Low Doses Of Acetylcholine Research Articles

Release of nitric oxide and prostaglandin H2 to acetylcholine in skeletal muscle venules.

The role of endothelium in regulating venular resistance is not well characterized. Thus we aimed to elucidate the endothelium-derived factors involved in the mediation of responses of rat gracilis muscle venules to acetylcholine (ACh) and other vasoactive agents. Changes in diameter of perfusion pressure (7.5 mmHg)- and norepinephrine (10(-6) M)-constricted venules (approximately 225 microns in diam) to cumulative doses of ACh (10(-9) to 10(-4) M) and sodium nitroprusside (SNP, 10(-9) to 10(-4) M), before and after endothelium removal or application of various inhibitors, were measured. Lower doses of ACh elicited dilations (up to 42.1 +/- 4.7%), whereas higher doses of ACh resulted in smaller dilations or even constrictions. Endothelium removal abolished both ACh-induced dilation and constriction. In the presence of indomethacin (2.8 x 10(-5) M), a cyclooxygenase blocker, or SQ-29548 (10(-6) M), a thromboxane A2-prostaglandin H2 (PGH2) receptor antagonist, higher doses of ACh caused further dilation (up to 72.7 +/- 7%) instead of constriction. Similarly, lower doses of arachidonic acid (10(-9) to 10(-6) M) elicited dilations that were diminished at higher doses. These reduced responses were, however, reversed to substantial dilation by SQ-29548. The nitric oxide (NO) synthase blocker, N omega-nitro-L-arginine (L-NNA, 10(-4) M), significantly reduced the dilation to ACh (from 30.6 +/- 5.5 to 5.4 +/- 1.4% at 10(-6) M ACh). In contrast, L-NNA did not affect dilation to SNP. Thus ACh elicits the release of both NO and PGH2 from the venular endothelium.

Does sympathetic activation blunt nitric oxide-mediated hyperemia in the human forearm?

To determine if the vasodilating substance nitric oxide (NO) interferes with the ability of sympathetic nerves to regulate blood flow in humans, forearm blood flow (FBF) was measured during brachial artery infusions of acetylcholine (ACh) to evoke endothelial NO release, and during infusions of the NO donor nitroprusside (NTP) in five healthy volunteers. Sympathetic activity was increased by application of lower body suction and antagonized by brachial artery infusions of phentolamine. In the control condition, FBF was 2.4 +/- 0.4 ml/100 ml per min and rose by 16.9 +/- 3.6 ml/100 ml per min during ACh at 16 micrograms/min and by 17.0 +/- 4.3 ml/100 ml per min at 64 micrograms/min. With suction, FBF was 1.7 +/- 0.6 ml/100 ml per min (p < 0.05 versus control) and rose by 11.4 +/- 3.2 ml/100 ml per min during ACh at 16 micrograms/min (p < 0.05 versus control). After phentolamine, FBF was 3.8 +/- 0.5 ml/100 ml per min at baseline (p < 0.05 versus control) and the increases in flow with ACh at either 16 or 64 micrograms/min were identical to control. During the control NTP trial, FBF rose by 6.3 +/- 1.1 ml/100 ml per min with NTP at 2.5 micrograms/min and by 12.1 +/- 1.4 ml/100 ml per min at 10 micrograms/min. Suction blunted and phentolamine augmented the increases in flow with NTP by approximately 50% (p < 0.05). Due to the unexpected results with ACh, the effects of suction and pharmacological sympathectomy with both phentolamine and bretylium on ACh-mediated dilation were evaluated with lower doses of ACh (8 and 32 micrograms/min) in six additional studies. Again, altered sympathetic activity had inconsistent effects on the rise in FBF with ACh administration. The effects of altered sympathetic activity on the blood flow responses to NTP indicate that sympathetic activity can modulate NO-mediated vasodilation, suggesting that NO does not have a major sympatholytic effect in the human forearm. That sympathetic activity did not consistently alter ACh-mediated vasodilation suggests that vasodilating mechanisms, in addition to NO release, can be activated by arterial administration of ACh in the human forearm.

Sites of nitric oxide (NO) actions in control of circular muscle motility of the perfused isolated canine ileum

In the isolated intra-arterially perfused canine ileum, N omega -nitro-L-arginine (L-NNA, 3 x 10(-4) M) increased tonic and phasic motor activity of the circular muscle. As has previously been shown, L-NNA enhanced contractions to electrical field stimulation at sites proximal to the serosal electrodes and converted initial relaxation when present at distal sites to contraction. L-NNA shifted the acetylcholine dose-response curve to the left and amplified the response to low-dose acetylcholine. Following L-NNA, addition of 10(-5) M sodium nitroprusside (NO donor) returned the tonic and phasic activity, the electrical field stimulation responses, and the acetylcholine dose-response curve to control values. Tetrodotoxin (TTX, 10(-6) M) increased tone (less than L-NNA) and abolished responses to both electrical field stimulation and motor activity induced by prior L-NNA. Subsequent L-NNA did not alter TTX-induced tonic motor responses. TTX also shifted the acetylcholine dose-response curve leftward and increased the responses to low-dose acetylcholine. After TTX, sodium nitroprusside returned the low-dose acetylcholine responses to control values and, after L-NNA, failed to restore them to control values. After L-NNA and TTX, sodium nitroprusside restored responses to low-dose acetylcholine to control values, Thus, removal of inhibition of the release of excitatory neurotransmitters, not removal of actions of NO on the muscle, accounted for the increases in tonic and phasic activity from L-NNA. Uninhibited release of excitatory transmitters augmented circular muscle responses to low-dose acetylcholine. TTX eliminated effects of excitatory transmitters, allowing exogenous NO to reduce low-dose acetylcholine contractions. No treatment affected the maximum responses to acetylcholine, produced by a contractile mechanism independent of muscle excitability and unaffected by exogenous NO or release of neurotransmitters.

Increased basal tone and hyperresponsiveness to acetylcholine and ergonovine in spasm-related coronary arteries in patients with variant angina

We compared the basal coronary artery tone and the constrictive responses to acetylcholine (Ach) and ergonovine (Erg) among three groups of patients: 31 patients (group 1) with variant angina in whom spasm was provoked by low doses of acetylcholine (intracoronary 20 μg) or ergonovine (intravenous 50 μg); 35 patients (group 2) in whom spasm was provoked by higher doses of acetylcholine (intracoronary 100 μg) or ergonovine (intravenous cumulative dose of 350 μg); and 26 control subjects. Patients with variant angina in whom spasm was provoked by low doses of acetylcholine or ergonovine, had a higher incidence of coexisting angiographic evidence of coronary artery disease, multi-vessel spasm and more frequent episodes of angina. The basal coronary artery tone at the spastic and non-spastic sites of the spasm-related artery was significantly elevated in group 1 compared to group 2 (44 ± 17 vs. 14 ± 11% and 26 ± 14 vs. 16 ± 10%, respectively, P < 0.05), but not in the non-spasm related artery. The magnitude of vasoconstrictive responses to acetylcholine and ergonovine at the non-spastic sites was also greater in group 1 than in group 2 and the control groups (acetylcholine: 40 ± 20 vs. 26 ±11, 27 ± 12%; ergonovine: 37 ± 18 vs. 12 ± 8, 13 ± 10%, respectively, P < 0.05). However, the basal coronary artery tone was not elevated at the spastic and non-spastic sites in group 2 compared to that in the control subjects. These findings suggest that the basal coronary artery tone is increased in patients with variant angina, with increased frequency of angina suggestive of higher disease activity at the spastic and non-spastic sites of the spasm-related artery, and this may be related to pathogenesis of coronary artery spasm.

Nizatidine accelerates gastric emptying of a solid meal in rats.

Nizatidine, a new histamine-2-receptor antagonist, stimulates gastrointestinal motility in dogs and gastric emptying of liquids in rats. Effect of nizatidine on gastric emptying of a solid meal was investigated using a novel gastric emptying model in rats. Male Wistar rats (weighing 200-300 g) were supplied with powdered food containing 30 w/w% barium 14 hr before the beginning of the experiment and x-ray photography of rat stomach was taken under light ether anesthesia. Gastric emptying was assessed by percentage of a decrease in area 30 min after drug was injected intraperitoneally. There was a positive correlation between the area of the gastric outline and the weight of the gastric contents (r = 0.94, P < 0.01). Ether anesthesia itself did not affect gastric emptying. Nizatidine increased gastric emptying dose-dependently (emptied percentage; vehicle: 4.9 +/- 1.5%, 1 mg/kg: 7.2 +/- 0.4%, 3 mg/kg: 10.4 +/- 2.0%, 10 mg/kg: 16.7 +/- 4.9%, 30 mg/kg: 25.7 +/- 7.4%). N-Desmethyl nizatidine (NDM) also stimulated gastric emptying, but nizatidine S-oxide, cimetidine, an famotidine had no significant effects on gastric emptying. Nizatidine and neostigmine, but not NDM, at a subthreshold dose accelerated gastric emptying treated with a low dose of acetylcholine (0.1 mg/kg). Atropine (2 mg/kg, -30 min) did not modulate the gastroprokinetic action of nizatidine, but blocked that of NDM. These findings suggest that this noninvasive method may allow measurement of gastric emptying of solids accurately and that nizatidine and NDM facilitate gastric emptying probably mediated by a direct and/or an indirect (acetylcholinesterase inhibition) cholinergic mechanism.

The role of non-quantal release of acetylcholine in regulation of postsynaptic membrane electrogenesis

In mammalian nerve-muscle preparations treated with an anticholinesterase, the acetylcholine (ACh) released non-quantally (NQR) reaches the postsynaptic receptors and causes a small depolarization of the membrane potential at the endplate region of the muscle fibres. Increase in quantal release potentiates the NQR and vice versa, the amplitude and the kinetic parameters of quantal miniature endplate currents (MEPCs) change during manipulation of NQR, indicating direct interaction between both types of release. Repetitive binding of ACh to postsynaptic receptors which prolongs the time course of MEPCs in anti-cholinesterase-treated endplates leads within 1-2 h to progressive desensitization in the presence of non-quantal release and to the subsequent shortening of the quantal responses. We have also investigated the effect of procedures known to modulate non-quantal acetylcholine release, on the small, but obvious, difference in the resting membrane potential between the endplate zone and other areas of the mouse muscle fibre. The resting membrane potential at the endplate zone with intact cholinesterase is more negative (by 2-4 mV) than in the endplate-free area. The experiments were performed to test the hypothesis that the hyperpolarization is caused by an electrogenic Na(+)-K+ pump operating during the action of ACh released in non-quantal form. Observations in favour of this idea are that both short-term denervation (which eliminates non-quantal but not quantal release) and ouabain abolish the local synaptic hyperpolarization and that subsequent application of low doses of ACh restores it. It follows, therefore, that the hyperpolarization is probably caused by a small but continuous ACh leakage from the nerve terminal.

Inhibition of Acetylcholinesterase Selectively Potentiates NG-Monomethyl-l-Arginine-Resistant Actions of Acetylcholine in Human Forearm Vasculature

1. NG-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor) inhibits vasodilator responses to acetylcholine but not methacholine in human forearm vasculature. To investigate whether this difference results from the relative susceptibility of these agonists to hydrolysis by acetylcholinesterase, we studied vasodilator responses to brachial artery administration of acetylcholine alone and in the presence of the acetylcholinesterase inhibitor edrophonium. 2. Vasodilator responses to constant-rate brachial artery infusions of acetylcholine were biphasic, with an initial peak response fading over 2 min to a plateau. Fade [(peak-plateau)/peak x 100%] was dose dependent (P < 0.02), ranging from 43 +/- 7% (mean +/- SEM) at low dose (16 nmol/min) to 9 +/- 8% at high dose (83 nmol/min). 3. Edrophonium (0.5 mumol/min intra-arterially) alone produced no change in forearm blood flow but increased blood flow responses to acetylcholine (P < 0.01), causing an approximately 10-fold reduction in the dose required to increase plateau blood flow by 10 ml min-1 100 ml-1. 4. Responses to low doses of acetylcholine alone (16 and 41 nmol/min) faded more (P < 0.01) than those to doses of acetylcholine with edrophonium chosen to produce similar plateau blood flows. Responses to acetylcholine (41 nmol/min) also faded more (P < 0.01) than those to methacholine (5 nmol/min), producing matched plateau flows. 5. Peak and plateau responses to acetylcholine (41 nmol/min) were reduced (P < 0.01) by similar amounts (47 +/- 15%, and 37 +/- 13% respectively, P = 0.39) by coinfusion of L/NMMA (4 mumol/min).(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of protein kinase C does not cause desensitization in rat and rabbit mandibular acinar cells.

We have examined whether activation of protein kinase C by phorbol esters decreases the responsiveness of rat and rabbit mandibular, and rat lacrimal, acinar cells to muscarinic stimulation. Intracellular free calcium concentration ([Ca2+]i) was measured in isolated single acini and cell clusters by fura-2 microspectrofluorimetry. Accumulation of inositol phosphates was measured in acinar cell suspensions. All three cell types showed very similar changes in [Ca2+]i in response to acetylcholine (ACh), although mobilization of Ca2+ required somewhat higher ACh concentrations in rat lacrimal acinar cells than in mandibular acinar cells. There was no evidence for different dose dependencies of the peak and plateau phases of the [Ca2+]i response. The ACh-evoked [Ca2+]i increase in rabbit mandibular acinar cells exhibited desensitization, since it declined in magnitude when cells were stimulated repeatedly with a maximal dose of agonist. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) rapidly and irreversibly decreased the ACh-evoked [Ca2+]i signals in rat lacrimal acinar cells and reduced ACh-stimulated inositol phosphate accumulation. This inhibitory effect of TPA was most marked in cells stimulated with low doses of ACh, implying that TPA treatment shifted the ACh dose response curve to higher ACh concentrations. In contrast to the results obtained with lacrimal acinar cells, TPA had no effect on the [Ca2+]i and inositol phosphate responses to ACh in either rat or rabbit mandibular acinar cells. These results suggest that, although ACh-evoked [Ca2+]i signals, and hence presumably the stimulus-response coupling machinery, are very similar between different acinar cell types, acinar cells show marked differences in their sensitivity to phorbol esters. The insensitivity of mandibular acinar cell [Ca2+]i signals to TPA also suggests that the secretory tachyphylaxis observed in perfused rat and rabbit mandibular salivary glands is unlikely to be a consequence of negative feedback mediated by protein kinase C.

Apically localized IP3 receptors control chloride current in airway gland acinar cells

We examined the role of inositol 1,4,5-trisphosphate (IP3) receptors in acetylcholine (ACh)-induced chloride (Cl-) current in acinar cells of human and feline airway submucosal glands, using whole cell patch-clamp analysis. ACh (10 nM-1 microM) induced an initial Cl- current followed by a K+ current, and lower doses of ACh (1-10 nM) often induced oscillations of both currents, which were mimicked by the application of intracellular IP3. Neither isoproterenol (-10 microM) nor raising intracellular adenosine 3,5-cyclic monophosphate induced any current. Caffeine (20-50 mM) and intracellular ryanodine (1-100 microM) induced a K+ current alone without Cl- current. Monoclonal antibodies to the IP3 receptor abolished both ACh-induced K+ and Cl- currents. Immunohistochemical analysis revealed the localization of IP3 receptors on both the cytosol and some regions of the endoplasmic reticulum beneath the apical membrane of acinar cells. These results indicate that apically localized IP3 receptors control Cl- secretion from airway submucosal gland cells.

Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh.

Lower esophageal sphincter (LES) basal tone and contraction in response to maximally effective doses (Emax) of acetylcholine (ACh) may be mediated by different intracellular transduction pathways. In the basal state resting tone, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation and levels of diacylglycerol (DAG) (C. Hillemeier, K. N. Bitar, and P. Biancani, unpublished data) are higher in LES circular muscle than in esophageal muscle, which does not maintain tone. In vitro resting tone and spontaneously elevated formation of Ins(1,4,5)P3 in LES circular muscle strips decrease in a dose-dependent manner in response to the phospholipase C antagonist 1-[6-([(17-beta)-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione (U-73122). Basal Ins(1,4,5)P3 formation, however, is submaximal, since it can be increased by cholinergic stimulation. These data suggest that LES tone is associated with partial activation of phospholipase C. We therefore tested submaximal doses of Ins(1,4,5)P3 and DAG in permeabilized LES muscle cells and found that they act synergistically; their interaction depends on calcium release and is mediated through a protein kinase C (PKC)-dependent pathway. In contrast, we have previously shown that contraction induced by Emax of ACh is mediated through calmodulin-dependent mechanisms (14). To investigate these differences, we tested high and low doses of ACh. Contraction induced by high doses of ACh was inhibited by calmodulin but not by PKC antagonists, as previously reported, but low ACh doses were preferentially inhibited by PKC antagonists. Similarly, low Ins(1,4,5)P3 concentrations activated a PKC-dependent pathway, whereas contraction induced by Emax of Ins(1,4,5)P3 was calmodulin dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chronic and subacute parathyroidectomy on blood pressure and resistance artery contractility in the spontaneously hypertensive rat.

Chronic parathyroidectomy (PTX) attenuates blood pressure and increases vascular force generation. The mechanism is not understood. We tested the hypothesis that PTX-induced changes in blood pressure and vessel contractility result from rapid changes in serum parameters induced by PTX, and the hypothesis that the endothelium-derived factors contribute to PTX-induced enhancement of force generation in the spontaneously hypertensive rat (SHR). Chronic PTX or sham surgery was performed on 5-week-old male SHR, and subacute PTX was performed on 13- to 14-week-old SHR. The following experimental parameters were measured 5-6 weeks after chronic and 2 days after subacute PTX: systolic blood pressure, serum Ca2+, 1,25-dihydroxyvitamin D3 (vitD) and mesenteric resistance artery contractility. Chronic PTX significantly reduced blood pressure, body weight and serum Ca2+ compared with sham surgery; subacute PTX reduced only serum Ca2+ and vitD. Compared with sham surgery, the mesenteric resistance artery following chronic PTX showed increased active stress responses to norepinephrine and serotonin that were not associated with changes in free intracellular Ca2+, but were abolished by endothelial denudation. Subacute PTX did not affect the active stress response to norepinephrine or endothelin. Low-dose acetylcholine induced relaxation that was attenuated in the chronic, but not the subacute, PTX subgroup compared with the sham-operated subgroup. High-dose acetylcholine induced contraction that was significantly greater in the chronic, but not subacute, PTX rats than in sham-operated rats. Indomethacin abolished the acetylcholine-induced contraction in all groups, but did not improve the impaired relaxation response of the chronic PTX subgroup. The PTX-induced decrease in blood pressure and increase in force generation do not result from acute changes initiated by reduction of serum parathyroid hormone, vitD or Ca2+. Moreover, the PTX-induced increase in force generation results from a decrease in the production of a non-cyclo-oxygenase endothelium-dependent relaxing factor.

Evidence of impaired endothelium-dependent coronary vasodilatation in patients with angina pectoris and normal coronary angiograms.

A group of patients has been described who have chest pain resembling angina and positive exercise tests, but normal coronary angiograms and no coronary-artery spasm. This constellation of features has sometimes been called syndrome X or microvascular angina. We attempted to determine whether endothelium-dependent vasodilatation of the coronary vasculature was impaired in patients with this syndrome. We infused the endothelium-dependent vasodilator acetylcholine and the endothelium-independent vasodilators papaverine and isosorbide dinitrate into the left coronary artery of 9 patients and 10 control subjects. The diameter of the left anterior descending coronary artery was assessed by quantitative angiography, and changes in coronary blood flow were estimated with the use of an intracoronary Doppler catheter. Acetylcholine, given in doses of 1, 3, 10, and 30 micrograms per minute, increased coronary blood flow in a dose-dependent manner in both groups. However, the mean (+/- SD) acetylcholine-induced increases in coronary blood flow were significantly less (P < 0.001) in the patient (8 +/- 14, 37 +/- 37, 59 +/- 67, and 103 +/- 77 percent, respectively) than in the controls (62 +/- 52, 186 +/- 93, 341 +/- 128, and 345 +/- 78 percent, respectively). The changes in coronary blood flow in response to 2 mg of isosorbide dinitrate (236 +/- 66 percent vs. 280 +/- 56 percent) and 10 mg of papaverine (366 +/- 168 percent vs. 411 +/- 92 percent) did not differ significantly between the patients and controls. The administration of papaverine resulted in myocardial lactate production in the patients but not in the controls. The three lower doses of acetylcholine caused a similar degree of dilatation of the left anterior descending coronary artery in the two groups, and the highest dose caused a similar degree of constriction in the two groups. Isosorbide dinitrate and papaverine caused a similar degree of dilatation in both groups. These findings suggest that endothelium-dependent dilatation of the resistance coronary arteries is defective in patients with anginal chest pain and normal coronary arteries, which may contribute to the altered regulation of myocardial perfusion in these patients.

Usefulness of intracoronary injection of acetylcholine as a provocative test for coronary artery spasm in patients with vasospastic angina

In order to examine both the sensitivity and specificity of coronary artery spasm induced by intracoronary injection of acetylcholine in patients with vasospastic angina, incremental doses of acetylcholine (20, 30, and 50 micrograms) were injected directly into each coronary artery in 21 patients with variant angina (group A), in 28 patients with other types of vasospastic angina (group B), and in 20 patients without any significant coronary artery disease (group C). Coronary artery spasm was defined as severe vasoconstriction (greater than or equal to 90% of reduction in luminal diameter) with chest pain and/or ischemic changes in the electrocardiogram. Intracoronary injection of acetylcholine induced spasm of at least one coronary artery in 20 patients (95%) of group A, in 27 patients (96%) of group B, and in only 2 patients (10%) of group C. The low dose of acetylcholine (20 micrograms) induced coronary spasm more frequently in group A patients (81%) than in group B patients (43%) (P less than 0.05). ST-segment elevation associated with anginal attacks was significantly (P less than 0.05) more frequent in group A (71%) than in group B (39%). When acetylcholine was injected separately into the left and right coronary arteries, spasm of both coronary arteries was observed in 7 out of 14 of group A (50%), in 8 out of 22 of group B (36%), and in none of the 20 of group C. We concluded that intracoronary injection of acetylcholine is a sensitive and reliable method for the induction of coronary spasm in patients with vasospastic angina as well as in those with variant angina.

Cholinergic Vasodilatation of Coronary Resistance Vessels in Dogs, Baboons and Goats

Intracoronary infusion of acetylcholine produces a prompt increase in coronary blood flow in dogs, but a paradoxical decrease has been reported in baboons and cattle. The action of acetylcholine was reexamined in anesthetized dogs, baboons and goats, with the coronary circulation pump perfused at constant pressure and the heart rate held constant with electrical pacing. Intracoronary infusions of low doses of acetylcholine produced coronary vasodilatation and an increase in coronary venous oxygen tension without a change in cardiac contractility (dP/dt) or myocardial oxygen consumption in all three species. High doses of acetylcholine produced coronary vasodilatation only in dogs, but resulted in a decrease in cardiac contractility and myocardial oxygen consumption accompanied by a decrease in coronary flow in baboons and goats. It is concluded that low doses of acetylcholine produce coronary vasodilatation in all three species, and that the decrease in coronary blood flow observed in baboons and goats at high doses is probably due to local metabolic vasoconstriction secondary to the negative inotropic effects of acetylcholine.

Biphasic response to acetylcholine in human veins in vivo: the role of the endothelium

1. The dose-response to acetylcholine has been examined in dorsal hand veins of healthy volunteers before and after removal of the endothelium. 2. Measurements were made in single dorsal hand veins during local infusions of acetylcholine. The vein was irrigated with distilled water to remove the endothelium. Dilator studies were performed in vessels preconstricted by a continuous infusion of noradrenaline. 3. In the endothelium-intact vessel the dose-response to acetylcholine was biphasic; low doses produced venodilatation with higher doses causing venoconstriction. 4. Dilatation to low doses of acetylcholine was abolished by prior irrigation with distilled water, consistent with denudation of the endothelium by this process. Irrigation augmented the constriction seen in response to higher doses of acetylcholine. 5. This is the first demonstration of an endothelium-dependent biphasic dose-response to acetylcholine in man. The results raise questions as to the possible physiological actions of endogenous acetylcholine and as to the use of the acetylcholine dose-response curve as a marker of endothelial function.

Ageing suppresses endothelium-dependent relaxation and generates contraction mediated by the muscarinic receptors in vascular smooth muscle of normotensive Wistar-Kyoto and spontaneously hypertensive rats.

We evaluated the effects of ageing and hypertension on endothelium-dependent relaxation and contraction of vascular smooth muscles. Aortic rings with and without endothelium from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR), divided into young, adult and old groups, were examined for relaxation in response to acetylcholine, adenosine triphosphate (ATP) and sodium nitroprusside. Relaxation responses to acetylcholine and ATP in SHR were either equal to or greater than those in WKY. Relaxations induced by ATP were not altered by ageing, while the degree of acetylcholine-induced endothelium-dependent relaxations was in an inverse order of age in both SHR and WKY. In old SHR and WKY and adult SHR, lower doses of acetylcholine caused relaxations, but increases in the dose of acetylcholine induced contraction. Acetylcholine-induced contractions were abolished by indomethacin or endothelium rubbing. We conclude that endothelium-dependent relaxations are not diminished by spontaneous hypertension but the relaxations mediated by the muscarinic receptors are reduced with ageing. Further, acetylcholine causes endothelium-dependent contractions by releasing cyclo-oxygenase products, not only in SHR but also in aged normotensive WKY.

Supersensitivity to acetylcholine of canine sinus and AV nodes after parasympathetic denervation.

Supersensitivity of the sinus (SAN) and atrioventricular (AVN) nodes to acetylcholine (ACh) after parasympathetic denervation has not been demonstrated conclusively. In this study, we denervated the SAN and AVN by surgically removing parasympathetic ganglia and painting the area with phenol. Sham dogs underwent thoracotomy without denervation. Four to 9 days later, vagal denervation was proved by supramaximal bilateral vagal stimulation, which prolonged the sinus cycle length (SCL) only 32 +/- 7% (mean +/- SE) and the AVN conduction time (AH interval) 15 +/- 7% in denervated dogs. We tested for supersensitivity by obtaining dose-response curves to ACh (1 ml, 10(-8.0) to 10(-4.0) M in 10(0.5) steps) infused over 15 s into the sinus nodal and posterior septal arteries in open chest-denervated (Den) dogs and in sham-operated (Sham) dogs that were anesthetized with alpha-chloralose. ACh concentration (Log[ACh], M) required to prolong SCL 50, 100, and 300% was -5.7 +/- 0.1, -5.6 +/- 0.1, and -5.4 +/- 0.1 in 10 Sham dogs vs. -6.4 +/- 0.1 (P less than 0.001), -6.3 +/- 0.1 (P less than 0.001) and -6.1 +/- 0.1 (P less than 0.001) in 11 Den dogs. ACh concentration necessary to produce second degree and complete AV block was -5.7 +/- 0.1 and -5.3 +/- 0.1 in 11 Sham dogs vs. -6.3 +/- 0.1 (P less than 0.001) and -5.8 +/- 0.1 (P less than 0.01) in 10 Den dogs. Because significantly lower doses of ACh prolonged SCL or produced AV block in Den compared with Sham dogs, we conclude that dogs with vagally denervated SAN and AVN develop a supersensitive response to ACh.

The pacemaker current in the sinus node

The cardiac 'pacemaker' current is recorded in isolated sino-atrial node cells during hyperpolarizations at voltages from -40/-50 mV to -100/-110 mV, which corresponds to the range where diastolic depolarization occurs. if is a hyperpolarizing-activated current, carried by Na and K, and in the pacemaker voltage range is inward. These properties allow if to serve as a tool to generate and control the 'pacemaker' depolarization phase of the action potential in sino-atrial node cells. The current if has long been shown to mediate the accelerating action of catecholamines in the heart. More surprisingly, recent experiments show that low doses of acetylcholine exert on if a strong inhibitory action. This new finding modifies the view that the slowing of pacemaker activity caused by acetylcholine is essentially due to activation of a K-current.

The fade of the response to acetylcholine at the rabbit isolated sino-atrial node.

1. The effect of acetylcholine (ACh) on the frequency of spontaneous action potentials, recorded with glass microelectrodes from small preparations of the sino-atrial node of the rabbit, has been investigated. 2. On application of ACh there was a rapid increase in cycle length (the interval between successive action potentials) but then, despite the continued presence of the ACh, cycle length decreased once again; i.e. the response to ACh 'faded' in the presence of ACh. This fade of the chronotropic response to ACh has been characterized. 3. To observe fade it was found to be important to use a fast flow bath in order to increase the ACh concentration at the preparation abruptly. In other words, the response to ACh was sensitive to the rate of increase in the ACh concentration at the tissue. This may explain why in one study of the sino-atrial node of the guinea-pig the authors failed to observe fade. 4. The concentration dependence of fade was studied in two ways: either as the fade in the response with different doses of ACh, or as the decrease in the response to a fixed test dose applied after a variable conditioning dose. Both methods indicated that fade increased with concentration over the range of roughly 10(-7) to 10(-5) M-ACh. 5. The onset of fade, i.e. the decline in cycle length during an exposure to ACh, was monophasic with low doses of ACh and biphasic (with fast and slow phases) with higher doses. The mean half-times of the fast and slow phases of fade were 10 +/- 2 s and 240 +/- 30 s (mean +/- S.E.M.; n = 5). However, in some preparations, rather than a smooth decrease in cycle length, there were discontinuities and cycle length could decrease in a stepwise fashion or even transiently alternate between two distinct states. 6. Time was required for recovery from fade because a response to a test dose applied soon after a conditioning dose was depressed. Recovery was studied by applying test doses at different times after conditioning doses. Recovery occurred with a half-time of 40 +/- 8 s (mean +/- S.D.; n = 8) after a short exposure (30 s) to ACh, but was slower (half-time, 250 +/- 20 s; n = 6) after a longer exposure (5 min). 7. Immediately after washing off ACh, cycle length decreased below its original value, although it then returned to this value over several minutes.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of acetylcholine and sodium cyanide on cat carotid baroreceptors.

1 The effects of intracarotid (i.a.) injections of acetylcholine (ACh) and sodium cyanide (NaCN) on baroreceptor activity recorded from the sinus nerve have been investigated in cats anaesthetized with pentobarbitone.2 Two types of baroreceptor unit were recorded. The predominant type discharged at least 3 to 4 spikes per pulse wave at normal BP; they are referred to as ;polyspike' units and may have been associated with A fibres. The other type discharged a maximum of 1 to 3 spikes per pulse wave, even at high BP; they are referred to as ;few-spike' units and may have been from C fibres.3 NaCN had no direct effect on either type of baroreceptor unit, even when injected in high doses (2.04 to 5.1 mumol i.a.) which cause maximal chemoreceptor stimulation, and it is concluded that as far as the cat's carotid baroreceptors and chemoreceptors are concerned, NaCN is a specific chemoreceptor stimulant.4 ACh had no direct effect on polyspike baroreceptor units unless very high doses (1.83 mumol i.a.) were injected, when there was occasionally a transient slight increase in discharge. This effect appeared to be secondary to muscle contraction caused by ACh since it was not seen when an adequate neuromuscular-blocking dose of gallamine had been administered.5 ACh stimulated the few-spike type of baroreceptor unit, an effect which was dose-related and lasted for up to 3 s; the threshold dose for baroreceptor stimulation was higher than that needed to excite chemoreceptor units. The increased discharge also occurred during experiments in which gallamine had been administered. Only five of these units were recorded during the investigation, despite an intensive search for them.6 There was a delayed increase in baroreceptor sensitivity following the administration of ACh in doses (37 to 366 nmol i.a.) which had no immediate direct effect on polyspike baroreceptor discharge. The effect was evidently not secondary to changes in sympathetic nerve activity to the sinus region since it was observed during an experiment in which the ganglioglomerular nerves had been cut. Whether the increased sensitivity resulted from direct or indirect actions of ACh remains to be determined.7 It is concluded that low doses of ACh or other drugs with nicotinic properties are unlikely to evoke baroreceptor reflexes on intracarotid injection, although they may cause delayed changes in baroreceptor sensitivity. Higher doses of ACh do not directly affect baroreceptor polyspike (A fibre) units, but transient baroreflex changes might result from stimulation of baroreceptor few-spike (C fibre) units. It is most unlikely that NaCN has any direct effect on baroreceptor reflex activity when injected into the carotid artery in doses used to elicit chemoreceptor reflexes.