P.A. Kemp

Regional and cardiac haemodynamic responses to glyceryl trinitrate, acetylcholine, bradykinin and endothelin-1 in conscious rats: effects of NG-nitro-l-arginine methyl ester

1 Conscious Long Evans rats, chronically instrumented for cardiovascular measurements, were challenged with i.v. bolus doses of glyceryl trinitrate (40 nmol kg−1), acetylcholine (1.2 nmol kg−1), bradykinin (3.2 nmol kg−1), or endothelin‐1 (0.25 nmol kg−1). Under control conditions these doses produced similar falls in mean arterial blood pressure (glyceryl trinitrate, −20 ± 3 mmHg; acetylcholine, −24 ± 2 mmHg; bradykinin, −21 ± 3 mmHg; endothelin‐1, −25 ± 3 mmHg), associated with renal, mesenteric and hindquarters vasodilatations (except for endothelin‐1 which caused mesenteric vasoconstriction). 2 In the presence of NG‐nitro‐l‐arginine methyl ester (l‐NAME, 10 mg kg−1), a potent inhibitor of nitric oxide biosynthesis and endothelium‐dependent vasorelaxation in vitro, the hypotensive responses to glyceryl trinitrate, acetylcholine, and endothelin‐1 were increased, although that to bradykinin was not. However, comparing the differences between the response to glyceryl trinitrate and that to any other agonist in the absence and presence of l‐NAME showed that there were relative attenuations of the hypotensive responses to bradykinin and endothelin‐1, but not to acetylcholine, in the presence of l‐NAME. 3 This comparative analysis showed that the renal and hindquarters vasodilator responses to bradykinin and endothelin‐1 were attenuated in the presence of l‐NAME, but the renal, mesenteric and hindquarters vasodilator responses to acetylcholine were not. However, when l‐NAME was administered in the presence of pentolinium, captopril and the vasopressin V1‐receptor antagonist, d(CH2)5[Tyr‐(Et)]DAVP, (to abolish baroreflex and neurohumoral mechanisms), there was attenuation of the renal and mesenteric vasodilator effects of acetylcholine relative to those seen with glyceryl trinitrate. Under those conditions only the renal vasodilator effects of bradykinin and endothelin‐1 were attenuated. 4 In separate experiments in conscious Long Evans rats, direct measurement of cardiac haemodynamics showed that the hypotensive responses to glyceryl trinitrate, acetylcholine, bradykinin and endothelin‐1 were entirely attributable to rises in total peripheral conductance since both in the absence and presence of l‐NAME there were no reductions in cardiac index in response to these substances. 5 The results indicate that measurement of systemic arterial blood pressure alone in conscious rats does not permit reliable quantitation of the influence of l‐NAME on regional vasodilator responses to glyceryl trinitrate, acetylcholine, bradykinin or endothelin‐1. Furthermore, these substances exert effects in different vascular beds that may be differentially influenced by baroreflex mechanisms, neurohumoral mechanisms, or both. Moreover, except in the case of the renal vasodilator response to endothelin‐1 (which was abolished in the presence of l‐NAME), even when l‐NAME caused attenuation of the vasodilator effects of acetylcholine or bradykinin (relative to glyceryl trinitrate), substantial responses remained. It is feasible that such responses in vivo are nitric oxide‐independent.

Effects of NG-nitro-L-arginine methyl ester on vasodilator responses to acetylcholine, 5'-N-ethylcarboxamidoadenosine or salbutamol in conscious rats.

1 Conscious, Long Evans rats (n = 16), chronically instrumented for the measurement of regional haemodynamics were given 3min, randomized infusions of two doses of sodium nitroprusside (1.5 and 15 μg min−1), acetylcholine (0.4 and 4 μg min−1), 5′‐N‐ethylcarboxamidoadenosine (NECA; 45 and 450 ng min−1), and salbutamol (24 and 240 ngmin−1) in the absence and in the presence of NG‐nitro‐l‐arginine methyl ester (l‐NAME; 1 mg kg−1 h−1), a potent inhibitor of nitric oxide biosynthesis. 2 Sodium nitroprusside caused hyperaemic vasodilatation in the mesenteric, and common carotid vascular beds. These effects were enhanced in the presence of l‐NAME, as was the hypotension. 3 Acetylcholine caused hyperaemic vasodilatation in the renal, internal carotid and common carotid vascular beds. These effects were attenuated in the presence of l‐NAME, but the hypotension was unaffected. 4 NECA caused hyperaemic vasodilatation in the renal, mesenteric, hindquarters, internal carotid and common carotid vascular beds. However, only the hindquarters and internal carotid responses were diminished in the presence of l‐NAME and the hypotension was unchanged. 5 Salbutamol caused hyperaemic vasodilatation in the hindquarters vascular bed only. This effect was reduced in the presence of l‐NAME, but the hypotension was unchanged. 6 The results indicate marked regional variations in the sensitivity of vasodilator responses to l‐NAME that can depend on the vasodilator agent chosen and the dose employed. It is clear from these findings also that measurement of mean arterial blood pressure alone cannot provide adequate information on which to judge the involvement of l‐NAME‐sensitive mechanisms in vasodilator responses in vivo.

Antagonistic effect of human α-CGRP [8–37] on the in vivo regional haemodynamic actions of human α-CGRP

Abstract In conscious rats, infusion of human α-CGRP [8–37] (30 nmol/kg/min) caused small, reversible reductions in hindquarters flow and vascular conductance only, whereas at a dose of 300 nmol/kg/min there was a tachycardia and an increase in mean arterial blood pressure, together with renal, mesenteric and hindquarters vasoconstrictions. Human α-CGRP (0.03 nmol/kg/min) caused tachycardia, hypotension, and transient renal, but sustained hindquarters, vasodilatation; these changes were accompanied by mesenteric vasoconstriction. Infusion of human α-CGRP [8–37] (30 nmol/kg/min) during administration of human α-CGRP (0.03 nmol/kg/min) abolished the effects of the latter but these re-appeared when the human α-CGRP [8–37] infusion was stopped. This dose of human α-CGRP [8–37] did not affect cardiovascular responses to isoprenaline. These results indicate that human α-CGRP [8–37] is an effective antagonist of the cardiovascular actions of human α-CGRP in vivo .

The effects of phosphoramidon on the regional haemodynamic responses to human proendothelin [1-38] in conscious rats.

1 Cardiovascular responses to human proendothelin [1–38], in the absence and presence of phosphoramidon, were studied in conscious Long Evans rats, chronically instrumented for the continuous recording of heart rate, systemic arterial blood pressure and renal, mesenteric and hindquarters blood flows. 2 A dose of 0.1 nmol kg−1 human proendothelin [1–38] caused a slight pressor effect (maximum 5 ± 2mmHg), but a clear bradycardia (maximum −29 ± 7 beats min−1). Renal haemodynamics were unchanged but there was mesenteric vasoconstriction and a vasodilatation followed by a vasoconstriction in the hindquarters. 3 A dose of 1.0 nmol kg−1 human proendothelin [1–38] caused a gradual hypertension (maximum 42 ± 4 mmHg at 10 min) and a profound bradycardia (–149 ± 10 beats min−1 at 30 min). There were gradual but marked, renal and hindquarters vasoconstrictions, whereas there was a substantial mesenteric vasoconstriction that was relatively rapid in onset. 4 In 2 animals, administration of human proendothelin [1–38] at a dose of 10 nmol kg−1 caused an initial hypotension followed by a rapidly‐developing pressor effect; there were renal and mesenteric vasoconstrictions and vasodilatation followed by vasoconstriction in the hindquarters. These changes were very similar to those seen following injection of endothelin‐1 (0.1 nmol kg−1). 5 Phosphoramidon (2 μmol kg−1) had no cardiovascular effects itself and it did not affect significantly the pressor or mesenteric vasoconstrictor effects of human proendothelin [1–38], but it reduced the bradycardia and renal and hindquarters vasoconstrictor responses. A higher dose of phosphoramidon (10 μmol kg−1) caused significant attenuation of all the responses to human proendothelin [1–38], but a substantial mesenteric vasoconstrictor response still occurred under these conditions. 6 The results are consistent with the involvement of phosphoramidon‐sensitive enzyme systems in the conversion of human proendothelin [1–38] to endothelin‐1 in vivo. In addition, considering the different patterns of responses to human proendothelin [1–38] in the effector tissues studied (heart, and renal, mesenteric and hindquarters vascular beds), and the differential degrees of inhibition of them by phosphoramidon, it is likely that the effects of human proendothelin [1–38] were due to its local (rather than systemic) conversion to endothelin‐1 by processes with differing degrees of susceptibility to phosphoramidon.

Active immunization with angiotensin I peptide analogue vaccines selectively reduces the pressor effects of exogenous angiotensin I in conscious rats

Male, Sprague‐Dawley rats were actively immunized with novel angiotensin vaccines, and their pressor responses to exogenous angiotensin I (AI) and angiotensin II (AII) were assessed in vivo. Serum antibody titres were also measured. The most effective vaccine consisted of an AI analogue conjugated with a tetanus toxoid carrier protein and adjuvanted with aluminium hydroxide. When this vaccine was injected on days 0, 21 and 42, pressor responses to AI on day 63 were significantly inhibited (maximum, 8.9 fold shift), but responses to AII were unaffected. The anti‐angiotensin antibody titre was increased 32,100 fold, and, uniquely, these antibodies also cross‐reacted with angiotensinogen. These findings indicate that active immunization against AI may be a useful approach for treating cardiovascular disorders involving the renin‐angiotensin system.

Enhancement of the hypotensive and vasodilator effects of endotoxaemia in conscious rats by the endothelin antagonist, SB 209670.

In conscious, chronically‐instrumented rats, the non‐selective endothelin antagonist, SB 209670 (10 μg kg−1 min−1), caused marked enhancement of the fall in mean arterial blood pressure during infusion of lipopolysaccharide (LPS) for 24 h (LPS alone=‐6 ± 3 mmHg; LPS + SB 209670 = −30 ±2 mmHg). This effect was accompanied by a conversion of the mesenteric vasoconstriction to a substantial mesenteric vasodilatation and an augmentation of the hindquarters vasodilatation, seen with LPS alone. Notably, the marked renal hyperaemic vasodilatation during LPS infusion was not affected significantly by SB 209670. These results indicate that endothelin, directly and/or indirectly, plays a pivotal role in the cardiovascular sequelae of endotoxaemia in conscious rats, and prevents marked hypotension, particularly by opposing mesenteric vasodilatation.

Regional haemodynamic effects of cyclosporine A, tacrolimus and sirolimus in conscious rats

The observation that the immunosuppressants, cyclosporine A (CsA) and tacrolimus, have pressor effects, but sirolimus does not, has led to an hypothesis that generalised sympathoexcitation, resulting from inhibition of calcineurin by CsA and tacrolimus underlies their pressor effects, because sirolimus does not inhibit calcineurin. It is unknown if sirolimus has haemodynamic actions not accompanied by a pressor effect, and whether or not the pressor effects of CsA and tacrolimus are accompanied by similar haemodynamic changes. Therefore, the first aim of our studies was to investigate these possibilities in conscious, chronically‐instrumented, male, Sprague‐Dawley rats. CsA (5.9 mg kg−1 bolus i.v.) caused rapid‐onset, prolonged hypertension, tachycardia and mesenteric vasoconstriction. There was a slower onset renal vasoconstriction, but no significant change in hindquarters vascular conductance; all the effects of CsA were significantly greater than those of vehicle. CsA given by infusion (over 30 min or 2 h) caused changes qualitatively similar to those above. Repeated administration of CsA over 4 days did not enhance its cardiovascular effects. Pretreatment with the angiotensin (AT1) receptor antagonist, losartan, and the endothelin (ETA and ETB) receptor antagonist, SB 209670, reduced the pressor and mesenteric vasoconstrictor effects of CsA. Additional administration of the α‐adrenoceptor antagonist, phentolamine, completely inhibited the cardiovascular effects of CsA. Tacrolimus (450 μg kg−1 bolus i.v.) caused similar peak pressor and tachycardic effects to CsA, but these were much slower in onset, and were maximal when there were no significant regional vasoconstrictions, indicating that the pressor effect was probably due to a rise in cardiac output. However, although propranolol reversed the tachycardic effect of tacrolimus, it did not influence the pressor response. Sirolimus (450 μg kg−1 bolus i.v.) had no tachycardic action, and only a modest, transient pressor effect, accompanied by equally brief reductions in renal, mesenteric, and hindquarters vascular conductances. The differences between the regional haemodynamic profiles of equipressor doses of CsA and tacrolimus, and the finding that sirolimus has significant cardiovascular actions, indicate that generalised sympathoexcitation, resulting from calcineurin inhibition (with CsA and tacrolimus), is unlikely to be the sole explanation of their pressor effects.

Comparative regional haemodynamic effects of the nitric oxide synthase inhibitors, S-methyl-l-thiocitrulline and l-NAME, in conscious rats

The regional haemodynamic effects of the putative nNOS inhibitor, S‐methyl‐L‐thiocitrulline (SMTC), were compared with those of the nonselective NOS inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME), in conscious, male Sprague–Dawley rats. SMTC (0.3 mg kg−1 bolus) produced a significant, short‐lived, pressor effect associated with renal, mesenteric and hindquarters vasoconstriction; the same dose of L‐NAME did not affect mean blood pressure (BP), although it caused bradycardia and mesenteric vasoconstriction. At the highest dose tested (10 mg kg−1), L‐NAME produced a significantly greater bradycardia and fall in mesenteric vascular conductance than SMTC, although the initial pressor response to SMTC was greater, but less sustained, than that to L‐NAME. Infusion of SMTC or L‐NAME (3 mg kg−1 h−1) induced rises in BP and falls in renal, mesenteric and hindquarters vascular conductances, but the effects of L‐NAME were greater than those of SMTC, and L‐NAME also caused bradycardia. The renal vasodilator response to acetylcholine was markedly attenuated by infusion of L‐NAME, but unaffected by SMTC. The hindquarters vasodilatation induced by salbutamol was attenuated by L‐NAME, but not by SMTC. The mesenteric vasodilator response to bradykinin was modestly enhanced by SMTC, but not by L‐NAME. The depressor and renal, mesenteric and hindquarters vasodilator responses to sodium nitroprusside were enhanced by L‐NAME, whereas SMTC modestly enhanced the hypotensive and renal vasodilator effects of sodium nitroprusside, but attenuated the accompanying tachycardia. The results are consistent with the cardiovascular effects of low doses of SMTC being attributable to nNOS inhibition.

Haemodynamic effects of human α-calcitonin gene-related peptide following administration of endothelin-1 or NG-nitro-L-arginine methyl ester in conscious rats

1 We investigated the peripheral haemodynamic effects of human α‐calcitonin gene‐related peptide (CGRP) following administration of endothelin‐1 or NG‐nitro‐l‐arginine methyl ester (l‐NAME), an inhibitor of nitric oxide production, in conscious, chronically‐instrumented, Long Evans rats. 2 Infusion of endothelin‐1 (3 nmol kg−1 h−1) caused hypertension, bradycardia and renal, mesenteric and hindquarters vasoconstrictions. Co‐infusion of human α‐CGRP (1.5 nmol kg−1 h−1) reduced the hypertension and abolished the hindquarters vasoconstriction caused by endothelin‐1 but the renal and mesenteric vasoconstrictor actions of endothelin‐1 were not affected. 3 Infusion of human α‐CGRP (15 nmol kg−1 h−1) in the presence of endothelin‐1 caused hypotension and hyperaemic vasodilatation in the hindquarters; the mesenteric vasoconstrictor effects of endothelin‐1 were diminished, but there was only a transient reversal of the renal vasoconstrictor effects of endothelin‐1. 4 Pretreatment with the non‐peptide angiotensin II receptor antagonist, DuP 753 (10 mg kg−1), caused slight hypotension associated with renal, mesenteric and hindquarters vasodilatations, but DuP 753 did not affect responses to endothelin‐1 infusion. However, under these conditions co‐infusion of human α‐CGRP (15 nmol kg−1 h−1) caused a sustained reversal of the renal vasoconstrictor effects of endothelin‐1. 5 These results indicate that the failure of human α‐CGRP to cause sustained reversal of the renal vasoconstrictor effects of endothelin‐1 in the absence of DuP 753 was due to activation of the renin‐angiotensin system (possibly as a consequence of the hypotension). 6 In the second experiment, l‐NAME (10 mg kg−1) caused renal, mesenteric and hindquarters vasoconstrictions similar to those seen in the presence of endothelin‐1. However, the renal vasoconstrictor effects of l‐NAME were reversed completely by human α‐CGRP (15 nmol kg−1 h−1), even though the latter caused hypotension comparable to that seen in the presence of endothelin‐1. These results are consistent with a lack of functional activation of the renin‐angiotensin system by human α‐CGRP in the presence of l‐NAME. 7 The vasoconstrictor effects of l‐NAME on the hindquarters were completely reversed by infusion of human α‐CGRP, but hindquarters flow and vascular conductance did not rise above baseline levels. Hence these results indicate the hindquarters hyperaemic vasodilator effects of human α‐CGRP seen in the presence of endothelin‐1 were contributed to by nitric oxide‐mediated mechanisms.

Human α-calcitonin gene-related peptide (CGRP)-(8–37), but not-(28–37), inhibits carotid vasodilator effects of human α-CGRP in vivo

Abstract Human α-calcitonin gene-related peptide-(8–37) alone (up to doses of 30 nmol kg −1 min −1 ) had no significant effects on blood pressure, heart rate or common or internal carotid haemodynamics, although it caused significant, reversible, inhibition of the hypotensive, tachycardic, and common and internal carotid vasodilator effects of human α-CGRP (0.03 nmol kg −1 min −1 ) in conscious, Long Evans rats. Human α-CGRP-(28-37) up to doses of 300 nmol kg −1 min −1 had no cardiovascular effects itself and did not influence responses to human α-CGRP. These results are consistent with the carotid haemodynamic effects of human α-CGRP being due largely to activation of the CGRP 1 -receptor subtype.