Acetylcholine-induced Dilation Research Articles

Measurement of endothelial cytosolic calcium concentration and nitric oxide production reveals discrete mechanisms of endothelium-dependent pulmonary vasodilatation.

Nitric oxide is an endothelium-derived relaxing factor. Conversion of L-arginine to nitric oxide follows mediator-induced elevation of endothelial cytosolic calcium concentration. However, not all endothelium-dependent vasodilatation is caused by endothelium-derived relaxing factor, and few studies have correlated changes in vascular tone with measurement of free cytosolic calcium concentration or nitric oxide. The effects of three endothelium-dependent vasodilators (acetylcholine, bradykinin, and A23187) on vascular tone and nitric oxide production were studied in proximal rat pulmonary artery rings. Changes in free cytosolic calcium concentration and nitric oxide production were also studied in bovine pulmonary artery endothelial cells. A23187 and bradykinin caused pulmonary vasodilatation, nitric oxide production, and elevation of endothelial calcium concentrations. Although acetylcholine caused endothelium-dependent vasodilatation, it reduced free cytosolic calcium concentration and failed to increase nitric oxide levels. Acetylcholine-induced dilatation was partially inhibited by meclofenamate but was unaffected by ouabain. Acetylcholine, unlike bradykinin and A23187, does not act through a nitric oxide-dependent mechanism in the rat pulmonary vasculature.

Prostanoids Contribute to Endothelium-Dependent Coronary Vasodilation in Guinea Pigs

This study characterizes the contribution of prostanoids to endothelium-dependent responses in two vascular regions of the guinea pig. We compared the mechanisms of relaxation responses to acetylcholine and adenosine triphosphate (ATP) in the coronary vasculature and in the abdominal aorta of the guinea pig. Endothelium-dependent responses were examined in an isolated, potassium-arrested guinea pig heart utilizing a modified Langendorff preparation. Coronary vessels were constricted with prostaglandin F2 alpha and dilated with acetylcholine (10(-9)-10(-6) mol) or ATP (10(-10)-10(-7) mol) before and after exposure to indomethacin (14 microM, n = 6) or ibuprofen (150 microM, n = 5). Helically cut strips of abdominal aorta (n = 6) were suspended in isolated tissue baths for measurement of isometric force. Relaxation to acetylcholine (5.5 x 10(-7) M) and ATP (10(-5) M) was quantified in strips contracted with norepinephrine before and after exposure to indomethacin (14 microM). In addition, the endothelium was damaged by exposing vessels to free radicals generated by electrolysis of the buffer (4 Hz, 9 V, 1 ms, 5 min). Following electrolysis of the buffer, relaxation responses to acetylcholine and ATP were significantly attenuated in both preparations. In the perfused heart, endothelium-dependent dilatation to acetylcholine, but not ATP were significantly inhibited in the presence of indomethacin or ibuprofen. In contrast, acetylcholine- and ATP-induced relaxation responses in the aorta were not altered by indomethacin. We conclude that prostaglandins contribute to acetylcholine-induced dilatation in the coronary bed but not in the abdominal aorta of the guinea pig. Furthermore, in the coronary bed, different endothelial factors mediate relaxation to acetylcholine and ATP.

EDRF-mediated dilatation in the rat isolated perfused kidney: a microangiographic study.

1. X-ray microangiographic techniques were used to study the influence of endothelium-derived relaxing factor (EDRF) on vasomotion in the isolated, intact, buffer-perfused kidney of the rat. The main renal (R0), segmental (R1) and interlobar (R2) arteries (control diameters ca. 600, 400 and 300 microns respectively) were studied quantitatively. 2. Inhibition of basal EDRF activity by haemoglobin (1 microM) did not elevate perfusion pressure or constrict R0, R1 and R2 in control preparations, implying a low level of spontaneous myogenic tone. In preparations preconstricted by 0.3 microM methoxamine, haemoglobin caused a further rise in perfusion pressure and amplified constrictor responses in R1 and R2 while also inducing 'paradoxical' dilatation of R0. 3. A spatially heterogeneous pattern of diameter responses (constriction of R2 and R1 with minimal dilatation of R0) was observed with two concentrations of methoxamine (0.3 microM and 3 microM). The magnitude of these responses was, however, smaller with 3 microM than 0.3 microM methoxamine, even though it increased perfusion pressure to a greater extent (88 mmHg cf. 24 mmHg). This 'paradoxical' behaviour indicates more pronounced constriction of distal arteries (which could not be resolved quantitatively) with 3 microM methoxamine. 4. In contrast to the heterogeneity of constrictor responses induced by methoxamine, the dilator action of acetylcholine was spatially homogeneous: log IC50 values calculated from the diameter changes induced in R0, R1 and R2 were similar and, moreover, equivalent to that calculated from the corresponding alterations in perfusion pressure. The fall in perfusion pressure induced by an approximately median effective concentration of acetylcholine (0.3 microM) was completely reversed by haemoglobin, consistent with the involvement of EDRF, although, reversal of the acetylcholine-induced dilatation of R0, R1 and R2 was not observed. 5. The results are consistent with the idea that constriction of distal vessels can attenuate and even directionally reverse intrinsic constrictor responses in the proximal R0, RI and R2 'feed' arteries by producing an overriding increase in 'upstream' pressure. This effect explains the paradoxical dilatation of Ro induced by haemoglobin in the presence of 0.3 microM methoxamine, the smaller magnitude of the diameter changes induced in R0, RI and R2 by 3 microM as compared to 0.3 microM methoxamine, and the failure of haemoglobin to reverse the acetylcholine-induced dilatation of R0, R1 and R2.

Functional and anatomical recovery of endothelium after denudation of coronary artery.

Endothelium-related coronary dilation after balloon denudation was compared with the extent of luminal lining of the regenerated endothelial cells. Under conditions of asepsis, a pair of 10-MHz piezoelectric crystals, an electromagnetic flow probe, and a cuff occluder were placed on the left circumflex coronary artery. Seven to 10 days after this instrumentation, the dogs were anesthetized and the endothelium was removed with the use of a balloon catheter. Immediately after balloon denudation, coronary dilations following reactive hyperemia (reactive dilation) and acetylcholine 3 micrograms/kg iv were reduced to 19 +/- 5 (P less than 0.01) and 10 +/- 4% (P less than 0.01) of control, respectively. In 2, 5, and 7-10 days after denudation, reactive dilation in the conscious dogs was 24 +/- 7 (n = 16), 71 +/- 10 (n = 11), and 75 +/- 9% (n = 5) of control, respectively. Acetylcholine-induced coronary dilation in conscious state was 52 +/- 12 (n = 9), 116 +/- 17 (n = 7), and 94 +/- 24% (n = 2) of control in 2, 5, and 7-10 days after denudation, respectively. There was a positive linear relation between the extent of reactive dilation and acetylcholine-induced dilation from 2 to 10 days after denudation (r = 0.734). The amount of reactive hyperemia, nitroglycerin-induced coronary dilation, and ergonovine-induced coronary constriction remained constant during the experimental period. The area covered by the endothelial cells at the denuded site was 8 +/- 3 (n = 5), 18 +/- 9 (n = 6), 64 +/- 9 (n = 6), and 81 +/- 11% (n = 3) of the denuded area in 0, 2, 5, and 7-10 days after denudation, respectively, which linearly related to the percent recovery of reactive dilation (r = 0.912). Thus reactive dilation or acetylcholine-induced coronary dilation depended on the presence of endothelium, irrespective of the shape or alignment of the regenerated tissues. These findings may be clinically relevant because anatomical integrity of the endothelium might be assessed in vivo by reactive dilation or acetylcholine-induced coronary dilation.

Altered endothelial cell-mediated arterial dilation in dogs with D. immitis infection.

Vascular endothelial cells regulate arterial diameter in vivo and in vitro. Stimulation of endothelial cell muscarinic receptors by acetylcholine results in the production and release of a nonprostaglandin metabolite of arachidonic acid that causes vascular smooth muscle relaxation. We examined femoral artery endothelium-dependent vasodilator responses in normal dogs and dogs with heartworm (Dirofilaria immitis) infection. Endothelium-dependent vascular reactivity was attenuated in dogs with D. immitis infection studied in the spring but not in the fall. The dilator response was inversely related to the number of female worms but not related to the presence of circulating microfilariae. Indomethacin markedly depressed responses to acetylcholine in dogs with D. immitis but did not alter acetylcholine-induced dilation in normal dogs. These data suggest that D. immitis releases substances that alter distal arterial endothelial cell arachidonic acid metabolism. The seasonal pattern may reflect the onset of maximal reproductive activity in the spring and its decline as the vector season ends in the fall.

Reversal of acetycholine-induced coronary resistance vessel dilation by hemoglobin

In large arteries, acetylcholine-induced dilation is mediated by endothelium-derived relaxing factor (EDRF) and can be blocked by hemoglobin (Hb). However, the role of EDRF in the microcirculation is uncertain. Therefore, the effect of Hb on acetylcholine-induced dilation of resistance vessels was studied in isolated, constant-flow perfused rabbit hearts. Hb reversibly blocked the acetylcholine-induced lowering of perfusion pressure (control −27 ± 3%; during Hb 0 ± 4%) without inhibiting responses to other dilators, strongly suggesting that acetylcholine action in the microcirculation is mediated by EDRF.

Long-term nitroglycerin treatment: effect on direct and endothelium-mediated large coronary artery dilation in conscious dogs.

We examined the effect of nitroglycerin (GTN) tolerance on an important determinant of nitrate-antianginal action, large coronary artery dilation, in 11 chronically instrumented conscious dogs. In addition, endothelium-mediated coronary artery dilation was studied because this shares a common dilator pathway with the nitrates, i.e., activation of soluble guanylate cyclase. With long-term GTN (1.5 micrograms/kg/min iv for 5 days) the diameters of the left circumflex and anterior descending coronary arteries showed an initial increase of 8.2 +/- 0.3% and 10.8 +/- 0.9%, respectively, returning to control levels by the second to third day of treatment. On days 4 and 5, the dose-response relations for GTN-induced epicardial artery dilation were shifted (p less than .01) to 17- to 20-fold higher doses. However, there was no attenuation of epicardial artery dilation induced by SIN-1 (n = 7), another activator of guanylate cyclase, or of endothelium-mediated dilation assessed both as flow-dependent dilation (n = 7) and as direct intra-arterial acetylcholine-induced dilation (n = 4). In addition, there was no clear tolerance to the peripheral vascular actions of GTN responsible for reflex tachycardia and increased coronary flow. We conclude that a moderate degree of nitrate tolerance to epicardial artery dilation does not affect the responsiveness to other exogenous or endogenous activators of guanylate cyclase. However, this tolerance to epicardial artery dilation, together with the maintenance of peripheral vascular actions that can induce reflex tachycardia, result in a potentially unfavorable balance of GTN effects.

Vagal nerve stimulation causes noncholinergic dilatation of gastric arterioles.

Vagal nerve stimulation causes prompt dilatation of gastric submucosal arterioles (the vessels that control gastric mucosal blood flow) in rats. In vivo microscopy was used to determine whether this direct vasodilator effect of vagal nerve stimulation on rat gastric submucosal arterioles is mediated by cholinergic fibers. Acetylcholine and atropine were topically applied to the submucosa. The distal end of the severed vagus nerve was electrically stimulated (8 V, 2 ms, 6 Hz, 20 s) subdiaphragmatically. Diameter changes of the submucosal arterioles were videotaped and measured with an image-splitting technique on playback of the videotapes. Acetylcholine, 10(-7) to 10(-5) M, dilated the arterioles dose dependently. Atropine prevented the acetylcholine-induced dilatation, 10(-5) M, nearly completely inhibiting the dilatation. Vagal nerve stimulation dilated the arterioles promptly, and this dilatation was not blocked by 10(-5) M atropine, a dose that blocked the acetylcholine-induced dilatation. These results indicate that vagal nerve stimulation causes atropine-resistant, noncholinergic dilatation of gastric submucosal arterioles in rats.

Endothelium-Mediated Dilations Contribute to the Polarity of the Arterial Wall in Vasomotion Induced by α2-Adrenergic Agonists

We tested whether or not an endothelium-mediated dilation is involved in the response of intact arteries to alpha-adrenergic stimulation, by separately applying agonists to the luminal or adventitial side of the arterial wall. Cumulative dose-response curves of the alpha 1-agonists l-phenylephrine or cirazoline applied luminally in rat tail arteries and in side branches of canine femoral arteries were identical to those obtained by adventitial application in the intact arteries, and were not modified by removal of the endothelium (eliminating acetylcholine-induced dilations). Constrictions induced by the alpha 2-agonists UK-14,304 or azepexole applied luminally were significantly lower than those induced by adventitial application, and were augmented significantly by removal of the endothelium. Half-maximally precontracted arteries were dilated by addition of alpha 2-agonists to the luminal perfusate; these dilations were abolished by removal of the endothelium. It is concluded that the functional polarity of the vascular wall of these arteries in response to alpha 2-agonists results from the release of a dilatory signal from the endothelial cells, counteracting the direct contractile activation of the adjacent smooth muscle cells by the agonists.

Pial artery responses to norepinephrine potentiated by endothelium removal.

The effect of endothelium removal on pial artery constriction in response to norepinephrine (NE) was studied in vitro using a perfused vessel setup in which pressure increases indicate vasoconstriction. In deenodothelialized rabbit arteries, the reaction to extraluminal NE was found to be characterized by a much higher Emax (2.0 times) and a slight (but significant) leftward shift of the concentration-response curve (lower EC50) compared with control vessels. In cat arteries subjected to either extra- or intraluminal NE, the Emax was also substantially higher in deendothelialized preparations (4.4 and 5.1 times, respectively), but there was no significant difference in the EC50 values. Anatomical verification and functional tests (acetylcholine-induced dilatation) confirmed the presence and the absence of the endothelium in control and lesioned arteries, respectively. This modulatory influence of the endothelium may be of importance in cerebrovascular pathology.

Neurogenic muscarinic vasodilation in the cat. An example of endothelial cell-independent cholinergic relaxation.

Nerve-mediated and acetylcholine-induced dilator behavior of feline posterior auricular arteries was studied in vitro. We evaluated the muscarinic nature and endothelial cell-dependence of the vasodilations and attempted to determine if there are inhibitory muscarinic receptors located directly on the smooth muscle cells in this artery. Transmural nerve stimulation of arteries which were pretreated with guanethidine (5 X 10(-6)M) and constricted with prostaglandin F2 alpha (3 X 10(-6)M) caused a frequency-dependent, tetrodotoxin-sensitive relaxation of up to 50% of induced tone. Atropine (10(-7)M) blocked more than 95% of this response at all frequencies. Removal of the endothelium by rubbing the intimal surface did not affect the magnitude of the response, but prolonged it slightly. Neurogenic relaxations in rubbed preparations were atropine-sensitive, although less so than control at higher stimulation frequencies. Relaxation of this artery to the calcium ionophore A23187 was completely endothelial cell-dependent. However, exogenous acetylcholine caused dose-dependent relaxations both in control and rubbed preparations. We conclude that the posterior auricular artery is an example of a blood vessel which has muscarinic receptors located directly on its smooth muscle cells which, when activated by acetylcholine released from perivascular nerves, mediate a smooth muscle cell relaxation. This finding contrasts with models of the vascular smooth muscle cell which indicates an excitatory role for muscarinic receptors.