Gilles Hamon

Expression of laminin α2 chain during normal and pathological growth of myocardium in rat and human

Objectives: Fibrosis is a classical feature of cardiac hypertrophy. To date changes within the basal lamina during normal and pathological cardiac growth have been poorly investigated. The goal of the present study was to determine if the expression of the muscle specific subunit of merosin (laminin α2 chain) together with that of fibronectin (FN) is modified in the diseased human heart. Laminin α2 chain expression was also investigated during physiological and pathological cardiac growth in the rat. Methods: In ten normal human hearts and ten hearts with idiopathic dilated cardiomyopathy (IDCM), the laminin-α2 and FN mRNA levels were quantified by slot-blot using total RNA and the protein distribution was analysed using an immunofluorescence approach. In Wistar rats, laminin α2 and FN mRNA expression was analyzed using RNase protection assay (RPA) and slot-blot assays. Results: The amount of laminin α2 mRNA did not vary in normal and pathological human hearts whereas it was significantly decreased in renovascular hypertensive rats (−20%) P <0.05 versus normal tissue). The amount of fibronectin mRNA increased in IDMC patients (×2, P <0.05 versus normal tissue), but was unchanged in hypertensive rats. A negative correlation was found between the cardiac laminin-α2 level and the age of the patients whatever the cardiac status. During postnatal development in the rat, a similar decrease in cardiac laminin-α2 level was observed between 3 and 30 weeks of age. Finally, the immunofluorescent approach failed to detect any alteration in laminin α2 distribution within the human myocardium. Conclusion: These data indicate that an imbalance between myocyte hypertrophy and the level of laminin-α2 might contribute to alterations in sarcolemmal properties, which occur during the development of cardiac hypertrophy and its transition to cardiac failure.

Effect of chronic treatment with trandolapril or enalapril on brain ACE activity in spontaneously hypertensive rats.

The aim of the present study was to determine whether the new ACE inhibitor trandolapril was able to inhibit brain ACE activity in spontaneously hypertensive rats (SHRs). Therefore, we have measured ex vivo ACE activity in discrete brain areas of SHRs after a 2-week oral treatment with trandolapril (0.001, 0.01, 0.1 and 1 mg/kg/day). The effects of trandolapril were compared to those of enalapril (10 mg/kg/day), used as a reference compound. Enalapril induced a decrease in ACE activity in brain areas not protected by the blood brain barrier (subfornical organ and median eminence) and in cerebral cortex. Conversely, trandolapril at a dose of 0.01 mg/kg/day and above induced a dose-dependent inhibition of ACE activity in all brain areas assayed, including the supraoptic and paraventricular hypothalamic nuclei, septum, amygdala, hippocampus, cerebellar and cerebral cortex, nucleus of the tractus solitary and caudate nucleus. The inhibition was roughly similar in all brain areas studied. These data suggest that after chronic oral administration in SHRs, trandolapril or its metabolite, in contrast to enalapril or enalaprilat, was able to reach all brain areas of SHRs, including those protected by the blood brain barrier.

Cardiovascular Actions and Tissue-Converting Enzyme Inhibitory Effects of Chronic Enalapril and Trandolapril Treatment of Spontaneously Hypertensive Rats

The angiotensin I-converting enzyme (CE) inhibitors, trandolapril (RU 44570) and enalapril were administered for 2 weeks to SHR at doses (3 and 10 mg/kg/ day, p.o., respectively) that produced important and comparable inhibitions of plasma (84 and 88%), aorta (97 and 88%), and atrium (89 and 82%) CE activities. At these doses, the inhibitory effects of trandolapril and enalapril were nonetheless different on CE in heart ventricle (58 and 72%) and kidney (45 and 85%). In addition, although both drugs reduced blood pressure (BP) and heart hypertrophy, trandolapril was more potent despite a lower dose-ratio. All these parameters were reexamined 1, 3, and 8 days after drug withdrawal: BP returned to control levels within 3 days in enalapril-treated rats, whereas it remained low for at least 8 days in trandolapril-treated animals. The reduction of heart hypertrophy owing to trandolapril was still present 8 days after drug discontinuation. On cessation of treatment, plasma CE increased above controls, ventricle CE returned to control levels within 3 days, whereas atrial and aortic CE activities remained inhibited for 8 days in the enalapril group. In contrast, in trandolapril-treated rats, CE activities in serum and tissues were still inhibited after 8 days. These results demonstrate that at the doses used trandolapril is more potent and longer acting than enalapril.

MECHANISM OF ACTION OF ANGIOTENSIN II ON EXCITATION‐CONTRACTION COUPLING IN THE RAT PORTAL VEIN

1 The action of angiotensin II (At II) has been studied on the electrical and mechanical activity of the vascular smooth muscle of the rat portal vein. 2 At low concentrations (between 5 × 10−10 and 10−9m) At II induces an acceleration of spontaneous action potential (AP) discharge without change in the resting membrane potential. The frequency and size of the associated contractions are simultaneously augmented. Under these conditions the size of the spikes is not affected, thus suggesting that At II triggers the release of Ca from internal stores. 3 The increase in AP discharge rate produced by low concentrations of At II results from an acceleration of the pacemaker potential. Furthermore, in the presence of 10 mm tetraethylammonium (TEA), there is an acceleration of the repolarizing phase of AP. 4 Ouabain (10−3 m) inhibits the increase in rhythmic activity induced by low concentrations of At II (in the presence of 10 mm TEA), thus suggesting that the Na‐K pump is directly or indirectly involved in this action of the peptide. 5 At higher concentrations, At II produces a concentration‐dependent depolarization with an EC50 of 1.2 × 10−8m and a maximum of 10−7m. The associated contraction has an EC50 of 3.3 × 10−8 m and a maximum of 3 × 10−7 m. 6 Ouabain (3 × 10−3 m) depolarizes the cell membrane. Under these conditions, At II (10−7 m) has a slight depolarizing effect, but it still produces a large tonic contraction. 7 It is concluded, that At II acts on different steps of excitation‐contraction coupling, depending on the concentration. At low levels, the peptide mainly accelerates spike discharge, through a mechanism involving the Na‐K pump. At higher concentrations, At II depolarizes the cell membrane. The contraction is then activated by the influx of Ca2+ due to secondary AP discharge and the release of Ca2+ from intracellular stores. Pharmacomechanical coupling has an important role in the triggering of contractions both at high and at low concentrations of At II.

Compared properties of trandolapril, enalapril, and their diacid metabolites.

Summary The effects of 14-day trandolapril or enalapril treatment of spontaneously hypertensive rats (SHRs) were studied on blood pressure and angiotensin-converting enzyme (ACE) activity measured ex vivo in various organs. Both ACE inhibitors caused dose-dependent decreases in blood pressure and ACE activity, trandolapril being 30− and 400− to 1,000-fold more active than enalapril on blood pressure and ACE activity, respectively. However, comparison of ACE inhibitory activities of the diacid forms of trandolapril and enalapril, i.e., trandolaprilat and enalaprilat, measured in vitro on various tissues, showed that trandolaprilat was only three- to fivefold more active than enalaprilat. To understand the reasons for such discrepancies between ex vivo effects of ACE inhibitors and in vitro actions of their diacid metabolites, we measured the lipophilicities of the compounds and investigated the possibility that trandolapril could display an ACE inhibitory effect by itself. Trandolaprilat was found to be far more lipophilic than enalaprilat, as shown by reverse-phase high-performance liquid chromatography studies performed at pH 7.4 (log kw7.4 = 1.487 vs. 0.108). In addition, trandolapril was practically as active in vitro as its diacid metabolite (IC50 = 2.5 vs. 1.35 nM) in inhibiting ACE activity in the aorta, whereas enalapril was practically devoid of any effect (IC50 = 240 nM). Measurements of relative affinities of inhibitors or metabolites for purified human renal ACE showed that trandolapril displayed about 20% of the affinity of its diacid metabolite (IC50 = 15 vs. 3.2 nM): enalaprilat affinity (34 nM) was within the same range as those of trandolapril and trandolaprilat, whereas enalapril displayed a very low affinity for the purified enzyme (IC50 μ M). It appears that several factors, including differences in potency of diacid compounds in inhibiting ACE, lipophilicity, and potency of the prodrugs to directly inhibit ACE, may account for the greater potency of trandolapril treatment compared with that of enalapril in decreasing blood pressure and inhibiting tissue ACE activity.

Renal and haemodynamic responses to a novel kappa opioid receptor agonist, niravoline (RU 51,599), in rats with cirrhosis

Because renal water retention is a complication of cirrhosis, niravoline (RU 51,599), a novel kappa (κ) opioid receptor agonist which is known to cause a water diuresis under normal conditions, may be useful in the therapy of chronic liver diseases. Thus, the present study examined the effects of niravoline on renal function in rats with cirrhosis. Urine was collected during the 2 h period following the administration of vehicle (saline) in one group of animals or niravoline (3 mg/kg, i.v.) in auother group. Urinary and plasma osmolality were measured prior to and 2 h after niravoline in a third group of animals. Urine flow and natraemia were significantly higher after niravoline (147±12 μL/min and 153±2 mmol/L, respectively) than after vehicle (27 ± 7 μL/min and 146 ± 1 mmol/L, respectively). Niravoline significantly decreased urinary osmolality and significantly increased plasma osmolality and free water clearance. This substance did not significantly change urinary sodium excretion. In conclusion, this study shows that niravoline, a κ opioid receptor agonist, induced a water diuresis in rats with cirrhosis.

Effects of 4 weeks of treatment with trandolapril on renal hypertension and cardiac and vascular hypertrophy in the rat.

Summary The effects of the angiotensin-converting enzyme inhibitor trandolapril were studied using a Goldlatt (two-kidney, one-clip) rat model of renovascular hypertension after 4 weeks of oral treatment at 0.3 or 1 mg/kg/day. The effects of trandolapril on blood pressure and on cardiac and vascular hypertrophy were analyzed in comparison with the control group. Trandolapril produced a rapid, dose-dependent decrease in blood pressure, which plateaued after 2 weeks of treatment. Complete normalization of blood pressure was observed at a daily dose of 1 mg/kg. Dose-dependent inhibition of cardiac hypertrophy was also observed, heart: body weight ratio being decreased by 17 and 30% at 0.3 and 1 mg/kg, respectively, leading to a normalization of this parameter at the higher dose compared with normotensive controls. Similarly, trandolapril produced a marked decrease in vascular wall hypertrophy in both the mesenteric artery and the aorta. Indeed, complete normalization of media thickness was observed, compared with the normotensive control group, at 1 mg/kg of trandolapril. These results show that short-term treatment with trandolapril can induce complete regression of cardiac and vascular hypertrophy, which is associated with the development of renal hypertension.

Effect of chronic converting-enzyme inhibition on kidney function of senescent hypertensive rats.

Summary The age-related changes in the structure and the function of the kidney and the effect of chronic inhibition of angiotensin-converting enzyme (ACE) activity on these alterations were assessed in senescent, genetically hypertensive rats. Mean blood pressure was unchanged between 6 and 21 months, being 136 ± 10 and 135 ± 21 mm Hg, respectively. Hypertrophy of the glomeruli with a high incidence of glomerulosclerosis was reported in the 21-month-old animals. Renal blood flow, glomerular filtration rate, and filtration fraction were reduced between 6 and 21 months, whereas albuminuria and cGMP excretion were markedly enhanced with aging. Chronic ACE inhibition by administration of 0.3 mg/kg/day trandolapril from 18–21 months increased the life expectancy of the animals without affecting their mean blood pressure. The incidence of glomerular lesions and the excretion of enzymes that reflected the integrity of tubular and glomerular cells were not altered by ACE inhibition. On the other hand, the filtration fraction was restored in the 21-month-old treated animals, and the age-related albuminuria and rise in cGMP excretion were prevented by ACE inhibition. These results indicated that ACE inhibitor administered at the end of the life of senescent hypertensive rats was able to prevent some of the age-related changes in kidney function when glomerulosclerosis was already present.

Trandolapril dose-response in spontaneously hypertensive rats : effects on ACE activity, blood pressure, and cardiac hypertrophy

Summary The dose-related effects of trandolapril on serum angiotensin-converting enzyme (ACE) activity, blood pressure and cardiac hypertrophy were studied after 2-week oral treatment in adult spontaneously hypertensive rats. Trandolapril caused a dose-dependent decrease in mean blood pressure at doses of 0.03–3 mg/kg. Inhibition of serum ACE was demonstrated by even the lowest dose (-9% at 0.003 mg/kg), was about 40% at 0.03 mg/kg, and rose to 84% at 3 mg/kg. Regression of cardiac hypertrophy (heart: body weight) was seen at doses of trandolapril as low as 0.03 mg/kg (-5.1%), which was also the minimal effective antihypertensive dose. Clear dose-response curves were observed for trandolapril from 0.03 mg/kg upwards with respect to hypotensive effect and regression of cardiac hypertrophy, which were not seen with enalapril. Enalapril had no significant effect on plasma ACE activity except at the highest dose (10 mg/kg), despite demonstrating hypotensive effects with smaller doses. These results indicate that trandolapril is a more potent (by a factor of about 30) inhibitor of ACE than enalapril (only 34% inhibition at 10 mg/kg) and causes a greater degree of regression of cardiac hypertrophy.

Study of the vasodilating activity of salbutamol on dog coronary arteries. Unexpected effects of methylene blue.

The vascular relaxant effect of salbutamol and its dependence on the endothelium were studied in the isolated dog coronary artery, precontracted with prostaglandin F2 alpha. Salbutamol induced a concentration-dependent relaxation which was partially inhibited by removal of endothelial cells. Atenolol 10(-6) mol/l, a beta 1-selective antagonist, inhibited the relaxant effect of salbutamol both in the presence and in the absence of endothelium. Conversely, ICI 118,551 10(-6) mol/l, a beta 2-selective antagonist, antagonized the response to salbutamol only in intact vessels. Methylene blue amplified markedly the relaxation to salbutamol but only in denuded rings. Therefore, the vasodilating effect of salbutamol on large coronary arteries seems to result from the stimulation of both, beta 1-receptors on smooth muscle cells and beta 2-receptors on endothelial cells, demonstrating the existence of the two types of adrenoceptors in the wall of large dog coronary arteries. In addition, the effect obtained with methylene blue in this study shed some doubts on its specificity as a guanylate cyclase inhibitor.