J E March

Depressor and regionally-selective vasodilator effects of human and rat urotensin II in conscious rats.

The regional haemodynamic effects of rat or human urotensin II (U‐II) 3, 30, 300 and 3000 pmol kg−1, i.v.) were assessed in separate groups of conscious, unrestrained, male, Sprague‐Dawley rats (n=8 in each). Rat and human U‐II had similar effects. At a dose of 3 pmol kg−1, neither peptide had any significant action, while at a dose of 30 pmol kg−1, there was a transient mesenteric vasodilatation (significant only for rat U‐II). At doses of 300 and 3000 pmol kg−1, there were dose‐dependent tachycardias, and mesenteric and hindquarters hyperaemic vasodilatations. Thus, in conscious rats, the predominant cardiovascular action of rat and human U‐II is vasodilatation. This is in contrast to recent findings with human U‐II in non‐human primates, but is consistent with effects on human isolated resistance vessels.

Regional haemodynamic effects of human and rat adrenomedullin in conscious rats.

1 Male, Long Evans rats were chronically instrumented with pulsed Doppler flow probes and intravascular catheters to permit assessment of the regional haemodynamic responses to human and rat adrenomedullin, to compare the responses to human adrenomedullin to those of human α‐CGRP in the absence and presence of the CGRP1‐receptor antagonist, human α‐CGRP [8–37], and to determine the involvement of nitric oxide (NO)‐mediated mechanisms in the responses to human adrenomedullin, relative to human α‐CGRP. 2 Human and rat adrenomedullin (0.3, 1, and 3 nmol kg−1, i.v.) caused dose‐dependent hypotension and tachycardia, accompanied by increases in renal, mesenteric and hindquarters flows and vascular conductances. At the lowest dose only, the hypotensive and mesenteric vasodilator effects of rat adrenomedullin were significantly greater than those of human adrenomedullin. 3 Human α‐CGRP at a dose of 1 nmol kg−1 caused hypotension, tachycardia and increases in hindquarters flow and vascular conductance, but reductions in renal and mesenteric flows, and only transient vasodilatations in these vascular beds. These effects were substantially inhibited by human α‐CGRP [8–37] (100 nmol kg−1 min−1), but those of human adrenomedullin (1 nmol kg−1) were not; indeed, the mesenteric haemodynamic effects of the latter peptide were enhanced by the CGRP1‐receptor antagonist. 4 In the presence of the NO synthase inhibitor, NG‐nitro‐1‐arginine methyl ester (1‐NAME, 183 nmol kg−1 min−1), there was only a slight, but significant, inhibition of the hindquarters hyperaemic vasodilator effect of human adrenomedullin, but not that of human α‐CGRP. 5 These results indicate that the marked regional vasodilator effects of human (and rat) adrenomedullin are largely independent of NO and, in vivo, do not involve CGRP1‐receptors.

Cardiac and regional haemodynamics, inducible nitric oxide synthase (NOS) activity, and the effects of NOS inhibitors in conscious, endotoxaemic rats.

1 A reproducible model of the hyperdynamic circulatory sequelae of endotoxaemia in conscious, chronically‐instrumented Long Evans rats, was achieved with a continuous infusion of lipopolysaccharide (LPS, 150 μg kg−1 h−1) for 32 h. Over the first 2 h of LPS infusion, there was a transient hypotension and tachycardia, accompanied by a marked increase in renal flow and vascular conductance, although there were reductions in cardiac and stroke index. Between 4–8 h after the start of LPS infusion, there was slight hypotension and tachycardia, and a transient rise in mesenteric flow and conductance, but reductions in the hindquarters vascular bed; the hyperaemic vasodilatation in the renal vascular bed was maintained. At this stage, all cardiac haemodynamic variables and total peripheral conductance, were increased, but central venous pressure was reduced. By 24 h after the onset of LPS infusion, there was clear hypotension and tachycardia, accompanied by increases in renal and hindquarters flow and conductance, although mesenteric haemodynamic variables were not different from baseline. At this stage, cardiac and stroke index were substantially elevated, in association with marked increases in peak aortic flow, dF/dtmax and total peripheral conductance; these changes were well‐maintained over the following 8 h of LPS infusion. 2 By 2 h after the start of LPS infusion, only lung inducible nitric oxide synthase (iNOS) activity was increased, but at 6 h there were significant increases in iNOS activity in lung, liver, spleen, heart and aorta (43.3 ± 7.8, 28.8 ± 3.3, 50.8 ± 7.2, 3.04 ± 0.29, 3.76 ± 0.94 pmol min−1 mg−1 protein, respectively). However, by 24 h after the start of LPS infusion, iNOS activity was not elevated significantly in any tissue examined, and kidney iNOS activity did not change significantly during LPS infusion. Plasma nitrite/nitrate levels were increased after 2 h infusion of LPS (from 6.07 ± 1.23 to 29.44 ± 7.08 μmol l−1), and further by 6 h (228.10 ± 29.20 μmol l−1), but were less 24 h after onset of LPS infusion (74.96 ± 11.34 μmol l−1). Hence, the progressive hypotension, increasing cardiac function and developing hyperaemic vasodilatation in renal and hindquarters vascular beds between 8–24 h after the onset of LPS infusion, occurred when tissue iNOS activity and plasma nitrite/nitrate levels were falling. 3 Pretreatment with NG‐monomethyl‐L‐arginine (l‐NMMA, 30 mg kg−1 bolus, 30 mg kg−1 h−1 infusion) 1 h before LPS infusion did not prevent the early hypotension, but abolished the initial renal vasodilatation and the later (6–8 h) fall in mean arterial pressure (MAP), and the additional renal vasodilatation. However, under these conditions, mesenteric and hindquarters flows and conductances were substantially decreased. Similar, but less marked, effects were seen with L‐NMMA pretreatment at 10 mg kg−1 bolus, 10 mg kg−1 h−1 infusion, whereas at a lower dose of 3 mg kg−1 bolus, 3 mg kg−1 h−1 infusion, L‐NMMA pretreatment had little effect on responses to LPS. 4 Delaying treatment with L‐NMMA (10 mg kg−1 bolus, 10 mg kg−1 h−1 infusion) until 4 h after the start of LPS infusion prevented the late hindquarters vasodilatation and attenuated the late renal vasodilatation, but still reduced mesenteric flow. When treatment with L‐NMMA was delayed until 24 h after the start of LPS infusion, renal and hindquarters vasodilatations were only slightly affected, but mesenteric flow was still compromised. Delayed treatment with L‐NAME (3 mg kg−1 h−1 starting 24 h after onset of LPS infusion) caused substantial inhibition of the renal vasodilatation, but also caused marked reduction in mesenteric and hindquarters flows and indices of cardiac performance. 5 These findings indicate that iNOS activity is not directly responsible for the widespread vasodilatation seen after 24 h infusion of LPS in conscious rats. If our observations can be extrapolated to the clinical situation, they indicate that non‐selective NOS inhibition could have detrimental effects in endotoxaemic patients with signs of a hyperdynamic circulation.

Regional haemodynamic responses to infusion of lipopolysaccharide in conscious rats: effects of pre‐ or post‐treatment with glibenclamide

To determine the putative contribution of KATP‐channels to the haemodynamic sequelae of endotoxaemia, three experiments were carried out in different groups of conscious, chronically‐instrumented, unrestrained, male Long Evans rats. In the first experiment, pretreatment with the KATP‐channel antagonist, glibenclamide, abolished the initial hypotension, but not the renal vasodilatation caused by LPS infusion. Subsequently, however, in the presence of glibenclamide and LPS there was a significant increase in mean arterial blood pressure, and a bradycardia, in contrast to the fall in mean arterial blood pressure and the tachycardia seen in the presence of vehicle and LPS. The pressor and bradycardic changes in the presence of glibenclamide and LPS were accompanied by significant reductions in hindquarters flow and vascular conductance, and these were significantly greater than those seen in the presence of vehicle and LPS, or glibenclamide and saline. Administration of glibenclamide 6 h after the onset of saline and LPS infusion, or 6 h after the onset of saline and LPS infusion in the presence of the AT1‐receptor antagonist, losartan, and the ETA‐, ETB‐ receptor antagonist, SB 209670, in the absence or presence of dexamethasone, caused a significant increase in mean arterial blood pressure and reductions in renal, mesenteric and hindquarters conductances, although the latter was the only vascular bed in which there was a reduction in flow. The results are consistent with a contribution from KATP‐channels to the vasodilatation caused by LPS, particularly in the hindquarters vascular bed.

Acute and chronic cardiac and regional haemodynamic effects of the novel bradycardic agent, S16257, in conscious rats.

1 We carried out experiments to assess the cardiac and regional haemodynamic effects of single or repeated injections of the novel bradycardic agent, S16257, (7,8‐dimethoxy 3‐{3‐{[(lS)‐(4,5‐dimethoxy‐benzocyclobutan‐l‐yl)methyl]methylamino}propyl}1,3,4,5‐tetrahydro‐2H‐benzapin 2‐one), in conscious rats. 2 In the first experiment, male Long Evans rats were chronically instrumented for the measurement of cardiac or regional haemodynamics (n = 9 in each group), and, on separate experimental days, were randomized to receive i.v. bolus injections of vehicle (5% dextrose) or S16257 at a dose of 1 mg kg−1. 3 In animals instrumented for the measurement of cardiac haemodynamics (n = 9), following injection of vehicle, there were no immediate changes, and 7–8 h later there were slight reductions in heart rate and mean arterial blood pressure only. Injection of S16257 caused an immediate, transient, pressor effect but thereafter there were reductions in heart rate, mean arterial blood pressure, cardiac index and total peripheral conductance, together with increases in stroke index and peak aortic flow. The integrated decreases in heart rate, mean arterial blood pressure, cardiac index and total peripheral conductance and increases in stroke index, peak aortic flow, dF/dtmax and central venous pressure following S16257 were all significantly greater than the changes after vehicle injection. After injection of S16257, the fall in heart rate and fall in cardiac index were not linearly related. 4 In animals instrumented for the measurement of regional haemodynamics (n = 9). the bradycardic effect of i.v. S16257 was accompanied by reductions in renal, mesenteric and hindquarters blood flows and vascular conductances that were greater than the changes seen following injection of vehicle, but only for the first 1 h. Considering animals instrumented for the measurement of cardiac and regional haemodynamics together, the bradycardic effect of S16257 was greater the higher the resting heart rate. 5 In the second experiment, animals chronically instrumented for the measurement of cardiac or regional haemodynamics (n = 9 in each group) were given s.c. injections of S16257 (1 mg kg−1) on four consecutive days. The general patterns of change in cardiac and regional haemodynamics following s.c. injection of S16257 were as described above for i.v. injection, although the rates of onset of effects were slower. The bradycardic effect of S16257 was less on the first, than on the subsequent, three days. 6 Overall, these results indicate that the bradycardic action of S16257 is not associated with any signs of negative inotropic action. Only the initial depressor effect of i.v. S16257 is associated with reductions in renal, mesenteric and hindquarters flow and vascular conductance significantly greater than those seen after vehicle injection. With repeated s.c. injection of S16257, there are no signs of desensitization to its bradycardic actions, nor impairment of regional perfusion. If these results extrapolate to the clinical setting, it seems likely that S16257 will have beneficial bradycardic effects, with no concurrent undesirable actions on other aspects of cardiovascular function.

Complex regional haemodynamic effects of anandamide in conscious rats

Experiments were carried out in conscious, chronically instrumented, male, Sprague‐Dawley rats to delineate the regional haemodynamic effects of the putative endogenous cannabinoid, anandamide, (0.075 – 3 mg kg−1), and to dissect some of the mechanisms involved. At all doses of anandamide, there was a significant, short‐lived increase in mean arterial blood pressure associated with vasoconstriction in renal, mesenteric and hindquarters vascular beds. The higher doses (2.5 and 3 mg kg−1), caused an initial, marked bradycardia accompanied, in some animals, by a fall in arterial blood pressure which preceded the hypertension. In addition, after the higher doses of anandamide, the hindquarters vasoconstriction was followed by vasodilatation. Although some of the effects described above resembled those of 5‐HT (25 μg kg−1), the bradycardia and hypotensive actions of the latter were abolished by the 5HT3‐receptor antagonist, azasetron, whereas those of anandamide were generally unaffected. None of the cardiovascular actions of anandamide were influenced by the CB1‐receptor antagonist, AM 251, but its bradycardic effect was sensitive to atropine, and its hindquarters vasodilator action was suppressed by the β2‐adrenoceptor antagonist, ICI 118551. The results differ, in several aspects, from those previously reported in anaesthetized animals, and underscore the important impact anaesthesia can have on responses to anandamide.

Regional haemodynamic effects of angiotensin II (3–8) in conscious rats

1 It has been reported that angiotensin II (AII) (3–8) causes endothelium‐dependent renal cortical vasodilatation, in anaesthetized rats, through interaction with a novel receptor that shows no affinity for the AT1‐receptor antagonist, losartan. Therefore in order to get a fuller profile of the regional haemodynamic effects of AII (3–8) in conscious rats we assessed its renal, mesenteric and hindquarters vascular effects, and compared them to the responses elicited by AII and AIII. 2 AII and AIII (1.25, 12.5 and 125 pmol kg−1) caused dose‐dependent pressor and renal and mesenteric vasoconstrictor effects. At doses up to 125 pmol kg−1, AII (3–8) was without any cardiovascular effects, but with doses of 1.25 and 12.5 nmol kg−1 there were dose‐dependent increases in mean arterial blood pressure and reductions in renal and mesenteric flows and vascular conductances. The responses to AII (3–8) (12.5 nmol kg−1) were abolished by losartan (20 μmol kg−1). 3 Since it has been found that pretreatment with l‐arginine can reveal a vasodilator effect of AII (3–8) on rabbit pial arterioles, we assessed responses to AII (3–8) (12.5 nmol kg−1) before and 5 min after onset of a primed infusion of l‐arginine (1.4 mmol kg−1 bolus, 1.4 mmol kg−1 h−1 infusion). Responses to AII (3–8) were unchanged by l‐arginine. 4 The results are consistent with AII (3–8) being a less effective agonist than AII (or AIII) at the AT1‐receptor, but provide no evidence for AII (3–8) interacting with a novel receptor that shows no affinity for losartan.

Effects of bosentan (Ro 47-0203), an ETA-, ETB-receptor antagonist, on regional haemodynamic responses to endothelins in conscious rats

1 Regional haemodynamic responses to endothelin (ET)‐1, −2 and −3 and big ET‐1 (all at 500 pmol kg−1) were assessed in the same conscious Long Evans rats (n = 8) in the absence or presence of the mixed ETA‐, ETB‐receptor antagonist, Ro 47–0203 (bosentan; 30 mg kg−1). 2 Bosentan blocked the initial depressor, tachycardic and hindquarters hyperaemic vasodilator effects of ET‐1, −2 and −3, and substantially curtailed the primary renal and secondary hindquarters vasoconstrictor responses. Bosentan did not inhibit the initial mesenteric vasoconstrictor action of ET‐1, but reduced the duration of the later mesenteric vasoconstriction. In contrast, bosentan delayed the rate of onset, and reduced the duration, of the mesenteric vasoconstrictor actions of ET‐2 and ET‐3. The most likely explanation of this finding is that ET‐1, but not ET‐2 or ET‐3, triggered a covert mesenteric vasodilator mechanism which was antagonized by bosentan. 3 Bosentan blocked all the effects of big ET‐1, and, in a separate group of rats (n = 7), blocked all the haemodynamic effects of a lower dose of ET‐1 (50 pmol kg−1), with the exception of a slight mesenteric vasoconstriction. 4 The most straightforward explanation of the results is that the major haemodynamic effects of ET‐1, −2 and −3, and all the effects of big ET‐1, are mediated through ETA‐ and/or ETB‐receptors that are effectively antagonized by bosentan.

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.

Assessment of the effects of endothelin‐1 and magnesium sulphate on regional blood flows in conscious rats, by the coloured microsphere reference technique

There is evidence to suggest that magnesium (Mg2+) is beneficial in the treatment of a number of conditions, including pre‐eclampsia and acute myocardial infarction. The mode of action of Mg2+ in these conditions is not clear, although the vasodilator properties of Mg2+ are well documented both in vitro and in vivo. Previously, we demonstrated that i.v. infusion of magnesium sulphate (MgSO4) alone, or in the presence of vasoconstrictors, caused increases in flow and conductance in the common carotid, internal carotid and hindquarters vascular beds, in conscious rats. Therefore, the objective of the present study was to investigate the regional and subregional changes in haemodynamics in response to the vasoconstrictor peptide endothelin‐1 (ET‐1) and MgSO4 in more detail, using the coloured microsphere reference technique. Infusion of ET‐1 and MgSO4 had similar effects on heart rate and mean arterial pressure as in our previous study. Infusion of ET‐1 caused a rise in mean arterial pressure and a fall in heart rate, and infusion of MgSO4 returned mean arterial pressure to control levels with no effect on heart rate. The responses to MgSO4 in the presence of ET‐1 showed considerable regional heterogeneity with blood flow increasing (e.g. skeletal muscle), decreasing (e.g. stomach) or not changing (e.g. kidney). Of particular interest was the finding that MgSO4 caused increases in flow in the cerebral and coronary vascular beds. This, and our previous studies, have shown that MgSO4 can reverse vasoconstriction in a number of vascular beds, and indicate that this compound may have therapeutic benefit in conditions associated with vasospasm.