Brent C. Williams

The correction of autonomic dysfunction in cirrhosis by captopril

BACKGROUND/AIMS Vagal dysfunction is reported in about 70% of patients with cirrhosis, irrespective of aetiology, as detected by cardiovascular reflex tests. We have previously shown that RR-variability on 24-h ECG is a more sensitive marker of vagal dysfunction in cirrhosis. Angiotensin II inhibits vagal function in animals, and it is elevated in cirrhosis and may be the cause of the vagal dysfunction. Our aim was to observe the effect of captopril on vagal dysfunction in cirrhosis. METHODS Eight patients with cirrhosis (biopsy proven, male two, female six, mean age 54.25) had 24-h ECG RR-variability performed. They then received captopril 25 mg t.d.s. for 48 h. The 24-h ECG was repeated on therapy. RESULTS Mean blood pressure remained unchanged: baseline 89.8 +/- 4.8 mmHg (mean +/- sem) versus 91.8 +/- 5.9 mmHg, p = not significant. Median baseline RR-variability was 791 (range 18-5344) counts/24 h and increased in all but one patient, with captopril, to 1548 (56-4824) p = 0.008. Three increased into the normal range. CONCLUSION The vagal dysfunction of cirrhosis is caused by neuromodulation by angiotensin II and is not due to a neuropathy.

In vivo and in vitro evidence of altered nitric oxide metabolism in the spontaneously diabetic, insulin‐dependent BB/Edinburgh rat

Altered vasoreactivity may contribute significantly to the pathogenesis of diabetic vascular complications. This study investigated the effect of (a) insulin‐treated diabetes, and (b) chronic in vivo administration of Nω‐nitro‐l‐arginine methyl ester (l‐NAME), a nitric oxide (NO) synthase inhibitor, on mean arterial pressure and in vitro vascular reactivity to noradrenaline in mesenteric arterial bed preparations from spontaneously diabetic, insulin‐dependent and treated BB rats, the best animal model of insulin‐dependent mellitus (IDDM) currently available. Four groups of animals from the Edinburgh colony (BB/E) of spontaneously diabetic BB rats were studied: age‐matched (mean±s.e.mean=156±2d) non‐diabetic (glycated haemoglobin=3.8±0.1%) and insulin‐treated diabetic (glycated haemoglobin=6.2±0.5%; duration of diabetes=56±4 d) groups were either l‐NAME treated (oral dose=27±1 mg kg−1 d−1; duration of treatment from 30 until 153 days of age) or untreated. Although our diabetic BB/E rats do not achieve overall normoglycaemia, individual adjustment of the daily insulin dose administered to every diabetic rat achieves better glycaemic control than previous groups studying altered vascular reactivity and endothelial dysfunction in this animal model of diabetes. Mean arterial pressure (measured directly via indwelling carotid arterial cannulae) was not significantly different between non‐diabetic (116±3 mmHg; n=10) and diabetic (122±2 mmHg; n=12) BB/E rats. l‐NAME treatment significantly (P<0.001) increased mean arterial pressure in both groups (165±6 mmHg; n=9 and 142±4 mmHg; n=6 respectively) but the degree of hypertension observed in l‐NAME‐treated diabetic rats was significantly (P<0.01) attenuated compared to non‐diabetic rats treated with l‐NAME. Mesenteric arterial bed preparations were cannulated under anaesthesia, excised and intralumenally perfused ex vivo with noradrenaline (0.2–20 μm). Basal perfusion pressures were not significantly different in mesentery preparations from non‐diabetic (27.0±2.6 mmHg) and diabetic (27.1±3.2 mmHg) BB/E rats. There was no significant difference in maximal response above basal perfusion pressure (MAX) or pEC50, defined as the negative log of the agonist concentration required to give 50% of the maximal response above basal perfusion pressure, to noradrenaline in untreated non‐diabetic (166±7 mmHg and 5.74±0.05 respectively) and diabetic (170±11 mmHg and 5.59±0.05) BB/E rats. In vivo treatment of non‐diabetic and diabetic BB/E rats with l‐NAME had no significant effect on basal perfusion pressure (25.9±4.8 mmHg and 28.5±3.9 mmHg respectively). l‐NAME treatment in vivo increased (P<0.001) MAX to noradrenaline of non‐diabetic rats (224±8 mmHg) but did not affect the value for diabetic rats (178±14 mmHg). l‐NAME treatment did not alter the pEC50 values in either group (5.71±0.05 and 5.65±0.05). Consistent with previous studies using vascular preparations from spontaneously diabetic BB rats, mesentery preparations from diabetic BB/E rats (n=12) exhibited a significantly reduced vasodilator response to acetylcholine (F value=4.4, P<0.05) across the concentration range studied compared to non‐diabetic BB/E rats (n=12) although there was no significant difference in maximal relaxation (diabetic 53.1±4.3% vs non‐diabetic 55.7±5.5%) or pEC50, (diabetic 6.92±0.25 vs non‐diabetic 7.49±0.22). There was no significant (F value=0.8, P>0.1) difference in the response to GTN between preparations from non‐diabetic and diabetic rats (maximal relaxation: 49.6±3.7% vs 48.5±4.3%; pEC50: 7.84±0.12 vs 7.89±0.22 respectively). In conclusion, vascular responsiveness to noradrenaline is not impaired in spontaneously diabetic BB/E rats with significantly better glycaemic control than those used in previous studies. However, following chronic l‐NAME treatment, diabetic BB/E rats exhibit attenuated hypertension and an absence of enhanced vascular responsiveness to noradrenaline in vitro compared to similarly treated non‐diabetic rats. These results, together with the significantly impaired endothelium‐dependent vasodilatation and unchanged endothelium‐independent vasodilatation in vitro of preparations from diabetic BB/E rats, are consistent with the hypothesis that functional changes in the synthesis and metabolism of NO (rather than altered vascular responsiveness to NO) occur in diabetes. Our results indicate that good glycaemic control alone is insufficient to prevent these abnormalities in NO availability and further studies to characterize the origin of these changes are necessary.

Direct and indirect effects of carbenoxolone on responses to glucocorticoids and noradrenaline in rat aorta.

Background In the kidney carbenoxolone impairs inactivation of glucocorticoids and facilitates their access to mineralocorticoid receptors by inhibiting 11β-hydroxysteroid dehydrogenase (11β-OHSD). 11β-OHSD is also expressed in vascular smooth muscle, and, in humans, carbenoxolone potentiates vasoconstrictor sensitivity to cortisol and noradrenaline. Objective To establish in vitro whether the vascular effects of carbenoxolone are mediated by inhibition of 11β-OHSD. Methods Noradrenaline-induced vasoconstriction was measured in helical de-endothelialized rat aortic strips following 2–5 h exposure to one or more of: carbenoxolone, corticosterone, a mineralocorticoid-receptor antagonist (spironolactone) and a glucocorticoid- and progesterone-receptor antagonist (RU 38486). Results Carbenoxolone potentiated noradrenaline-induced vasoconstriction in aortae from adrenalectomized rats, an effect which was prevented by spironolactone but not by RU 38486. By contrast, when corticosterone was added or when aortae from non-adrenalectomized rats were studied, carbenoxolone attenuated noradrenaline-induced vasoconstriction. Conclusions Carbenoxolone has a direct effect, independent of 11β-OHSD, which potentiates noradrenaline-induced vasoconstriction and might be mediated by activation of mineralocorticoid receptors. Carbenoxolone also has an indirect effect, attenuating noradrenaline-induced vasoconstriction dependent on corticosterone and, therefore, mediated by inhibition of 11β-OHSD. Although experiments with carbenoxolone must be interpreted with caution because of its direct effect, the present data confirm that 11β-OHSD modulates vascular sensitivity to glucocorticoids and noradrenaline. Therefore, 11β-OHSD activity might influence blood pressure by effects in both the kidney and the vasculature.

Catecholamine stimulation of cortisol secretion by 3-day primary cultures of purified zona fasciculata/reticularis cells isolated from bovine adrenal cortex

Primary cultures of zona fasciculata/reticularis cells derived from bovine adrenal cortex secreted cortisol and corticosterone in response to isoprenaline, noradrenaline and adrenaline on the third day of culture. The potency order was isoprenaline greater than noradrenaline, adrenaline with an ED50 for all three agonists within the range 1-5 x 10(-8) M. A dose-dependent increase in medium content of cyclic AMP was also observed. Secretion of cortisol in response to these catecholamines was specifically blocked by propranolol but unaffected by phentolamine. The beta-agonist effect on cortisol secretion was specifically and progressively reduced, in a time- and dose-dependent manner, by pre-incubation of the cells with adrenaline.

Endothelium-derived hyperpolarizing factor and potassium use different mechanisms to induce relaxation of human subcutaneous resistance arteries

This investigation examined the hypothesis that release of K+ accounts for EDHF activity by comparing relaxant responses produced by ACh and KCl in human subcutaneous resistance arteries. Resistance arteries (internal diameter 244±12 μm, n=48) from human subcutaneous fat biopsies were suspended in a wire myograph. Cumulative concentration‐response curves were obtained for ACh (10−9 – 3×10−5 M) and KCl (2.5 – 25 mM) following contraction with noradrenaline (NA; 0.1 – 3 μM). ACh (Emax 99.07±9.61%; −LogIC50 7.03±0.22; n=9) and KCl (Emax 74.14±5.61%; −LogIC50 2.12±0.07; n=10)‐induced relaxations were attenuated (P<0.0001) by removal of the endothelium (Emax 8.21±5.39% and 11.56±8.49%, respectively; n=6 – 7). Indomethacin (10 μM) did not alter ACh‐induced relaxation whereas L‐NOARG (100 μM) reduced this response (Emax 61.7±3.4%, P<0.0001; n=6). The combination of ChTx (50 nM) and apamin (30 nM) attenuated the L‐NOARG‐insensitive component of ACh‐induced relaxation (Emax: 15.2±10.5%, P<0.002, n=6) although these arteries retained the ability to relax in response to 100 μM SIN‐1 (Emax 127.6±13.0%, n=3). Exposure to BaCl2 (30 μM) and Ouabain (1 mM) did not attenuate the L‐NOARG resistant component of ACh‐mediated relaxation (Emax, 76.09±8.92, P=0.16; n=5). KCl‐mediated relaxation was unaffected by L‐NOARG+indomethacin (Emax; 68.1±5.6%, P=0.33; n=5) or the combination of L‐NOARG/indomethacin/ChTx/apamin (Emax; 86.61±14.02%, P=0.35; n=6). In contrast, the combination of L‐NOARG, indomethacin, ouabain and BaCl2 abolished this response (Emax, 5.67±2.59%, P<0.0001, n=6). The characteristics of KCl‐mediated relaxation differed from those of the nitric oxide/prostaglandin‐independent component of the response to ACh, and were endothelium‐dependent, indicating that K+ does not act as an EDHF in human subcutaneous resistance arteries.

Preservation of vascular function in rat mesenteric resistance arteries following cold storage, studied by small vessel myography

The use of isolated blood vessels to investigate the physiological and pharmacological control of the vasculature is limited by the requirement to use freshly isolated vessels. Hence, the aim of this study was to determine whether vascular smooth muscle and endothelial cell function could be preserved in resistance arteries by storing them in physiological salt solution (PSS) at 4°C. Third order mesenteric resistance arteries (mean internal diameter 237±6 μm) were dissected from the mesenteric bed of male Cob‐Wistar rats. The vessel segments were mounted in a small vessel myograph for measurement of isometric tension, and equilibrated at their optimum resting force. Contractile responses to noradrenaline (NA; 1×10−9–3×10−5 M), phenylephrine (PE, 1×10−9–3×10−5 M), potassium chloride (KCl; 2.5–140 mM) and endothelin (ET‐1, 1×10−11–3×10−7 M) and relaxant responses to acetylcholine (ACh; 1×10−9–3×10−5 M) and 3‐morpholinosydnonimine (SIN‐1; 1×10−9–1×10−4 M) were obtained in arteries, immediately after dissection (day 0) and following one to four days storage (day 1–day 4). All arteries produced concentration‐dependent contractions in response to each of the vasoconstrictors. There were no significant differences in the magnitude or sensitivity (pD2) of the vasoconstrictor responses between fresh and stored vessels. Arteries precontracted with NA to approximately 80% of the maximum response, relaxed in a concentration‐dependent manner in response to ACh and SIN‐1. Vessel storage for up to three days resulted in no change in response to ACh or SIN‐1. Vessels analysed after four days of storage demonstrated a significant increase in sensitivity to ACh and SIN‐1 (−logIC50 (M) values; ACh; day 0, 7.46±0.13 vs day 4, 7.97±0.11, P<0.01 and SIN‐1; day 0, 4.87±0.10 vs day 4, 5.52±0.08, P<0.01). There was also a significant increase in the maximum relaxant response to ACh after four days of storage (% relaxation; day 0, 92.65±2.84 vs day 4, 100.36±0.36, P<0.05). These results demonstrate that small resistance arteries remain viable if stored in PSS at 4°C for up to four days, with no loss in endothelial cell function. The altered sensitivity to the vasodilators on day 4 suggests that vessels should only be stored for up to three days following dissection for analysis of functional responses.

Angiotensin II-stimulated cortisol secretion is mediated by a hormone-sensitive phospholipase C in bovine adrenal fasciculata/reticularis cells

Conditions have been established for the incorporation of [3H]inositol ([3H]Ins) into the phosphoinositides of cultured bovine adrenal zona fasciculata/reticularis (ZFR) cells. Stimulation of these prelabelled cells with angiotensin II (10(-11)-10(-7) M AII) resulted in the dose-dependent (max. 16-fold at 10(-7) M AII), time-dependent formation of water-soluble radiolabelled products which show the same chemical and chromatographic properties as [3H]InsP, [3H]InsP2 and [3H]InsP3 standards. The results of the time-course studies of the changes in these products are consistent with the view that AII rapidly (less than 15 s) induces the activation of a polyphosphoinositide-specific phospholipase C. The action of this phospholipase on the polyphosphoinositides is sustained throughout 15 min of stimulation. The dose dependency of this response correlates closely with cortisol output and is reduced (to 52%, P less than 0.00005), but not abolished, in the absence of extracellular Ca2+. To our knowledge these results are the first clear demonstration that AII stimulates a polyphosphoinositide-specific phospholipase C in bovine ZFR cells.

Acetylcholine stimulates cortisol secretion through the M3 muscarinic receptor linked to a polyphosphoinositide-specific phospholipase C in bovine adrenal fasciculata/reticularis cells

Zona fasciculata/reticularis (ZFR) cells, isolated from the bovine adrenal cortex, secreted cortisol in response to acetylcholine (AcCh). The response was present in freshly isolated cells and in cells maintained in primary culture, reaching a maximum after 48-72 h and thereafter declining. Cells maintained in primary culture for 72 h secreted cortisol with an ED50 at 1.2 x 10(-6) M. The potent inhibition of AcCh-stimulated secretion by atropine, and the relative ineffectiveness of nicotine or nicotinic antagonists, were consistent with a predominantly muscarinic response to AcCh in these cells. A selective M1-receptor agonist, McN-A-343, had no effect on cortisol secretion whereas the M3 antagonist, hexahydro-sila-difenidol, produced a dose-dependent inhibition of AcCh-stimulated cortisol secretion. These findings are consistent with AcCh mediating its effects on cortisol secretion through an M3 receptor. While AcCh had no effect on cAMP formation, a dose-dependent increase in [3H]phosphoinositols (identified using high-performance liquid chromatography (HPLC)) occurred in a manner that was not dependent on an influx of extracellular Ca2+. Detailed HPLC analysis of the formation of 3H-labelled phosphoinositols and glycerophosphoinositols from pre-labelled cells over the period 0-15 min showed that the earliest significant rise was in Ins(1,4,5)P3 at 5 s, followed by later rises in InsP1, InsP2 and Ins(1,3,4)P3. Additional studies using cells loaded with fura-2 indicator revealed a 1.6-fold increase in [Ca2+]i from a mean resting value of 75 nM in response to 10(-4) M AcCh. Furthermore, the rise in Ca2+ was not abolished by lowering extracellular Ca2+ to resting cytosolic levels, suggesting the mobilisation of an intracellular pool. These observations indicate that AcCh promotes rapid activation of a Ca2(+)-independent and polyphosphoinositide-specific phospholipase C, and that the Ins(1,4,5)P3 formed releases Ca2+ from an intracellular pool. The stimulation by AcCh of this signal transduction mechanism is consistent with our conclusion, based on the effects of the selective muscarinic agonist and antagonist on cortisol secretion, that the AcCh receptor is of the M3 subtype. We conclude that AcCh, acting through an M3 receptor coupled to phospholipase C, regulates cortisol secretion at the cellular level in bovine adrenal ZFR cells.

The sensitivity of human blood platelets to the aggregating agent ADP during different dietary sodium intakes in healthy men

SummaryWe have investigated the effect of varying sodium intake on the renin-angiotensin system, ADP-induced patelet aggregationin vitro, and blood 5-HT concentrations in 9 male volunteers.Systolic blood pressure was slightly reduced during a low sodium diet, whereas the diastolic pressure remained unchanged. Plasma renin activity and aldosterone concentration both fell significantly when sodium intake was increased; plasma angiotensin lI concentraion also fell, but not significantly.There was a significant fall in haematocrit after an increased sodium intake, but there was no change in the whole-blood platelet count after correcting for this. There were no significant changes in either total (i. e. PRP) or platelet 5-HT concentrations.The extent of platelet aggregation induced by 5 and 20 μmol · 1−1 of ADP increased significantly when dietary sodium intake was increased. When compared with low or normal sodium intakes, lower concentrations of ADP were required to produce 50% of maximum aggregation after a high sodium intake. The 5-HT2, receptor antagonist ketanserin (1 μmol · 1−1in vitro) reduced the extent of aggregation induced by 5 μmol · 1−1 ADP after the volunteers had taken a high sodium diet, whereas the angiotensin 11 receptor anatgonist saralasin (1 nmol-1−1) increased the rate of aggregation after the low sodium diet.Thus, during a high sodium intake, human platelets become more sensitive to the aggregating agent ADP It is possible that this effect is mediatedvia platelet 5-HT2 receptors, since ketanserin abolished the increase in salt-induced aggregation seen with 5 μmol · 1−1 ADP.

Intravenous captopril treatment in patients with severe cardiac failure.

The effect of intravenous captopril was studied in 26 patients with severe chronic heart failure. Fourteen patients received a 25 mg intravenous bolus dose and 12 patients were given a series of incremental intravenous doses over the range 0.3125-45 mg. After the 25 mg bolus dose there was a rapid reduction in systemic vascular resistance and systemic blood pressure. The effect was greatest five minutes after the dose when cardiac output was increased by 20%. Mean right atrial pressure and pulmonary end diastolic pressure fell more slowly and reached their nadir 60 minutes after administration. Plasma free captopril concentration was significantly correlated with percentage reduction in systemic vascular resistance 15 minutes after the bolus injection, but was not correlated with either changes in right atrial or pulmonary artery pressures. With the series of incremental doses there was a progressive fall in systemic vascular resistance until a cumulative dose of 5.0 mg was reached; beyond this there was no further significant change. The rapid response to intravenous captopril indicates that it may be useful in the treatment of patients with severe heart failure who require intensive treatment. After intravenous injection of captopril haemodynamic responses in patients with heart failure were greatest at plasma concentrations of 100 g/ml to 150 ng/ml. This is considerably higher than the plasma free captopril concentrations found after conventional oral doses of captopril.