantitative effects of many vasoactive stimuli at various levels of the microvasculature remain unknown. To determine the site of constriction or dilation to serotonin and vasopressin in the coronary microcirculation, we studied microvascular responses in the left ventricle of anesthetized cats (n = 36). To compensate for motion due to contraction of the heart, the epicardium was visualized with stroboscopic epi-illumination controlled by a computer to flash once per cardiac cycle in mid-diastole, making the vessels appear stationary. Serotonin (16 micrograms/kg/min) or vasopressin (0.5 units/min) was infused into the left atrium while maintaining aortic pressure constant with a snare on the descending aorta or inferior vena cava. Myocardial blood flow was measured with radioactive microspheres. During infusion of serotonin, aortic pressure and heart rate did not change, but myocardial perfusion increased 90 +/- 38% (mean +/- SEM) from a control value of 159 +/- 27 ml/min.100 g. Arteries and arterioles larger than 90 microns constricted in response to serotonin (control 159 +/- 12 microns; percent change -18 +/- 3; range -41 to 10%) while arterioles less than 90 microns dilated to serotonin (control 54 +/- 7 microns; percent change 22 +/- 9; range -10 to 62%). During infusion of vasopressin, aortic pressure and heart rate did not change, and myocardial perfusion decreased 16 +/- 7% (control, 147 +/- 18 ml/min.100 g).(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin and prostaglandin H(2)/thromboxane A(2) enhance myogenic constriction in hypertension by increasing Ca(2+) sensitivity of arteriolar smooth muscle

The myogenic response of skeletal muscle arterioles is enhanced in hypertension because of the release of endothelin (ET) and prostaglandin H(2) (PGH(2))/thromboxane A(2) (TxA(2)) from the endothelium. We hypothesized that ET and PGH(2)/TxA(2) modulate Ca(2+) signaling in arteriolar smooth muscle and thereby enhance myogenic constriction. Thus, simultaneous changes in intracellular Ca(2+) concentration in smooth muscle ([Ca(2+)](i)), measured by fura 2 microfluorometry (expressed as Ca(2+) fluorescence ratio [R(Ca)]), and diameter were obtained as a function of intraluminal pressure (P(i)) in isolated cannulated gracilis muscle arterioles (diameter approximately 120 micrometer) of normotensive Wistar rats (WR) and spontaneously hypertensive rats (SHR). In the absence of extracellular Ca(2+), increases in P(i) from 20 to 160 mm Hg increased the passive diameter of arterioles without changes in R(Ca). In the presence of extracellular Ca(2+) and endothelium, increases in P(i) elicited similar increases in R(Ca) (30+/-7% for control and 33+/-8% for SHR at 160 mm Hg) but a significantly (P<0.05) greater constriction of SHR arterioles compared with WR arterioles (at 160 mm Hg, 55+/-4% versus 38+/-2%, respectively, of passive diameter). In the absence of the endothelium, P(i)-induced changes in the R(Ca) and diameter of SHR and WR arterioles did not differ significantly. Also, a step increase in P(i) (from 80 to 140 mm Hg) elicited a similar increase in R(Ca) but greater constrictions in SHR versus WR arterioles. In the presence of the TxA(2) receptor inhibitor SQ29,548 and the ET(A) receptor inhibitor BQ123, there was no difference between responses of SHR and WR arterioles. In WR arterioles, increasing concentrations of KCl elicited a significant increase in R(Ca) (38+/-7% at 80 mmol/L) and completely constricted the arterioles. In contrast, constrictions to ET (52+/-7% at 3x10(-12) mol/L) and the TxA(2) agonist U46619 (40+/-8% at 3x10(-9) mol/L) were not accompanied by increases in R(Ca) at submaximal concentrations. Collectively, these findings suggest that in hypertension, endothelium-derived ET and PGH(2)/TxA(2) increase the Ca(2+) sensitivity of the contractile apparatus of arteriolar smooth muscle; thus, the similar increases in [Ca(2+)](i) in response to the elevation of intraluminal pressure elicit greater myogenic constriction.