Sheila M. Gardiner

Control of regional blood flow by endothelium-derived nitric oxide.

The regional hemodynamic consequences of inhibiting vascular endothelial nitric oxide generation with NG-monomethyl-L-arginine (L-NMMA) were studied in conscious Long-Evans rats. Experiments were carried out in groups of chronically instrumented rats with intravascular catheters and pulsed Doppler probes to monitor regional blood flow. L-NMMA (0.3-300 mg/kg) caused a dose-dependent, long-lasting (5-90 minutes), and enantiomerically specific increase in mean blood pressure and also caused bradycardia. The increase in blood pressure was accompanied by a dose-dependent and long-lasting vasoconstriction in the internal carotid, mesenteric, renal, and hindquarters vascular beds that could be attenuated, in a concentration-dependent manner, by L-arginine but not by D-arginine. In contrast, L-arginine did not affect the pressor or vasoconstrictor effects of vasopressin. These results indicate that nitric oxide production by vascular endothelial cells contributes to the maintenance of blood pressure and to the control of the resting tone of different vascular beds in the conscious rat.

Regional and cardiac haemodynamic effects of NG-nitro-L-arginine methyl ester in conscious, Long Evans rats

1 Regional haemodynamic responses to i.v. bolus doses (0.1–10.0 mg kg−1) of NG‐nitro‐l‐arginine methyl ester (l‐NAME) were measured in conscious, Long Evans rats (n = 8) chronically instrumented with renal, mesenteric and hindquarters pulsed Doppler flow probes and intravascular catheters. 2 l‐NAME caused dose‐dependent pressor effects associated with renal, mesenteric and hindquarters vasoconstrictions. The mesenteric vascular bed showed earlier onset with more rapid, and greater, maximum vasoconstrictions than the renal or hindquarters vascular beds; however, the hindquarters vasoconstriction was more persistent. d‐NAME was without significant effects (n = 2). 3 Primed infusion of l‐arginine (100 mg kg−1 bolus followed by 100 mg kg−1 h−1 infusion), starting 10 min after an i.v. bolus injection of l‐NAME (10 mg kg−1), caused significant reversal of the pressor responses, and renal and mesenteric vasoconstrictions, but not of the hindquarters vasoconstriction. Primed infusions of l‐arginine (100 mg kg−1, 100 mg kg−1 h−1) starting 5 min after l‐NAME (1 mg kg−1) additionally caused some reversal of the hindquarters vasoconstriction, but this effect was transient. 4 Primed infusion of l‐arginine (100 mg kg−1, 100 mg kg−1 h−1) starting 30 min before i.v. bolus injection of l‐NAME (10 mg kg−1) caused significant attenuation of the pressor effects and the renal and mesenteric vasoconstrictions but not of the hindquarters vasoconstriction. 5 In a separate group of rats (n = 8) chronically instrumented with thoracic aortic electromagnetic flow probes for the measurement of cardiac haemodynamics, i.v. bolus injection of l‐NAME (10 mg kg−1) produced significant reductions in total peripheral conductance, cardiac output, stroke volume, peak thoracic aortic flow and the maximum rate of rise of aortic flow; these were coincident with the maximum pressor and vasoconstrictor effects. 6 These results, collectively, are consistent with l‐NAME interfering with l‐arginine‐nitric oxide pathways that have important influences on regional vascular conductances in vivo. The pressor effect resulting from l‐NAME‐induced vasoconstrictions is offset by a substantial reduction in cardiac function that may depend on direct and/or indirect effects of l‐NAME on the heart.

Regional haemodynamic changes during oral ingestion of NG‐monomethyl‐l‐arginine or NG‐nitro‐l‐arginine methyl ester in conscious Brattleboro rats

Homozygous Brattleboro (i.e. vasopressin‐deficient) rats were chronically instrumented with pulsed Doppler probes and intravascular catheters to permit continuous monitoring of regional haemodynamics. Over a 9 h period, rats drinking water showed no systematic changes in heart rate or mean arterial blood pressure although renal, mesenteric and hindquarters vascular conductances fell. These changes showed diurnal rhythyms, probably related to the nocturnal habits of rats. In separate groups of animals spontaneous oral ingestion of NG‐monomethyl‐l‐arginine (l‐NMMA; 1mg ml−1) or NG‐nitro‐l‐arginine methyl ester (l‐NAME; 0.1 mg ml−1) caused marked hypertension but no significant bradycardia. Compared to control animals, rats drinking l‐NMMA for 9 h showed significantly greater mesenteric and hindquarters vasoconstrictions, and rats drinking l‐NAME showed greater vasoconstrictions in all 3 vascular beds.

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.

A potential role for Akt/FOXO signalling in both protein loss and the impairment of muscle carbohydrate oxidation during sepsis in rodent skeletal muscle

Sepsis causes muscle atrophy and insulin resistance, but the underlying mechanisms are unclear. Therefore, the present study examined the effects of lipopolysaccharide (LPS)‐induced endotoxaemia on the expression of Akt, Forkhead Box O (FOXO) and its downstream targets, to identify any associations between changes in FOXO‐dependent processes influencing muscle atrophy and insulin resistance during sepsis. Chronically instrumented male Sprague–Dawley rats received a continuous intravenous infusion of LPS (15 μg kg−1 h−1) or saline for 24 h at 0.4 ml h−1. Animals were terminally anaesthetized and the extensor digitorum longus muscles from both hindlimbs were removed and snap‐frozen. Measurements were made of mRNA and protein expression of selected signalling molecules associated with pathways regulating protein synthesis and degradation and carbohydrate metabolism. LPS infusion induced increases in muscle tumour necrosis factor‐α (8.9‐fold, P < 0.001) and interleukin‐6 (8.4‐fold, P < 0.01), paralleled by reduced insulin receptor substrate‐1 mRNA expression (−0.7‐fold, P < 0.01), and decreased Akt1 protein and cytosolic FOXO1 and FOXO3 phosphorylation. These changes were accompanied by significant increases in muscle atrophy F‐box mRNA (5.5‐fold, P < 0.001) and protein (2‐fold, P < 0.05) expression, and pyruvate dehydrogenase kinase 4 mRNA (15‐fold, P < 0.001) and protein (1.6‐fold, P < 0.05) expression. There was a 29% reduction in the muscle protein: DNA ratio, a 56% reduction in pyruvate dehydrogenase complex (PDC) activity (P < 0.05), and increased glycogen degradation and lactate accumulation. The findings of this study suggest a potential role for Akt/FOXO in the simultaneous impairment of carbohydrate oxidation, at the level of PDC, and up‐regulation of muscle protein degradation, in LPS‐induced endotoxaemia.

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.

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.

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.

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 .