José Luis García

Regional differences in the arterial response to vasopressin : role of endothelial nitric oxide

1 . The isometric response to arginine‐vasopressin (10−10‐10−7 m) was studied in 2 mm long rabbit arterial segments isolated from several vascular beds (cutaneous, pial, renal, coronary, muscular, mesenteric and pulmonary). 2 . Vasopressin induced contraction in central ear (cutaneous), basilar (pial), renal, coronary and saphenous (muscular) arteries, but had no effect in mesenteric and pulmonary arteries; the order of potency for the contraction was: ear > basilar > renal > coronary > saphenous arteries. 3 . Treatment with the blocker of nitric oxide synthesis NG‐nitro‐L‐arginine methyl ester (L‐NAME; 10−6‐10−4 m) increased significantly (P < 0.05) the contraction to vasopressin in ear (148% of control), basilar (150% of control), renal (304% of control), coronary (437% of control) and saphenous (235% of control) arteries. Removal of the endothelium increased significantly (P < 0.05) the contraction to vasopressin in basilar (138% of control), renal (253% of control), coronary (637% of control) and saphenous (662% of control) arteries, but not in ear artery. Mesenteric and pulmonary arteries in the presence of L‐NAME or after endothelium removal did not respond to vasopressin, as occurred in control conditions. 4 . The specific antagonist for V1 vasopressin receptors d(CH2)5Tyr(Me)AVP (3 × 10−9‐10−7 m) was more potent (pA2 = 9.3–10.1) than the antagonist for both V1 and V2 vasopressin receptors desGly‐d(CH2)5‐D‐Tyr(Et)ValAVP (10−7‐10−6 m) (pA2 = 7.4–8.4) to block the contraction to vasopressin of ear, basilar, renal and coronary arteries. 5 . The specific V2 vasopressin agonist [deamino‐Cys1, D‐Arg8]‐ vasopressin (desmopressin) (10−10‐10−7 m) did not produce any effect in any of the arteries studied, with or without endothelium. 6 . In arteries precontracted with endothelin‐1, vasopressin or desmopressin did not produce relaxation. 7 . These results suggest: (a) most arterial beds studied (5 of 7) exhibit contraction to vasopressin with different intensity; (b) the vasoconstriction to this peptide is mediated mainly by stimulation of V1 vasopressin receptors, and (c) endothelial nitric oxide may inhibit the vasoconstriction to this peptide, especially in coronary and renal vasculatures.

Cerebral blood flow and cerebrovascular reactivity after inhibition of nitric oxide synthesis in conscious goats.

1 The role of nitric oxide in the cerebral circulation under basal conditions and after vasodilator stimulation was studied in instrumented, conscious goats, by examining the action of inhibiting endogenous nitric oxide production with NG‐nitro‐l‐arginine methyl ester (l‐NAME). 2 In 6 unanaesthetized goats, blood flow to one brain hemisphere (electromagnetically measured), systemic arterial blood pressure and heart rate were continuously recorded. l‐NAME (35 mg kg−1 by i.v. bolus) decreased resting cerebral blood flow by 43 ± 3%, increased mean arterial pressure by 21 ± 2%, and decreased heart rate by 41 ± 2%; cerebrovascular resistance increased by 114 ± 13% (P < 0.01); the immediate addition of i.v. infusion of l‐NAME (0.15–0.20 mg kg−1 during 60–80 min) did not significantly modify these effects. Cerebral blood flow recovered at 72 h, arterial pressure and cerebrovascular resistance at 48 h, and heart rate at 6 days after l‐NAME treatment. 3 A second treatment with l‐NAME scheduled as above reproduced the immediate haemodynamic effects of the first treatment, which (except bradycardia) reversed with l‐arginine (200–300 mg kg−1 by i.v. bolus). 4 Acetylcholine (0.01–0.3 μg), sodium nitroprusside (3–100 μg) and diazoxide (0.3–9 mg), injected into the cerebral circulation of 5 conscious goats, produced dose‐dependent increases in cerebral blood flow, and decreases in cerebrovascular resistance; sodium nitroprusside (30 and 100 μg) also caused hypotension and tachycardia. 5 The reduction in cerebrovascular resistance from resting levels (in absolute values) to lower doses, but not to the highest dose, of acetylcholine was diminished, to sodium nitroprusside was increased, and to diazoxide was unaffected after l‐NAME, compared to control conditions. The effects on cerebrovascular resistance to acetycholine normalized within 24 h and to sodium nitroprusside within 48 h after l‐NAME treatment. 6 This study provides information about the evolution of the changes in cerebral blood flow and cerebrovascular reactivity after inhibition of endogenous nitric oxide in conscious animals. The results suggest: (a) endogenous nitric oxide is involved in regulation of the cerebral circulation by producing a resting vasodilator tone, (b) the cerebral vasodilatation to acetylcholine is mediated, at least in part, by nitric oxide release, and (c) inhibition of nitric oxide production induces supersensitivity of cerebral vasculature to nitrovasodilators.

Cooling effects on nitric oxide production by rabbit ear and femoral arteries during cholinergic stimulation

1 Ear (cutaneous) and femoral (deep) arteries from rabbit were perfused at 37°C and 24°C (cooling) and the production of nitrite, as an index of nitric oxide production, was measured under basal conditions and cholinergic stimulation. 2 In both types of arteries under control conditions, the basal production of nitrite was similar at 24°C and 37°C. Compared with the control conditions, the basal production of nitrite was significantly lower in ear and femoral arteries without endothelium or treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME, 10−4m) but it was similar in those treated with atropine (10−6m). 3 At 37°C, methacholine (10−7‐10−5 m) increased the production of nitrite in ear and femoral arteries; this increase persisted during 30–60 min and was practically abolished by L‐NAME (10−4m), atropine (10−6m), or removal of the endothelium. In ear arteries the total nitrite production to activation with methacholine was higher at 24°C than at 37°C due to this production persisted increased for a longer period (> 150 min), whereas in femoral arteries it was lower at 24°C than at 37°C. 4 It is suggested that: (a) the endothelium of rabbit ear and femoral arteries produce nitric oxide under basal conditions, which is increased by cholinergic stimulation, and (b) cooling potentiates endothelial nitric oxide production to cholinergic stimulation in cutaneous arteries, whereas it inhibits this production in deep arteries.

Effects of nitric oxide synthesis inhibition on the goat coronary circulation under basal conditions and after vasodilator stimulation

1 The role of nitric oxide in the coronary circulation under basal conditions and when exposed to various vasodilator stimuli was studied in instrumented, anaesthetized goats, by examining the action of inhibiting endogenous nitric oxide production with NG‐nitro‐l‐arginine methyl ester (l‐NAME). 2 In 12 goats, left circumflex coronary blood flow (electromagnetically measured), systemic arterial blood pressure and heart rate were continuously recorded. l‐NAME (3–4, or 8–10 mg kg−1 injected i.v.) decreased resting coronary blood flow by 20 and 28%, increased mean arterial pressure by 23 and 30% and increased coronary vascular resistance by 47 and 65%, respectively, without affecting heart rate, or blood gases or pH. These haemodynamic effects were reversed by l‐arginine (200‐ 300 mg kg−1 by i.v. injection, 5 goats). 3 Acetylcholine (0.001–0.1 μg), sodium nitroprusside (0.01–0.3 mg), and diazoxide (0.1–3 mg), injected intracoronarily in 6 goats, produced dose‐dependent increases in coronary blood flow; sodium nitroprusside (0.1–0.3 mg) also caused hypotension and tachycardia. 4 During the effects of l‐NAME, the coronary vasodilatation to acetylcholine was attenuated, to sodium nitroprusside was increased, and to diazoxide was unaffected, in comparison with control conditions. The hypotensive effects of sodium nitroprusside were also increased during treatment with l‐NAME. 5 Graded coronary hyperaemic responses occurred after 5, 10 or 20 s of coronary occlusion. The magnitude of hyerpaemia for each occlusion duration was increased during treatment with l‐NAME, in comparison to control. 6 The results suggest: (a) endogenous nitric oxide is involved in regulation of coronary circulation by producing a basal vasodilator tone, (b) acetylcholine‐induced coronary vasodilatation is mediated, in part, by nitric oxide, and (c) inhibition of basal endogenous nitric oxide production induces supersensitivity of coronary vessels to nitrovasodilators and enhances hyperaemic responses after short periods of ischaemia of the myocardium.

Role of the endothelium in the response to cholinoceptor stimulation of rabbit ear and femoral arteries during cooling

1 The role of the endothelium in the effects of cooling on the response to cholinoceptor stimulation of the rabbit central ear (cutaneous) and femoral (non‐cutaneous) arteries was studied using 2 mm long cylindrical segments. 2 Concentration‐response curves for acetylcholine (10−9‐10−5 m), methacholine (10−9‐10−5 m) and sodium nitroprusside (10−9‐10−4 m) were isometrically recorded in arteries under conditions, with and without endothelium or following pretreatment with the nitric oxide‐synthesis inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME, 10−6‐3 x 10−4 m) at 37°C and at 24°C (cooling). 3 Ear and femoral arteries showed endothelium‐dependent relaxation to acetylcholine and methacholine at 37°C and 24°C. The extent of relaxation of the control ear arteries, but not of the control femoral arteries, to acetylcholine and methacholine increased during cooling. 4 l‐NAME (10−6−3 × 10−4 m) reduced in a concentration‐dependent way the response of ear arteries to acetylcholine at both 37°C and 24°C, this reduction being more potent at 37°C. l‐Arginine (10−5‐10−3 m) reversed in a concentration‐dependent manner the inhibitor effects of 10−5 m l‐NAME at both temperatures. 5 Sodium nitroprusside caused a concentration‐dependent relaxation in both arteries that was endothelium‐independent. However, the extent of relaxation to this nitrovasodilator in ear and femoral arteries was lower at 24°C. 6 These results suggest that cooling augments the reactivity of cutaneous (ear) arteries, but not that of non‐cutaneous (femoral) arteries to cholinoceptor stimulation by endothelium‐mediated mechanisms. Cooling could therefore facilitate the stimulated release of endothelial nitric oxide in cutaneous vessels.

Coronary vasoconstriction produced by vasopressin in anesthetized goats. Role of vasopressin V1 and V2 receptors and nitric oxide.

To examine the role of vasopressin V1 and V2 receptors, nitric oxide and prostanoids in the coronary vascular effects of [Arg8]vasopressin, coronary blood flow was measured with an electromagnetic flow transducer placed around the left circumflex (23 goats) or anterior descending (11 goats) coronary artery and vasopressin (0.03-1 microg) was intracoronarily injected in 34 anesthetized, open-chest goats. Basal mean values for coronary blood flow, mean systemic arterial pressure and heart rate, were 34 +/- 2.38 ml/min, 89 +/- 3.34 mmHg and 80 +/- 3.06 beats/min, respectively. Vasopressin produced dose-dependent decreases in coronary blood flow and the maximal reduction of this flow, attained with 1 microg of vasopressin, was 14 +/- 1.49 ml/min (42 +/- 2.64% of basal flow) (P < 0.01). Desmopressin (0.03-1 microg; 8 goats) did not affect significantly coronary blood flow. The intracoronary infusion of the antagonist for vasopressin V1 receptors d(CH2)5Tyr (Me) arginine vasopressin (2 microg/min per kg, 6 animals) significantly diminished the effects of vasopressin on coronary blood flow (the effects of 1 microg of vasopressin were reduced by 28%, P < 0.05). The mixed antagonist for vasopressin V1 and V2 receptors desGly-d(CH2)5-D-Tyr(Et)Val arginine vasopressin (0.2, 0.7 and 2 microg/min per kg, 9 animals) decreased in a dose-dependent manner the effects of vasopressin on coronary blood flow (the effects of 1 microg of vasopressin were decreased by 61% with 2 microg/min per kg, P < 0.01). Intracoronary infusion of saline (vehicle, 3 goats) did not change the effects of vasopressin on coronary blood flow. Intravenous administration of the inhibitor of nitric oxide synthesis N-omega-nitro-L-arginine methyl ester (L-NAME, 47 mg/kg, 9 animals) decreased resting coronary blood flow by 10% (P < 0.01) and augmented mean systemic arterial pressure by 20% (P < 0.01), without changing heart rate. During this treatment the reduction in coronary blood flow produced by vasopressin was higher than under control (the effects of 1 microg of vasopressin were increased by 28%, P < 0.01). Intravenous administration of the inhibitor of cyclooxygenase, meclofenamate (5 mg/kg, 7 animals), neither modified resting coronary blood flow, arterial pressure and heart rate nor the effects of vasopressin on this flow. These data indicate that vasopressin produces marked coronary vasoconstriction and suggest that: (a) it may be mediated by vasopressin V1 receptors, without involvement of vasopressin V2 receptors, (b) it is probably inhibited by nitric oxide under normal conditions and (c) it may be not modulated by prostanoids.

Role of Nitric Oxide and Potassium Channels in the Cholinergic Relaxation of Rabbit Ear and Femoral Arteries: Effects of Cooling

The main objective of this work was to study the role of potassium channels in the cholinergic relaxation of cutaneous arteries during cooling. Acetylcholine (10(-8)-10(-4) M) produced isometric concentration-dependent relaxation of precontracted segments of rabbit ear (cutaneous) and femoral (noncutaneous) arteries; this relaxation was higher at 24 degrees C (cooling) than at 37 degrees C in ear, but not in femoral, arteries. In both types of arteries, at 37 and 24 degrees C, the relaxation to acetylcholine was partially reduced by the inhibitor of nitric oxide synthase NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), and the relaxation that remained after L-NAME was higher at 24 degrees C than at 37 degrees C in ear, but not in femoral, arteries. At 37 and 24 degrees C, the persistent relaxation to acetylcholine after L-NAME was further reduced by smooth muscle depolarization with medium containing a high concentration of potassium (6 x 10(-2) M), and with the nonspecific inhibitors of potassium channels tetraethylammonium (10(-2) M) or 4-aminopyridine (5 x 10(-3) M) in both ear and femoral arteries. In ear arteries, the inhibitor of high conductance calcium-activated potassium channels charybdotoxin (10(-7) M), alone or combined with L-NAME, reduced the relaxation to acetylcholine at 24 degrees C, but not at 37 degrees C. In femoral arteries, charybdotoxin alone did not modify, but combined with L-NAME reduced, the relaxation to acetylcholine at either temperature. At 37 and 24 degrees C, the inhibitor of low conductance calcium-activated potassium channels apamin (10(-7) M), the inhibitor of ATP-dependent potassium channels glibenclamide (10(-5) M) and the cyclooxygenase inhibitor meclofenamate (10(-5) M), alone or combined with L-NAME, did not modify the relaxation of both ear and femoral arteries to acetylcholine. These results suggest: (1) the cholinergic relaxation of cutaneous (ear) and noncutaneous (femoral) arteries could be mediated by endothelial nitric oxide and by activation of potassium channels, and (2) cooling increases the relaxation of cutaneous arteries to cholinergic stimulation, which may be mediated, in part, by an increased response of potassium channels.

Cooling and response to adrenoceptor agonists of rabbit ear and femoral artery: role of the endothelium

1 The effects of cooling on the response of the rabbit central ear (cutaneous) and femoral (non‐cutaneous) arteries to stimulation of adrenoceptors and the role of the endothelium in these effects, were studied in 2 mm long cylindrical segments. 2 Concentration‐response curves for noradrenaline (10−9 − 3 × 10−4 m), phenylephrine (α1‐adrenoceptor agonist, 10−9 − 3 × 10−4 m) and B‐HT 920 (α2‐adrenoceptor agonist, 10−7 − 10−3 m) were recorded isometrically in arteries with and without endothelium at 37°C and at 24°C (cooling). To analyze further the endothelial mechanisms in the responses to adrenoceptor stimulation during cooling, the effects of the adrenoceptor agonists on ear arteries in the presence of NG‐nitro‐l‐arginine methyl esther (l‐NAME) (10−5 m) were also determined. 3 In every condition tested, the three adrenoceptor agonists produced a concentration‐dependent arterial contraction and the order of potency in ear and femoral arteries was noradrenaline ≥ phenylephrine > B‐HT 920. The response of ear and femoral arteries to phenylephrine or B‐HT 920 was blocked by prazosin (10−6 m). Yohimbine (10−6 m) decreased slightly the response of ear arteries and increased that of femoral arteries to B‐HT 920. 4 The sensitivity of both ear and femoral arteries to the three adrenoceptor agonists was significantly lower at 24°C than at 37°C. 5 In ear arteries, endothelium removal or treatment with l‐NAME did not influence the response at 37°C, but did increase it during cooling to adrenoceptor stimulation. In femoral arteries, endothelium removal increased the sensitivity to noradrenaline and, especially, to B‐HT 920 at 37°C, but did not affect the response at 24°C. 6 The results suggest that: (a) rabbit ear and femoral arteries are equipped mainly with α1‐adrenoceptors; (b) at 37°C, the contraction of the ear artery to adrenoceptor agonists is mostly endothelium‐independent, and in the femoral artery the contraction to α2‐adrenoceptor activation is endothelium‐dependent; (c) cooling inhibits the contraction to adrenoceptor agonists in both ear and femoral arteries: in the ear artery probably by increasing the availability of endothelial nitric oxide, but in the femoral artery by depressing the sensitivity of α‐adrenoceptors in the smooth musculature. 7 The results suggest that the endothelium may modulate the adrenoceptor response of cutaneous arteries during changes in temperature.

Role of nitric oxide in the effects of hypoglycemia on the cerebral circulation in awake goats.

This study was performed to examine the role of nitric oxide in the effects of hypoglycemia on the cerebral circulation. Hypoglycemia was induced with insulin and its effects on cerebral blood flow (measured with an electromagnetic flow transducer placed on the internal maxillary artery) were studied in awake goats under control conditions and after administration of the nitric oxide synthesis inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 47 mg/kg). Also, cerebrovascular reactivity to vasodilator stimuli was examined during insulin-induced severe hypoglycemia, before and after L-NAME treatment. In five animals under control conditions (glycemia = 90 +/- 7 mg/dl, cerebral blood flow = 64 +/- 4 ml/min, mean systemic arterial pressure = 102 +/- 4 mmHg, cerebrovascular resistance = 1.62 +/- 0.11 mmHg/ml per min and heart rate = 73 +/- 6 beats/min), insulin decreased glycemia: when hypoglycemia was moderate (glycemia = 46 +/- 2 mg/dl) or severe (glycemia = 26 +/- 1 mg/dl) cerebral blood flow increased by 25 +/- 4% and 47 +/- 6%, and cerebrovascular resistance decreased by 18 +/- 3% and 34 +/- 4%, respectively. Under basal conditions, L-NAME did not affect glycemia but reduced resting cerebral blood flow by 37 +/- 2%, increased mean arterial pressure by 33 +/- 2% and decreased heart rate by 28 +/- 3%; after L-NAME, both moderate and severe hypoglycemia did not alter significantly resting cerebral blood flow and cerebrovascular resistance. In five other goats, L-NAME, administered during severe hypoglycemia, abolished the increase in cerebral blood flow, and increased cerebrovascular resistance and mean arterial pressure over the control (normoglycemic) values. In these animals with severe hypoglycemia, acetylcholine (0.01-1 microg), isoproterenol (0.03-3 microg) and diazoxide (0.3-9 mg), injected into the internal maxillary artery, decreased cerebrovascular resistance in a dose-dependent manner, and this decrease was similar before and after L-NAME. Therefore, insulin-induced hypoglycemia may produce cerebral vasodilatation by releasing nitric oxide and may diminish the capacity of the cerebral vasculature to release nitric oxide in response to acetylcholine.

Peptidergic modulation of the sympathetic contraction in the rabbit ear artery: effects of temperature

The effects of neuropeptide Y, endothelin‐1, arginine‐vasopressin and angiotensin II on the vascular contraction to sympathetic nerve stimulation were studied in isolated segments, 2 mm long, from the rabbit central ear artery, a cutaneous vessel, during changes in temperature (24°–41°C). Transmural electrical stimulation (1–8 Hz, at supramaximal voltage) produced frequency‐dependent contraction, and this response, partially blocked by tetrodotoxin (1 μM) and phentolamine (1 μM), was reduced by cooling (30°C–24°C) and was not modified by warming (41°C), as compared to that recorded at 37°C. Pretreatment with neuropeptide Y (10, 30 and 100 nM) increased in a concentration‐dependent manner the vascular contraction to sympathetic stimulation at every temperature studied, but this potentiation was greater during cooling (34°C–24°C) than at 37°C or warming (41°C). Pretreatment with endothelin‐1 (3 and 10 nM) or vasopressin (0.1, 0.3 and 1 nM) increased in a concentration‐dependent manner the vascular contraction to sympathetic stimulation during cooling (34°C–24°C), but not at 37°C or warming (41°C). Pretreatment with angiotensin II (0.1, 0.3 and 1 μM) did not modify the contraction to sympathetic stimulation at any temperature studied. These results suggest that neuropeptide Y, endothelin‐1 and vasopressin, but not angiotensin II, modulate the cutaneous vasoconstriction to sympathetic nerve stimulation by potentiating this vasoconstriction during cooling.