Bioassay studies suggest that impaired endothelium-dependent relaxation in atherosclerotic arteries is due to a reduced release of biologically active endothelium-derived relaxing factor (EDRF). We tested the hypothesis that endothelial dysfunction is caused by deficiency of the EDRF precursor L-arginine. Aortae from normal and cholesterol-fed (1 %, 4 months) rabbits were excised and incubated for 1 h with 5 mM L-arginine. Pretreatment with L-arginine had no effect on the relaxation to acetylcholine in normal vessels and was without effect on the impaired response of atherosclerotic arteries to acetylcholine. This finding suggests that L-arginine deficiency is unlikely the underlying cause of impaired endothelium-dependent relaxation in the aorta of cholesterol-fed rabbits.
The aim of this study was to evaluate the effects of aging on endothelium-dependent and endothelium-independent relaxation of rabbit thoracic aorta from New Zealand white rabbits aged 4–6 and 7–12 months. The contractile response to noradrenaline (NA) decreased with increasing age, but NA [EC50] did not vary significantly. Acetylcholine (Ach)-induced relaxation of aortic rings precontracted with NA [EC50] did not change significantly with increasing age. The relaxation induced by ATP of aortic rings, precontracted with NA [EC50], was signficantly greater in young than in adult rabbits. This difference between young and adult animals became more evident in aortic rings deprived of endothelium: in adult animals, the ATP-induced relaxation of aortic rings with endothelium was significantly greater than in the rings without endothelium. The endothelium-independent relaxation by sodium nitrite (NaNO2) at lower concentrations was significantly greater in young than in adult rabbit aortic rings precontracted with NA [EC50]. Concluding, the age-induced changes in vascular response in male New Zealand white rabbits are related to an impaired mechanism at smooth muscle level.
The cerebral circulation is supplied with two vasodilator systems: the parasympathetic system storing vasoactive intestinal peptide, peptide histidine isoleucine, acetylcholine and in a subpopulation of nerves neuropeptid Y, and the sensory system, mainly originating in the trigeminal ganglion, storing substance P, neurokinin A and calcitonin gene-related peptide (CGRP). Recent knowledge of the innervation and effects of the dilator neuropeptides in the cerebral circulation is reviewed. Their role in the pathophysiology of subarachnoid hemorrhage and migraine has now received attention, with documentation of a clear linkage with the release of CGRP. In subarachnoid hemorrhage, other perivascular peptides are, to a lesser extent, involved.
The mesenteric vasculature of Dahl salt-sensitive (DS) rats on high-salt diet is supersensitive to nerve stimulation and to norepinephrine. The current experiments were undertaken to examine whether the enhanced sensitivity to nerve stimulation is due solely to the postsynaptic supersensitivity to norepinephrine, to increased sympathetic innervation, to altered transmitter release or to the presence of another transmitter acting as a potentiator. Catecholamine content and neuropeptide Y (NPY) presence were determined in tissues from young (approximately 5 weeks old) male Dahl rats exposed to 5 days of high (7%) or low (0.45%) salt diet. Catecholamine content from mesenteric artery, renal artery, caudal artery, right atrium, aorta, vas deferens and adrenal gland was quantified by high-pressure liquid chromatography with an electrochemical detector. A strain difference, independent of diet, between young DS and Dahl salt-resistant (DR) rats was seen only in adrenal epinephrine content. DS high-salt (+) rats displayed reduced norepinephrine content relative to DR+ in the mesenteric artery and right atrium. The release of norepinephrine from isolated mesenteric vasculature into the perfusate in response to transmural stimulation showed no significant differences between DS+ and DR+ preparations under basal, or deoxycorticosterone acetate (DOCA; 30 microM) perfusion conditions. The addition of 5 microM cocaine to the DOCA perfusion, while increasing total norepinephrine outflow in all preparations, failed to differentiate between DS+ and DR+. NPY immunofluorescence along mesenteric artery sections of DS+ and DR+ rats was not significantly different. Thus, in the tissues examined, enhanced responsiveness of vascular smooth muscle may not be explained by hypernoradrenergic innervation, elevated NPY innervation or altered release of transmitter.
The effects of various neurotransmitters on phosphoinositide hydrolysis, mobilization of Ca2+ and release of [3H]-norepinephrine ([3H]-NE) were studied in cultures of sympathetic neurons of chick embryos. [3H]-inositol-1,4,5-triphosphate ([3H]-IP3) was increased in sympathetic neurons by acetylcholine (ACh), muscarine and serotonin (5-HT). Dopamine and norepinephrine did not stimulate phosphoinositide hydrolysis. Intracellular concentration of free Ca2+ ([Ca2+]i) was measured in Indo-1-loaded sympathetic neurons at rest and after addition of test agents. Measurements were made in the cell body and growth cone regions since Ca2+ mobilization is known to be different in different regions of the sympathetic neurons. ACh (nicotinic component was blocked by hexamethonium) and 5-HT failed to increase the [Ca2+]i, in the cell body as well as in the growth cone. The spontaneous release of [3H]-NE was not affected by ACh and 5-HT. Caffeine increased the [Ca2+]i only in the cell body but not in the growth cone and had no effect on the release of [3H]-NE. These results suggest that an IP3-insensitive but caffeine-sensitive pool of Ca2+ is present only in the somatic region of sympathetic neurons and is not coupled to the transmitter release.
Activity of the efferent nerve supply to the vasculature results in local increases or decreases in the tone of the vascular smooth muscle cells with corresponding changes in diameter. This results in changes in pressure and flow, both of which, because they too influence the vascular wall, extend the influence potentially to the entire bed. As the vascular bed is sensitive to pressure - an increase causing vasoconstriction - and to flow - an increase causing variable amounts of contraction and relaxation - the final results must reflect their interaction. Thus, the direct changes in artery tone brought about by neural activity are modified and diffused throughout the entire regional arterial system by the concomitant changes in the flow and pressure of the blood.
Many endogenous and pharmacological vasodilators hyperpolarize vascular smooth muscle and this response appears to be due to an increased conductance to potassium ions. The hyperpolarization may contribute to the mechanism of dilation by causing voltage-dependent calcium channels to close. Recent evidence indicates that the response to hyperpolarizing vasodilators is mediated through activation of ATP-sensitive potassium (KATP) channels. Single KATP channels on isolated vascular smooth muscle cells are activated by cromakalim and calcitonin gene-related peptide (CGRP). This response is inhibited by glibenclamide. Cromakalim, CGRP and other vasodilators hyperpolarize and relax arteries in vitro and these responses are reversed by glibenclamide. The hypotensive effects of these agents in vivo are antagonized by glibenclamide. We propose that activation of KATP channels and the associated membrane hyperpolarization represents an important general mechanism of vasodilation.
Elucidation of the gene structure of several receptors known to mediate the signal of hormone or transmitter binding to intracellular effector systems through guanine-nucleotide-binding proteins (G proteins) has revealed that these receptors comprise a superfamily of related proteins. The hallmark of all G-protein-linked receptors is a presumed topography of 7 membrane-spanning loops, analogous to the structure of bacteriorhodopsin. Members of this gene superfamily contain regions, particularly with the hydrophobic domains, of homologous sequence. The expression of G-protein-linked receptors in heterologous cell systems has allowed for the study of the pharmacological and biochemical properties of individual receptor subtypes in a manner not previously possible with intact tissues containing multiple receptors. Site-directed mutagenesis experiments have identified many conserved amino acids which are involved in ligand binding, receptor activation by agonists and receptor-G protein coupling, and suggest that the conservation of receptor structure throughout this gene family may reflect a conservation of important functional domains within these proteins.
Inhibition of angiotensin-converting enzyme (ACE) shifts the limits of cerebral blood flow autoregulation toward lower blood pressure values. This effect seems to be mediated by blocking the formation of angiotensin II on the luminal side of the larger cerebral resistance vessels. Baseline cerebral blood flow (the flow within the autoregulatory limits) is not changed by acute or chronic ACE inhibition. An interaction between the vascular renin-angiotensin and the sympathetic nervous system is present. Activation of the latter inhibits the downwards shift of the upper limit of autoregulation following ACE inhibition.
Previous studies have demonstrated that vein storage in normal saline leads to significant mechanical morphological, and biochemical aberrations. However, little information is available regarding the functional damage that occurs. The purpose of this study was to evaluate the effect of saline storage on venous smooth muscle and endothelial function. Segments of ten external jugular veins from male New Zealand White rabbits were placed nondistended in either modified Krebs solution at 37 °C (Krebs-stored, KS) or heparinized normal saline at room temperature (saline-stored, SS) for 1 h. Segments 4 mm in length were then simultaneously studied in vitro under isometric tension. There was no difference in maximum tension or sensitivity to either bradykinin or histamine. Acetylcholine-induced relaxation in KS segments was not significantly different from relaxation in a historical cohort of nonstored segments (nonstored 87.4 ± 1.0% vs. KS 84.5 ± 2.0%; p = NS). However, there were significant attenuations in SS segment endothelium-dependent relaxation in response to both acetylcholine (KS 84.5 ± 2.0% vs. SS 76.4 ± 2.7%, p < 0.02) and aden-osine diphosphate (KS 47.9 ± 2.9% vs. SS 40.6 ± 3.7%, p < 0.002). Relaxant responses to sodium nitroprusside (endothelium-independent) were not significantly different in the two groups (KS 94.6 ± 1.6% vs. SS 95.7 ± 2.2%; p = NS). Electron microscopic evaluation of SS segments revealed endothelial cell disruption with cellular edema and loss of intact junctions. These results demonstrate that rabbit external jugular vein storage in normal saline at room temperature for 1 h (1) is associated with significant morphologic damage, (2) impairs endothelial-dependent relaxation, and (3) appears to have no effect on smooth muscle cell function. Additionally, KS-stored (37 °C) veins exhibit a level of endothelium-dependent relaxation that is equivalent to nonstored controls.
Rabbit abdominal aortas and human umbilical arteries are currently used as substrata for the study of platelet adhesion and aggregate formation under flow conditions. Using immunohistochemical and ultrastructural methods, we have analyzed both vessel surfaces. The reactivity towards platelets of the subendothelium (SE) exposed on these vessels after mechanical or enzymatic digestion (alpha-chymotrypsin) was morphometrically quantified and the nature of the interaction studied in the electron microscope. After mechanical damage, the ultrastructural study of rabbit aortas showed a clearly defined internal elastic lamina (IEL). In contrast, umbilical vessels lacked a consistent IEL and masses of amorphous material often located deeper in the media were the main constitutents of the SE. Immunohistochemical labeling of the von Willebrand factor bound to both types of vessel differed considerably. Quantification of platelet interactions after perfusion of citrated blood showed qualitative differences between mechanically damaged rabbit or human vessels. Enzymatic digestion produced a more thrombogenic surface on rabbit aortas (p less than 0.01 vs. nondigested), but decreased their reactivity towards platelets on umbilical arteries (p less than 0.01 vs. nondigested). The ultrastructural study of the interacting platelets revealed that aggregates, when present, were found on the extracellular matrix underlying endothelial cells of rabbit aortas, but interacting with fibrillar structures probably derived from cell elements of the media in the case of umbilical arteries. These findings indicate that rabbit aortas and umbilical arteries possess structural characteristics that result in different thrombogenic properties with respect to circulating platelets.
Morphometric studies conducted on the blood vessels of the spontaneously hypertensive rat have provided evidence that medial hypertrophy is a key characteristic of the vascular change which occurs in hypertension. In the present study, we determined whether 3-methylhistidine (3MH), a post-translationally modified amino acid which is found uniquely in the actin and myosin of muscle, could provide a biochemical marker of such change. Our results indicated that the concentrations of 3MH were selectively elevated in the blood vessels from the spontaneously hypertensive rat, when compared with concentrations in vascular tissues from the Wistar-Kyoto rat. The concentrations of 3MH in non-vascular tissues were similar in the two strains. Chronic captopril treatment prevented the development of hypertension in the spontaneously hypertensive rat and was associated with a reduction of the vascular concentrations of 3MH. We therefore conclude that blood vessel concentrations of 3MH are a useful biochemical index of the changes in vascular smooth muscle contractile protein which occur during the development of hypertension in the spontaneously hypertensive rat.
Sudden occlusion of the middle cerebral artery (MCA) in normotensive rats increases blood flow through anastomosing branches into the territory of the occluded artery. Three weeks after MCA occlusion, anastomoses to anterior cerebral branches are increased by more than 50% in luminal diameter. One month after MCA occlusion, blood flow and blood flow reserve to the territory of the occluded MCA are returned to normal levels. In stroke-prone spontaneously hypertensive rats (SHRSP), the anastomoses are significantly narrower and blood flow through the anastomoses is less than in normotensive rats. Tissue infarction invariably develops in the territory of the occluded MCA in SHRSP. We propose that the luminal width of the anastomosis is a major determinant of blood flow into the territory of the occluded artery and of the amount of tissue protected from infarction by collateral circulation.
This study investigated the release of nitric oxide (NO) from glyceryl trinitrate (GTN) and SIN-1 in Langendorff rabbit hearts. Infusion of either GTN (10-40 microM) or SIN-1 (0.45-4.5 microM) into the coronary inflow tract resulted in a decrease in coronary perfusion pressure and NO release (oxyhemoglobin technique) into the coronary effluent. NO release from SIN-1 occurred spontaneously whereas passage through the coronary circulation, i.e. active metabolism, was required for NO release from GTN. Removal of the coronary endothelium and blockade of endothelial NO formation did not affect NO release from GTN and SIN-1. In GTN-tolerant hearts, there was a considerable inhibition of GTN- but not SIN-1-induced NO formation and coronary vasodilation. These data suggest (1) that metabolic NO release from GTN occurs during passage of the coronary circulation and is independent of the presence of endothelium, and (2) reduced NO release is a major cause of nitrate tolerance.
The characteristics of carbon monoxide (CO)-induced, endothelium-independent relaxation of rabbit aorta were compared with those of nitric oxide (NO)-induced and light-induced relaxation and endothelium-dependent relaxation mediated by endothelium-dependent relaxing factor (EDRF). CO was less than one thousandth as potent as NO as a relaxant. Various findings, including an increase in cyclic GMP associated with CO-induced relaxation, led to the conclusion that CO - like NO, EDRF and light - produces relaxation as a result of its stimulation of guanylate cyclase. LY 83583, which generates superoxide, was a potent, fast-acting inhibitor of acetylcholine-induced endothelium-dependent relaxation and NO-induced relaxation, and a fairly potent, moderately fast-acting inhibitor of photorelaxation, but only a very weak inhibitor of CO-induced relaxation. The ability of LY 83583 as well as hemoglobin to inhibit photorelaxation is consistent with the hypothesis that on radiation a photo-induced relaxing factor is formed which can stimulate guanylate cyclase and which can be inactivated by superoxide and by hemoglobin.
Vascular smooth muscle sensitivity to norepinephrine (NE), measured by contractile responses in vitro, varied in different arteries of the rabbit and also in the same vessels in other species. There was a good correlation between variation in the affinity of NE for the alpha-1-adrenoceptor and tissue sensitivity. The variation was continuous and probably not indicative of different receptor subtypes. Solubilization of alpha-1-adrenoceptors from the membrane changed the affinity for specific ligands while reconstitution restored it. Taken together, these results suggest the presence of a factor(s) within the receptor microenvironment capable of modulating affinity and hence tissue sensitivity to NE. In some blood vessels, receptor number was correlated significantly with affinity of the alpha-1-adrenoceptor for NE also. In general, the contribution of receptor number was considerably less than the affinity for NE.
