Oestrogen modulates cardiac ischaemic remodelling through oestrogen receptor-specific mechanisms.

AIM: Observational and clinical studies suggest different responses upon sex hormone replacement therapy in ischaemic heart disease. Few studies, however, have examined the impact of oestrogen receptor-dependent mechanisms on the extent of injury after myocardial infarction (MI). Therefore, we set out to evaluate the effect of oestrogen (E2) replacement on infarct size and remodelling, and the respective role of the oestrogen receptors (ER)alpha and -beta in this process, using ERalpha- and ERbeta-deficient mice. METHODS: Wild type (WT) (ERalpha(+/+) and ERbeta(+/+)), ERalpha-deficient (ERalpha(-/-)) and ERbeta-deficient (ERbeta(-/-)) mice were ovariectomized and subsequently supplemented with E2 or placebo using subcutaneous 60-day release pellets. MI was induced by left coronary artery ligation. Two weeks following MI, haemodynamic function was assessed and infarct size was determined. RESULTS: There was no significant difference in infarct size between E2- or placebo-treated WT (ERalpha(+/+) and ERbeta(+/+)) mice. Surprisingly, E2 treatment did result in smaller infarct sizes in ERalpha(-/-) mice, but increased the infarct size in ERbeta(-/-) mice. Increase of the left ventricular mass post-MI was significantly larger in the E2-treated ERalpha(-/-) animals compared with placebo-treated animals. E2 treatment also significantly increased post-MI mortality in ERalpha(+/+), ERbeta(+/+) and ERalpha(-/-) animals, but not in ERbeta(-/-) mice. CONCLUSIONS: Although E2 modulates the infarct size in ERalpha(-/-), it also appears to be responsible for the higher mortality following MI. ERbeta appears to be the receptor involved in the modulating effects of E2 in the infarcted heart.

Comparison of the antiatherosclerotic effect of tibolone with that of estradiol and ethinyl estradiol in cholesterol-fed, ovariectomized rabbits.

OBJECTIVE: Tibolone is a synthetic steroid with tissue-specific estrogenic, progestogenic, and androgenic properties. The drug relieves climacteric symptoms and prevents osteoporosis but does not stimulate the endometrium. We have previously shown that in laboratory animals tibolone inhibits the atherogenesis induced by a high-cholesterol diet. Therefore, we compared the antiatherosclerotic effect of oral tibolone at different dose levels with that of oral 17beta-estradiol (E2) and ethinyl estradiol (EE). DESIGN: Atherosclerotic lesion formation (increase in vessel wall cholesterol deposition and fatty streak formation) was measured in ovariectomized rabbits after 20 weeks on an atherogenic diet (fed daily 80 g of a rabbit chow containing 0.4% cholesterol, 3.75% peanut oil, and 3.75% coconut oil) in eight groups: group 1, placebo (n = 35); group 2, control (n = 34) received normal rabbit chow; group 3, E2 group (E2 4 mg, n = 12); group 4, EE group (EE 60 microg, n = 10); and groups 5-8, tibolone (6 mg, n = 12; 2 mg, n = 13; 0.6 mg, n = 25; and 0.15 mg, n = 11, respectively). During the study, blood samples were obtained for the evaluation of plasma triglycerides, cholesterol, lipoproteins, and glutamate pyruvate transaminase. After 20 weeks, the animals were killed, and cholesterol concentration and the formation of fatty streaks in the wall of the aortic arch were evaluated. RESULTS: In the placebo group, the atherogenic diet induced a mean increase in total plasma cholesterol concentration from 1.1+/-0.1 mmol/L (control group) to 34.1+/-1.8 mmol/L (mean +/- SE). This resulted in an accumulation of cholesterol in the aortic arch from 48+/-4 (control group) to 608+/-44 nmol/mg protein and in the formation of fatty streaks (41.8+/-3.2% of the surface of the aortic arch was covered with fatty streaks). Tibolone had strong dose-dependent antiatherosclerotic effects. It reduced the accumulation of cholesterol in the aortic arch at doses of 6 to 0.15 mg by 99, 97, 87, and 57% and the formation of fatty streaks by 98, 97, 81, and 38%, respectively. E2 had only a marginal antiatherosclerotic effect, whereas EE showed an effect comparable to that of tibolone at doses of 2 to 0.6 mg. With EE, the accumulation of cholesterol in the vessel wall was reduced by 93% and the formation of fatty streaks by 73%. Mean plasma cholesterol concentrations were also reduced by tibolone (64, 70, 61, and 47%) and EE (57%). This reduction was mainly mediated via a reduction in beta-very-low-density lipoprotein cholesterol. Analysis, however, indicated that the observed antiatherosclerotic effects of tibolone and EE, at least partly, are due to a direct effect on the vessel wall and independent of the changes in plasma cholesterol. At equipotent antiatherosclerotic doses, EE showed a stronger uterotropic effect (measured as the increase in uterine weight) than tibolone. EE increased uterine weight from 0.57 g/kg body weight (BW) (control group) to 3.5 g/kg BW; tibolone at doses of 6, 2, 0.6, and 0.15 mg increased uterine weight to 2.5, 2.8, 2.2, and 1.3 g/kg BW, respectively. CONCLUSION: Tibolone can protect the arterial vessel wall against atherosclerotic lesions induced by a hypercholesterolemic diet. However, it has much less estrogenic effects on the uterus compared with EE at equipotent doses, indicating tissue selectivity for tibolone. The clinical implications of these findings require investigation.

Tibolone prevents atherosclerotic lesion formation in cholesterol-fed, ovariectomized rabbits.

Tibolone (Org OD14), a synthetic steroid with estrogenic and progestogenic/androgenic properties, is clinically effective for the treatment of climacteric symptoms and the prevention and treatment of osteoporosis in postmenopausal women. The effect on atherogenesis, however, is not known. In the current study, we investigated the effect of tibolone in comparison with that of estradiol and norethisterone acetate on atherogenesis in 140 ovariectomized New Zealand White rabbits that had been induced by an atherogenic diet (0.4% cholesterol, 20 weeks). Tibolone at 18, 6, or 2 mg/d orally completely prevented cholesterol accumulation and fatty streak formation in the aorta; the impairment of endothelium-dependent smooth muscle relaxation of the aorta; and complex lesion formation after endothelial denudation in the carotid artery. Tibolone also reduced the increased postovariectomy plasma lipid concentrations. Analysis of the results, however, indicated that a substantial part of the strong, beneficial effects were plasma lipid independent. Compared with subcutaneous estradiol decanoate (150 microgram once weekly) and oral 17beta-estradiol (4 mg/d), the effects of tibolone were more pronounced at equipotent uterotropic activity. Norethisterone acetate (1 mg/d) did not affect atherosclerotic lesion formation. There are no indications that the progestogenic/androgenic properties of tibolone counteracted its atheroprotective effect on the vessel wall. Therefore, tibolone has the intrinsic potential to be a compound that protects the arterial vessel wall against atherosclerotic processes.

Two new closely related rat models with relevance to arterial thrombosis--efficacies of different antithrombotic drugs.

Two thrombosis models in rats are described in which mixed type thrombi are formed at arterial and venous flow rates. The models, containing a silk thread in the aorta and vena cava, respectively, were characterised for the activity of three platelet inhibitors, three thrombin active site inhibitors and five glycosaminoglycans (GAGs). In the two models a similar highly platelet-dependent thrombus developed both in size and composition during the first 10 min after insertion of the silk thread. The thrombotic processes were self-limiting, thus maintaining blood flow, but persisted twice as long in the vena cava model. In both models the thrombus consisted for more than 65% of platelets. Thrombus development under arterial as well as under venous flow conditions was inhibited dose dependently by all tested compounds including aspirin and the synthetic alpha-methyl glycoside copy of the ATIII binding pentasaccharide within heparin, Org31540/SR90107A. Simultaneous fibrin deposition and platelet activation, which represents an essential element of arterial thrombosis, initially dominated-in both models. The gradual thrombus outgrowth, in the cava model, was more sensitive to factor Xa selective anti-coagulants, as is venous thrombosis.

In vitro and in vivo characterization of a calcium modulator of the diphenylalkylamine type with selective coronary dilatory properties.

UNLABELLED: VUF 8929 (N-¿2-[bis(p-fluorophenyl)methoxy]ethyl¿-(2-phenyl)ethylamine maleate, CAS 140890-71-7) is a diphenylalkylamine derivative structurally related to prenylamine. The calcium antagonistic properties of this compound have been studied in in vitro and in vivo systems. VUF 8929 has affinity for the voltage-operated calcium channel. Its pKD for the displacement of [3H]nitrendipine bound to cerebral rat cortex is 6.27 (+/- 0.17). The compound influences the [3H]nitrendipine binding through an allosteric interaction with a site adjacent to the dihydropyridine binding site. Competitive experiments with the additional presence of the phenylalkylamine gallopamil showed that this allosteric site is a property common to diphenyl- and phenylalkylamines. It was further observed that VUF 8929 has a high affinity for calmodulin as it shows high potency in inhibiting the calmodulin mediated activation of PDE. The inhibition of K+ (IC50 0.5 mumol/l)- and noradrenaline (IC50 1.3 mumol/l)-induced contractions of rabbit aorta rings was in the same concentration range as found for the calmodulin inhibitory activity. In vitro platelet aggregation was also inhibited in the same concentration range when washed platelets were used. Thus calmodulin antagonism may contribute to the observed effects on aorta ring contractions and platelets aggregation. Platelet aggregation, however, in media in which albumin was added or in platelet rich plasma was not affected. It is assumed that due to the high lipophilicity, common to many diphenylalkylamines, VUF 8929 has a strong binding to plasma proteins. This may also explain why orally administered VUF 8929 did not affect the alpha 2-induced pressor response in pithed rats and the ex vivo collagen induced aggregation response. The haemodynamic profile in anaesthetized dogs showed that intravenous injected VUF 8929 reduced the workload of the heart while coronary blood flow increases at a dose of 0.3 mg/kg. Reversible occlusion of the coronary artery, which leads to S-T segment elevation and local venous acidosis, were reduced by VUF 8929 indicating that the compound has anti-ischaemic properties. IN CONCLUSION: VUF 8929 is a calcium antagonist which has anti-ischaemic properties, reduces the workload of the heart and increases coronary flow. Due to these properties VUF 8929 is a potential cardioprotective agent.

Hemodynamic profiles of methoxamine and B-HT 933 in spinalized ganglion-blocked dogs.

The hemodynamic profiles of methoxamine and B-HT 933 during a stepwise-increased infusion in spinalized ganglion-blocked dogs show that both compounds induce a dose-dependent increase in arterial blood pressure due to an increase in total peripheral resistance. Methoxamine initially also increases cardiac output, which contributes to the increase in blood pressure. The increased cardiac output is the result of an increased stroke volume, because heart rate is unchanged. B-HT 933 increases neither cardiac output nor heart rate. Because neither compound stimulates the heart directly, the increase in stroke volume induced by methoxamine can be due only to an increased venous return. B-HT 933 appears to be devoid of an effect on the capacitance vessels, because stroke volume and cardiac output are unchanged. The present study suggests that under in vivo conditions, postjunctional alpha 2-adrenoceptors in the capacitance vessels of the dog are not functional.

Protective effects of prostaglandins against ulcerogenic activity of indomethacin during different stages of erosion development in rat stomach. Role of acid and bicarbonate secretion.

Prostaglandin E2 (PGE2) and PGF2 beta decreased the gastric erosive activity of orally administered indomethacin in a dose-dependent manner, when given as a continuous intravenous infusion in the conscious rat. PGE2 protected both during the initial stage of erosion induction and during the later outgrowth to larger erosions. Moreover PGE2 was able to stop the eroding process at any stage as long as the infusion continued. Both PGs were protective only in doses which also reduced the histamine-stimulated acid secretion. PGE2 protected the stomach against indomethacin-induced erosions even in the presence of exogenously administered acid. An infusion of PGE2 stimulated the secretion of bicarbonate in the stomach during some minutes but had no effect during prolonged infusion. These results suggest that, although effects on secretion of acid and bicarbonate were found, these effects cannot be the (only) explanation for the cytoprotective effects observed. Furthermore the protective effect of PGE2 is not confined to any specific stage of the development of indomethacin-induced gastric injury.

Protection by paracetamol against various gastric irritants in the rat.

Four nonsteroid anti-inflammatory drugs (NSAID), indomethacin, phenylbutazone, ibuprofen, and glafenine, caused erosions in the rat stomach in a dose-dependent manner. Paracetamol, which has been shown to protect against the gastric erosive activity of aspirin, reduced the gastric toxicity of indomethacin but was ineffective against the erosive activity of phenylbutazone and glafenine. Only the high erosion score of a large dose of ibuprofen was partly decreased by paracetamol. The gastric damaging effects of necrotizing concentrations of ethanol and sodium hydroxide were strongly reduced by paracetamol, but the erosive activity of hydrochloric acid was only slightly decreased by paracetamol. Thus, although paracetamol protected the gastric mucosa against various noxious agents, this drug was not able to protect against every type of gastric damage. Paracetamol might be protective by stimulating the biosynthesis of prostaglandins in the stomach wall.

The influence of orphenadrine or imipramine on the hypertensive response of physostigmine in the rat.

UNLABELLED: An intravenous infusion of orphenadrine or imipramine to artificially ventilated, urethane anaesthetized rats, completely blocked the physostigmine induced increase in blood pressure and the blood pressure increase induced by electrical stimulation of the posterior hypothalamus; effects mediated via the sympathetic nerve. The noradrenaline induced blood pressure increase was not changed during an infusion with orphenadrine but was markedly depressed during an infusion with imipramine. During an infusion with both orphenadrine or imipramine the pressor response induced by stimulation of the spinal cord were completely blocked in pithed rats. The pattern of the blockade was comparable with the blockade of the pressor response after hypothalamic stimulation. These results show that at least in rats both orphenadrine and imipramine prevents the central stimulatory sympathetic effects on the cardiovascular system by interfering with the sympathetic nervous system. The site of action is discussed. IN CONCLUSION: the present results show that although physostigmine may be helpful in the treatment of central anticholinergic effects caused by overdoses of orphenadrine and imipramine it is of no use for combating the direct toxic effects of both drugs on the cardiovascular system.

The influence of paracetamol on the erosive activity of indomethacin in the rat stomach.

Indomethacin induced erosions in the glandular part of the rat stomach in a dose-dependent manner. Gastric erosions became apparent about 15 min after administration of indomethacin and the damage was maximal at about 4 h. The erosive activity of indomethacin administered subcutaneously was similar to that after oral administration, confirming the data of other authors. The erosive activity of subcutaneously applied aspirin, however, was far less than that of oral administered aspirin and it was not dose dependent. In a dose-dependent manner, paracetamol reduced the incidence of gastric erosions induced with indomethacin; this effect was independent of the route of administration of either drug. Paracetamol was also effective when given 0.5 or 1 h before indomethacin. Orally administered paracetamol also reduced the incidence of gastric erosions induced with aspirin but after subcutaneous administration, paracetamol had no protective effect. The differences between the erosive activities of indomethacin and aspirin are discussed with emphasis on the differentiating influence of paracetamol on the incidence of gastric erosions. Direct contact with the mucosa is apparently more important for the erosive activity of aspirin than for that of indomethacin. Possible mechanisms by which paracetamol exerts its protective activity are proposed.

The influence of physostigmine on respiratory and circulatory changes caused by overdoses of orphenadrine or imipramine in the rat.

Orphenadrine or imipramine were given intravenously as an infusion to spontaneously breathing, anaesthetized rats until respiratory arrest, the primary cause of death for both drugs. Intravenous injections of physostigmine did not prolong survival. Artificial ventilation prolonged survival for orphenadrine and imipramine by about a factor 3 and the rats died from cardiogenic shock. The cardiotoxic properties of orphenadrine and imipramine express themselves as a progressing disturbance in stimulus formation and conduction, a decrease in dP/dt max and increase in left ventricular end-diastolic pressure and a decrease in cardiac output caused by the progressing decrease of heart rate. An intravenous injection of physostigmine did not prolong survival and had no favourable effect on the cardiotoxicity caused by orphenadrine and imipramine. Although physostigmine may be useful in the treatment of the anticholinergic syndrome it has, at least in the rat, no favourable effect on the respiratory insufficiency, due to overdoses of orphenadrine and imipramine. Moreover it is not effective in antagonizing the cardiotoxic effects of orphenadrine or imipramine.

The anti-inflammatory, analgesic and antipyretic activities of non-narcotic analgesic drug mixtures in rats.

The effects of non-narcotic analgesics have been examined, separately and in admixture, on carrageenan-induced hind paw oedema and on yeast-induced hyperalgesia and hyperthermia in adult rats. The efficacy of the drugs was evaluated using the kinetics of drug-receptor interaction. In addition, the hypothesis was tested that the anti-inflammatory, analgesic and antipyretic activities of the drug mixtures used equal the addition of the activities of the individual drugs and could be predicted from their intrinsic activities and affinities. Dose-dependent inhibition of paw oedema, hyperalgesia and hyperthermia was observed after oral administration of acetylsalicylic acid (aspirin), paracetamol, phenacetin (60, 125, 250 and 500 mg.kg--1), and caffeine (12.5, 25, 50 and 100 mg.kf--1). Over the dose-ranges used, the anti-inflammatory activities of paracetamol, phenacetin and caffeine tended to be smaller than that of aspirin. The dose producing a semi-maximal effect for caffeine was lower than that for aspirin which in turn was comparable to that for paracetamol or phenacetin. The analgesic activities of phenacetin and caffeine were classified as stronger than that of aspirin, whereas the efficacy of paracetamol was similar. Paracetamol and aspirin were comparable as antipyretics. The antipyretic activity of phenacetin was higher but that of caffeine was lower than that of aspirin. For caffeine the dose producing a semi-maximal effect was lower than that of aspirin. Within the dose-ranges used, low doses of mixtures of aspirin with either paracetamol, phenacetin or caffeine exhibited anti-inflammatory, analgesic and antipyretic activities which were not different from the activities expected on the basis of addition. Incidentally, at some of the higher dose levels potentiation of the activity of the drugs was found. Low doses of the triple combinations: aspirin + paracetamol + caffeine and aspirin + phenacetin + caffeine showed anti-inflammatory and antipyretic activities which were not different from those expected on the basis of addition, but the activities observed with higher doses of these combinations indicated potentiation. It is concluded that, in the rat, the anti-inflammatory, analgesic and antipyretic activities of dual and triple combinations of aspirin, paracetamol, phenacetin and caffeine at least equal the activities expected on the basis of addition.

Hypotensive response in spontaneously hypertensive rats following microinjection of alpha-methylnoradrenaline in the caudal brain-stem.

Local microinjection of 1.25 nmol (-)-alpha-methylnoradrenaline in the A2-region of the nucleus tractus solitarii (NTS) caused a decrease of blood pressure and heart rate in both spontaneous hypertensive rats (SHR) and Wistar-Kyoto (W/K) rats. Although the maximal responses in both strains did not differ, the decrease in blood pressure lasted longer in the SHR. These results do not support the concept of a diminished sensitivity of catecholaminergic receptors in the NTS of SHR to alpha-methylnoradrenaline.

Effect of catecholamine-receptor stimulating agents on blood pressure after local application in the nucleus tractus solitarii of the medulla oblongata.

The effect of various catecholamines and alpha-mimetics, given by microinjection in the A2-region of the nucleus tractus solitarii (NTS), on blood pressure was investigated in anesthetized male rats. A dose-dependent decrease of blood pressure and heart rate was induced by adrenaline as the most effective drug, followed by noradrenaline, dopamine, alpha-methylnoradrenaline and octopamine. Ablation of the rostral or caudal part of the NTS, or removal of the area postrema did not diminish the effect of alpha-methylnoradrenaline. Higher doses of noradrenaline and alpha-methylnoradrenaline caused an initial rise of blood pressure, while the blood pressure lowering effect of noradrenaline was diminished, and that of alpha-methylnoradrenaline and dopamine delayed. Isoprenaline and the (+)-stereoisomers of noradrenaline and alpha-methylnoradrenaline were ineffective. The hypotensive effect of dopamine was not prevented by systemic injection of the dopamine beta-hydroxylase inhibitor FLA 63. Prior application of haloperidol, yohimbine and phentolamine antagonized the hypotensive response to dopamine and alpha-methylnoradrenaline. Application of peripherally effective alpha-mimetics into the A2-region had no or little effect, while high doses increased blood pressure. Tyramine and clonidine caused some decrease of blood pressure. Clonidine also decreased blood pressure when it was applied in the area of the locus coeruleus. Application of isoprenaline in the locus coeruleus also decreased blood pressure while in contrast adrenaline, noradrenaline, dopamine and alpha-methylnoradrenaline increased blood pressure. The present data suggest that the catecholaminergic receptors in the A2-region of the NTS differ from the classic vascular alpha-receptor and that the NTS also may contain structures which can antagonize the decrease in blood pressure.

The area postrema and control of arterial blood pressure; absence of hypertension after excision of the area postrema in rats.

Acute surgical excision of the area postrema (AP) in the rat failed to affect arterial blood pressure or heart rate. The was no effect on cardiovascular reflex responses during diving or on the heart rate responses to acute decreases or increases of blood pressure caused by bradykinin or angiotensin, respectively. Electrolytic lesions of the AP in acute experiments caused variable damage to the nucleus tractus solitarii (NTS). In these rats large variations in blood pressure occurred. Excision of the AP in a chronic experiment failed to change blood pressure, heart rate, water intake or plasma renin activity. In contrast, bilateral electrolytic lesions of the NTS at the level of the AP caused a severe acute hypertension and completely blocked cardiovascular reflex responses. Hypertension also existed in rats with NTS lesions studied for a longer period of time. There experiments failed to confirm the hypothesis that the AP exerts a tonic inhibitory control of basal blood pressure. Hypertension previously reported after ablation of the AP may be explained by damage to the NTS.