A sensitive reverse transcriptase polymerase chain reaction assay for measuring the effects of dehydration and gestation on rat amounts of vasopressin and ocytocin mRNAs.

This study describes a competitive reverse transcriptase polymerase chain reaction (RT-PCR) method for assaying the amounts of vasopressin (AVP) and ocytocin (OT) mRNAs in the rat hypothalamus and uterus. Despite the low concentrations of these mRNAs, the RT-PCR method readily measured both AVP and OT mRNAs in the same sample. A common internal standard for both reactions was designed to quantify the reaction. Both AVP and OT mRNAs were readily quantified in a 75 ng sample of total RNA from the hypothalamus. Water deprivation stimulated AVP mRNA production 3-fold and OT mRNA production 1.7-fold in the hypothalamus. Gestation only influenced the amount of OT mRNA in the hypothalamus (3-fold increase) and uterus (38-fold increase). The amount of AVP mRNA in the hypothalamus remained unchanged and no AVP mRNA was detected in the uteri of either non-pregnant or pregnant rats. This competitive RT-PCR is a powerful tool that provides rapid and precise assays of AVP and OT mRNAs.

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.

Trandolapril decreases prevalence of ventricular ectopic activity in middle-aged SHR.

BACKGROUND: Although severe arrhythmias are still a major problem in patients with left ventricular hypertrophy (LVH), the relationship between ventricular remodeling and its regression or prevention, and the prevalence of ventricular premature beats (VPB) or more sustained arrhythmias are still poorly explored in hypertensive heart disease. METHODS AND RESULTS: Holter monitoring was used to quantify supraventricular premature beats and VPB and heart rate (HR) in middle-aged spontaneously hypertensive rats (SHR) and Wistar rats treated for 3 months with trandolapril (ACE inhibitor, 0.3 mg/kg per day). Hypertrophy and fibrosis were morphometrically determined. Statistical analysis was performed with the use of simple regression and multivariate data analysis (cluster and correspondence analysis). SHR have higher cardiac mass and fibrosis, more VPB, and a decreased HR. Cluster analysis demonstrated that trandolapril was only effective in SHR. Trandolapril significantly reduced cardiac hypertrophy, fibrosis, and VPB incidence and increased the HR. Simple regression analysis showed that VPB incidence correlated to both hypertrophy and fibrosis. Correspondence analysis evidenced a strong correlation between hypertrophy, fibrosis, and VPB, but only for severe hypertrophy, and the correlation disappeared for moderate hypertrophy. CONCLUSIONS: After trandolapril treatment, the regression of VPB incidence not only is linked to hypertrophy and fibrosis, but additional causal factors also are involved including the myocardial phenotype and new calcium metabolism. Our model of Holter monitoring in conscious middle-aged SHR and multivariate data analysis might be useful in correlating myocardial structural modifications and ectopic activity.

Morphometric detection of incipient glomerular lesions in diabetic nephropathy in rats. Protective effects of ACE inhibition.

BACKGROUND: Glomerulosclerosis is the main renal lesion complicating diabetes in humans and in experimental models. Angiotensin I-converting enzyme (ACE) inhibitors are effective in preventing the development of diabetic nephropathy. Incipient glomerular lesions were explored in streptozotocin-diabetic rats at a stage when glomerulosclerosis was not yet established. The modulation of such early glomerular lesions by a new ACE inhibitor (Trandolapril (T) at high or low doses was assessed. EXPERIMENTAL DESIGN: Five groups of rats were designed as follows: (a) nondiabetic control rats, (b) diabetic rats, (c) diabetic rats treated with 0.1 mg/kg/day of T, (d) diabetic rats treated with 1 mg/kg/day of T, and (e) nondiabetic rats treated with 1 mg/kg/day of T. The rats were killed at 1, 3, and 6 months after the beginning of the treatment. The kidneys were studied using a powerful morphometric technique at optical microscopic level with an image analyzer to measure the following glomerular parameters to assess the development of incipient glomerular lesions: (a) total glomerular surface area, (b) glomerular tuft surface area, (c) mesangial surface area, (d) ratio of the mesangial surface area to the glomerular tuft surface area, and (e) mean thickness of the Bowman's capsule. In parallel, albuminuria was measured. RESULTS: The results showed the development of glomerular hypertrophy in parallel with the increase in glomerular mesangial domain and in albuminuria with diabetes. They also demonstrated that ACE inhibitor given at a high dose is significantly effective in reducing glomerular hypertrophy and the expansion of the mesangial domain. ACE inhibitor given at a low dose tended to reduce glomerular hypertrophy and the expansion of the mesangial domain. Furthermore, ACE inhibitor at both doses completely abolished the albuminuria increase, maintaining the levels of albuminuria within the range of young nondiabetic rats. CONCLUSIONS: Using morphometric image analysis, incipient glomerular changes can be detected before glomerulosclerosis is patent in experimental diabetes. Moreover, they can be easily and reliably quantified by this technique, allowing comparison among experimental groups. These changes can be prevented by ACE inhibition.

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

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 = 50 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

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

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.

Effects of subchronic treatment with trandolapril and enalapril on cardiovascular morphologic alterations in the aged spontaneously hypertensive rat with heart failure.

The effects of a 3-month treatment period with the angiotensin-converting enzyme (ACE) inhibitors trandolapril (0.3 mg/kg/day, p.o.) and enalapril (10 mg/kg/day, p.o.) on hemodynamics, cardiac hypertrophy, and vascular structures were examined in old spontaneously hypertensive rats (SHRs) (24 months at the end of treatment) presenting with congestive heart failure. During the course of treatment, the mortality rate was lower in the two treated groups than in the control group. At the end of treatment, serum ACE activity was inhibited by 63 and 33% by trandolapril and enalapril, respectively, but the decrease in blood pressure they induced was not significant. The atrial natriuretic factor(ANF) plasma levels and cyclic GMP urine excretion were about 10-fold and 3-fold higher, respectively, in old SHRs than in old Wistar rats. These values were markedly decreased by both ACE inhibitors. The ventricular hypertrophy was greatly decreased by both compounds (-24% by trandolapril and -26% by enalapril). In the aorta, the media hypertrophy was significantly decreased and nuclear density increased to a similar extent by both ACE inhibitors. In the mesenteric artery, trandolapril treatment induced a complete regression of the media hypertrophy and a marked decrease in extracellular matrix surface. In addition, the collagen network appeared less dissociated in the treated animals. Similarly the nuclear density was increased and the surface of cell nuclei was decreased by trandolapril. Enalapril appeared much less potent on these parameters. These data demonstrate that treatment with trandolapril of aged SHRs presenting with heart failure results in an increase in survival of the animals and a marked regression of cardiac and vascular hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)

Regression of left ventricular hypertrophy by converting enzyme inhibition in 12-15-month-old spontaneously hypertensive rats: effects on coronary resistance and ventricular compliance in normoxia and anoxia.

The effects of trandolapril, a converting enzyme inhibitor (CEI), on left ventricular (LV) diastolic stiffness and coronary vascular resistance (CVR), were studied with an isolated heart preparation in 15-month-old spontaneously hypertensive rats (SHR). The hypertensive animals were treated for 3 months with trandolapril (0.3 mg/kg/day) (SHRT), and compared with untreated age-matched Wistar-Kyoto rats (WKY) and SHR. Trandolapril treatment resulted in 15% diminution in blood pressure (BP). In contrast, it completely normalized left ventricular (LV) weight. Untreated SHR, as compared with WKY, had a dilated LV and increased diastolic tissue stiffness. Trandolapril had no effect on either chamber or tissue stiffness. Five-minute anoxia resulted in the same dramatic increase in chamber stiffness in every experimental group. During anoxia, as during normoxia, tissue stiffness was still greater in SHR than in WKY. A major effect of CEI was to normalize the tissue stiffness of SHR under anoxia. Coronary vascular resistance (CVR) was increased in SHR as compared with WKY. Trandolapril improves CVR and significantly shifts the coronary pressure flow curve to the dilatory side. Both collagen concentration (approximately 2 mg/g) and the content in slow V3 myosin isoform, used as biologic markers of cardiac senescence, were the same in the three experimental groups, but higher than in young hearts. Trandolapril had no effect on these parameters. In semisenescent SHR, despite having rather slight effect on arterial pressure, trandolapril completely normalized LV weight. In addition, collagen content and its physiologic counterpart, tissue stiffness, were unaffected by 3-month treatment with trandolapril. Nevertheless, the anoxia-induced increase in LV tissue stiffness was improved by trandolapril in parallel with reduction in LV hypertrophy (LVH).

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.

Kappa agonists and vasopressin secretion.

The effects of opiates on vasopressin secretion have been controversial for many years. This is probably due to the existence of different types of opioid receptors and to the lack of specificity of the compounds used. Specific kappa agonists, which have been described recently, produce a marked diuretic effect without any associated increase in electrolyte elimination. They seem to exert their effects through an interaction with kappa receptors situated on nerve terminals and/or pituicytes. These receptors could be directly coupled to L-type calcium channels, their activation leading to a decrease in the effectiveness of action potentials to evoke vasopressin secretion from nerve terminals in the neurohypophysis. This mechanism of action may explain the decrease in plasma vasopressin levels induced by kappa agonists.

PAF receptor. 2. Quantitative hydrophobic contribution of the agonist's etheroxid chain.

In order to undertake a quantitative assessment of the contribution of the hydrophobic effect of the etheroxid chain to agonistic activity, it was necessary to include in the calculated data a highly lipophilic platelet-activating factor (PAF) analogue. The synthesis of such a compound--racemic 1-O-docosyl-2-O-acetylglycero-3-phosphocholine (C22 PAF)--is described. The regression analysis performed with data of 14 etheroxid analogues (reviewed by Godfroid et al., 1987), combined with data obtained with C22 PAF, is significant with respect to a parabolic evolution between lipophilicity of the chain and the logarithm of relative platelet stimulation. This result is characteristic of a hydrophobic interaction between the agonist and the PAF receptor.

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.

Comparison of the effects of the ace inhibitors trandolapril and enalapril on phlogogen induced foot pad oedema in the rat.

Two angiotensin converting enzyme (ACE) inhibitors, trandolapril and enalapril, were compared for their effects on rat food-pad oedema induced by carrageenin, bradykinin, dextran and platelet activating factor (PAF). Trandolapril (0.03-30.0 mg/kg, per os) potentiated carrageenin-induced oedemas. Enalapril produced the same effect at 3-10 fold higher doses (0.3-30.0 mg/kg per os). Both ACE inhibitors were equiactive in potentiating bradykinin-induced oedema. Neither compound affected dextran-induced oedema. In marked contrast PAF-induced oedema was reduced by both ACE inhibitors, trandolapril being approximately 10 fold more active than enalapril. The observed differences in potency between the two ACE inhibitors corresponded with their previously described actions on inhibition of plasma and tissue ACE and in inducing hypotension. The results suggest a crucial role of kinins in the oedemagenic response to carrageenin. The reason why the ACE inhibitors reduced PAF-induced oedema is not clear, but could involve peripheral vasodilation.

4-Hydroxy-3-quinolinecarboxamides with antiarthritic and analgesic activities.

A series of 4-hydroxy-3-quinolinecarboxamides has been synthesized and evaluated by the oral route as antiinflammatory agents in carrageenin-induced foot edema and adjuvant-induced arthritis and as analgesic agents in the acetic acid induced writhing test. Among the most active molecules, some have shown both analgesic and acute antiinflammatory activities. Others, such as compounds 24, 37, and 52, were only powerful peripherally acting analgesics. Compound 52, being active at 1 mg/kg (ED50), is the most potent compound in the series. Some analogues, substituted in the 2-position by an alcohol, ester, or amine function, displayed potent antiarthritic activity in the same range as that of piroxicam and were also active in acute tests of inflammation and nociception. They inhibited the activity of both cyclooxygenase and 5-lipoxygenase at micromolar concentrations. Compound 102 (RU 43526) showed potent antiarthritic activity (adjuvant-induced arthritis, ED50 = 0.7 mg/kg, po) and gastrointestinal tolerance (ED100 greater than 250 mg/kg, po) and thus it is presently undergoing an extensive pharmacological evaluation.

Structure-activity relationship in PAF-acether. 4. Synthesis and biological activities of carboxylate isosteres.

The synthesis and biological characterization of some 3-carboxylate isosteres of PAF-acether structurally modified in positions 1 (ether, carbamate), 2 (acetoyl, ethoxy), and 3 (chain length and polar head group) are reported. All derivatives present antagonist activities against PAF-acether-induced effects in vitro (platelet aggregation) and in vivo (bronchoconstriction and thrombocytopenia in guinea pig and, to a lesser extent, hypotension in rat). The functional modifications presented here do not modify dramatically the potency of antagonist activities, and there is no enantioselectivity. All of the isosteres are specific PAF-acether antagonists, except the 1-carbamoyl analogue, which is also potent against acetylcholine-induced hypotension and bronchoconstriction.

Bronchospasmolytic effects of RU 42173.

The bronchodilator properties of RU 42173, a new beta-adrenergic stimulant with an original structure, as a cyclic analogue of an arylethanolamine, have been evaluated on different in vitro and in vivo models and compared with those of salbutamol and isoprenaline. RU 42173 equipotently inhibited histamine-, acetylcholine-, and KCl-induced contractions in isolated guinea pig trachea or small bronchus and in isolated human bronchus. When administered to guinea pigs by the IV or aerosol route, RU 42173 dose-dependently inhibited bronchospasm induced by histamine, acetylcholine, and methacholine. It also inhibited PAF-induced bronchoconstriction and PAF-induced hyperreactivity to histamine. Moreover, RU 42173 had a rapid onset and prolonged duration of action. The potency of RU 42173 was similar to that of salbutamol.

Structure-activity relationship in PAF-acether. 3. Hydrophobic contribution to agonistic activity.

The synthesis of some selected PAF-acether homologues with an alkoxy-chain length from C1 to C20 in position 1 is described. All agonist activities are closely correlated among themselves and with the calculated fatty-chain hydrophobicity. After a discussion on recent published results and comparison with our data, we conclude that the ether oxide function is absolutely essential at the glycerol 1-position for potent agonist activity and that potency correlates well with hydrophobicity parameters. We indicate the importance of steric and configurational constraints.

Release of prostaglandins E and F in an algogenic reaction and its inhibition.

E and F prostaglandins were measured by radioimmunoassay in the peritoneal fluid of rats which had been injected with an irritant, acetic acid. The considerable increase recorded 5 min after the injection virtually disappeared in 90 min. For the first 15 min the PGE2 level was twice that of PGF2 alpha, the levels then equalized and after 90 min the PGE2 level was less than that of PGF2 alpha. This balance between PGEs, which are hyperalgesic, and PGF2 alpha, which has often been shown to be a PGE antagonist, could regulate defence mechanisms. An examination of cells collected by washing the peritoneum revealed a large decrease between 15 and 30 min after injection of the irritant and suggested that the prostaglandins could be produced by neutrophil polynuclear cells but also by destruction of macrophages. Various types of prostaglandin biosynthesis inhibitors (non-steroid anti-inflammatory agents, non-narcotic analgesics and some monoamino-oxidase inhibitors and antioxidants) prevented prostaglandin release. Their activity on release paralleled their activity on acetic acid-induced writhing.

Inhibition of prostaglandin biosynthesis by non-narcotic analgesic drugs.

The existence of a relationship between inhibition of prostaglandin biosynthesis and analgesic or anti-inflammatory activity was investigated in the case of the non-narcotic analgesics glafenine, floctafenine and clometacine, in comparison to indomethacin and acetylsalicylic acid. These compounds inhibit prostaglandin biosynthesis from arachidonic acid in a guinea-pig lung homogenate as strongly as indomethacin. On its biosynthesis in rat epididymal tissue stimulated by noradrenaline, glafenine equals indomethacin inhibitory potency, whereas floctafenine and clometacine are less active. Acetylsalicylic acid is the least active in both preparations. In vivo, prostaglandin biosynthesis induced in rat peritoneal fluid by injection of acetic acid is inhibited by the 5 drugs, ranked as follows: floctafenine greater than indomethacin greater than glafenine greater than clometacine greater than acetylsalicylic acid. The pharmacological profile of glafenine, floctafenine and clometacine is characterized by a relatively strong effect on acetic acid writhing and a relatively weak effect on carrageenin oedema, U.V. erythema and adjuvant arthritis. The inhibition of prostaglandin biosynthesis seems better correlated with their analgesic activity than with their anti-inflammatory effects. The results show that prostaglandins could play an important role in the genesis of tissulary pain in animals.