Effect of maturation and aging on beta-adrenergic signal transduction in rat kidney and liver.

The characteristics of the beta-adrenergic signal transduction system were analyzed in kidney and liver membrane preparations from neonatal (2-3 days), mature (2 months), and old (2 years) rats. When comparing kidneys from adult to neonatal rats, we found a higher beta-receptor density and a higher percentage of beta(1)-receptor subtype, lower immunoreactive G(salpha)-protein, a lower ratio between the high and low molecular weight splice variant of G(salpha), lower immunoreactive G(ialpha)-protein, and lower basal adenylate cyclase activity. When comparing livers from adult to neonatal rats, we found lower beta-receptor density and basal adenylate cyclase activity. Very few differences could be detected when comparing mature to old kidneys or livers. Stimulated adenosine 3',5'-cyclic monophosphate (cAMP) synthesis was tissue- and age-dependent. In liver, G-protein- and beta-receptor-stimulated cAMP synthesis mirrored basal adenylate cyclase activity and was highest in liver from neonatal animals. In contrast, cAMP synthesis was significantly more stimulated in kidneys from mature animals than from neonatal and senescent rats. We conclude that: (i) the stoichiometry of the components within the beta-receptor/G-protein/adenylate cyclase complex is not fixed but is both tissue- and age-dependent; (ii) adenylate cyclase enzyme activity is possibly but not necessarily the rate-limiting step in the beta-receptor-mediated synthesis of cAMP; and (iii) there is in vivo evidence for a preferential co-expression of the large splice variant of the G(s)-protein and beta(2)-receptor subtype. It is speculated that this could have important physiological consequences for the development of the kidney.

[Reports of suspected side effects of selective serotonin reuptake inhibitors in Belgium and The Netherlands]. / Meldingen van vermoedelijke bijwerkingen van selectieve serotonine-heropnameremmers in België en Nederland.

OBJECTIVE: To study the adverse drug reaction (ADR) profile of selective serotonin re-uptake inhibitors (SSRI) in Belgium and the Netherlands. DESIGN: Descriptive study. SETTING: The Belgian Pharmacovigilance Centre and the Dutch Drug Safety Unit. METHOD: All adverse reactions of fluoxetine, fluvoxamine, paroxetine and sertraline, reported between the moment of registration of these drugs and January 1st 1995, were assessed for causality. Possible, probable and certain adverse reactions were counted per drug and per country. RESULTS: At the national monitoring centres of Belgium and of the Netherlands adverse reactions were reported 78 and 537 times, respectively. Approximately 30% of the adverse reactions were well-established. Approximately 60% of the reactions were poorly documented or not serious. Less than 1% of the adverse reactions reported in Belgium and approximately 13% of the reactions reported in the Netherlands, were less established and/or serious. These reactions were: movement disorders and extrapyramidal reactions, convulsions, galactorrhoea, hyponatraemia and syndrome of inappropriate antidiuretic hormone secretion, and purpura. CONCLUSION: The ADR profile was compatible with the ADR profile in the medical literature, but not all ADR were similarly represented in the product information of the different SSRI. More adverse reactions were reported in the Netherlands than in Belgium.

[Generalized side effects with the use of carbamazepine]. / Gegeneraliseerde bijwerkingen bij gebruik van carbamazepine.

Since 1973, 42 cases of generalized adverse reactions to carbamazepine were reported to the Netherlands Centre for Monitoring of Adverse Reactions to Drugs and the Belgian Centre for Drug Monitoring. The organ involvement can be very diverse in an allergic reaction to carbamazepine. Serious and protracted disturbances in pulmonary diffusion capacity may be present even in the absence of changes on the chest X-ray. Analysis of the type IV mechanism involved suggests that caution is warranted with regard to administration of other drugs during the acute phase of the allergic reaction particularly if these drugs have a propensity to cause type IV allergic reactions themselves.

Desensitization of alpha 1-, beta- and glucagon receptors in rat hepatocytes: influence of ageing.

The alpha 1-agonist phenylephrine in hepatocytes from mature and senescent rats, and the beta-agonist isoproterenol in hepatocytes from senescent rats, elicited a time-dependent, homologous desensitization of alpha 1- and beta-receptor-mediated glycogenolysis respectively, which was maximal after 20 min of exposure to the agonists. Glucagon triggered desensitization of the glycogenolytic response to glucagon, isoproterenol and phenylephrine, which was maximal after less than 5 min; this was followed by resensitization of the glucagon response only. After 20 min of treatment with phenylephrine or isoproterenol, no change in cellular distribution of alpha 1- or beta-receptors was noticed, using sucrose gradient centrifugation. After a 1-h exposure to both agonists, a shift of both receptors to a light density fraction was found, reflecting receptor internalization. Neither the rate of functional desensitization, nor the degree of receptor internalization was altered upon ageing. We conclude that functional desensitization and internalization of adrenergic receptors in rat hepatocytes are separate events in time and are largely unaffected by the ageing process. Thus, despite the absence of a beta-receptor-mediated glycogenolytic response in hepatocytes from mature rats, isoproterenol triggers the internalization of beta-receptors.

No functional atypical beta-adrenergic receptors in human omental adipocytes.

Isoproterenol stimulates lipolysis in human omental adipocytes with an EC50 (concentration at which an agonist produces half-maximal stimulation) of 120 nM. CGP12177 (dl-4-3[(1,1-dimethylethyl) amino]-2-hydroxylpropoxy]1,3-dihydro-2H-benzimidazol-2-one hydrochloride), a potent beta 1-/beta 2- adrenergic receptor (AR) antagonist but being an agonist for atypical beta-AR, fails to stimulate lipolysis in these cells, even at a concentration as high as 0.1 mM. Since CGP12177 is a partial agonist, its failure to stimulate lipolysis may result from a poor stimulus-response coupling, so that it can not be excluded that atypical beta-AR are actually present and even functional in these cells. To evaluate this hypothesis, we estimated the potency of CGP12177 to inhibit the isoproterenol-stimulated lipolysis. This inhibition curve reflects a single class of sites and the IC50-value (concentration at which an antagonist produces half-maximal inhibition) of CGP12177 (3.8 nM) is in good agreement with what should be expected for beta 1-AR/beta 2-AR. Moreover, metoprolol and atenolol, two beta 1- AR- selective antagonists, shift the isoproterenol dose-response curve to the right with high potency as well. These potencies are similar to the ones found for beta 1-AR in the human heart but appreciably higher than those which should be expected for atypical beta-AR. The present study suggest that atypical beta-AR are not functional in human omental adipocytes.

Species and strain-related differences in the expression and functionality of beta-adrenoceptor subtypes in adipose tissue.

The beta-adrenoceptor subtypes which trigger lipolysis in white adipocytes vary markedly between calf and rats, and even between different rat strains. In calf adipocytes, CGP12177, a potent antagonist for beta 1- and beta 2-adrenoceptors (i.e., "classical beta-adrenoceptors") and a partial agonist for atypical beta-adrenoceptors, did not stimulate lipolysis, but inhibited with high affinity (IC50 = 0.66 nM) the lipolytic response to 10 nM isoproterenol. In adipocytes from both Wistar rats and Sprague-Dawley OFA rats, CGP12177 stimulated lipolysis to almost the same extent as isoproterenol. Low concentrations of CGP12177 (3 nM) inhibited part of the lipolytic response to 10 nM isoproterenol in the Sprague-Dawley OFA rat adipocytes, but not in Wistar rats at all ages tested (2-4 weeks, 2-4 months, 24-26 months). Hence, functional beta-adrenoceptors are only classical in calf adipocytes, only atypical in Wistar rat adipocytes and both classical and atypical in Sprague-Dawley OFA rat adipocytes. Binding experiments were performed with 150 pM [125I]CYP. On calf adipocyte membranes, competition binding curves with CGP12177 displayed one high affinity binding site (IC50 = 4.7 nM), whereas the curves for CGP20712 (beta 1-selective antagonist) and ICI118551 (beta 2-selective antagonist) were biphasic. In agreement with the functional data, these results indicate that only beta 1- and beta 2-adrenoceptors are present in calf adipose tissue. For both rat strains, only half of the displaceable [125I]CYP binding sites displayed high affinity for CGP12177 (IC50 = 6.8 to 7.5 nM), and competition binding studies with CGP20712 and ICI118551 indicated that they represent beta 1- and beta 2-adrenoceptors. The remaining [125I]CYP binding sites possessed an about 50 times lower affinity for CGP12177 (IC50 = 260 to 345 nM). They are likely to represent atypical beta-adrenoceptors. It is concluded that the presence and the physiological relevance of beta-adrenoceptor subtypes in adipose tissue may not only be species-related, but also strain-related.

Influence of aging on the alpha 1-receptor-mediated glycogenolysis in rat hepatocytes.

The involvement of the Ca(2+)-dependent and Ca(2+)-independent, insulin-sensitive pathway in the alpha 1-receptor-control of glycogenolysis was investigated in hepatocytes from young adult, mature adult, and senescent rats. Upon chelation of extracellular Ca2+, phenylephrine caused a similar increase in glucose output that was potently inhibited by insulin, indicating the presence of both pathways in each age group. From the age-related decreasing sensitivity of the Ca(2+)-dependent pathway toward verapamil and nifedipine, and toward insulin, we suggest that the contribution of Ca(2+)-fluxes in eliciting glycogenolysis through the Ca(2+)-dependent pathway decreases upon aging. Both pathways were inhibited by the protein kinase C (PKC) activator, 4 beta-phorbol 12-myristate 13-acetate (PMA); the inhibitory effect was decreased in hepatocytes from mature adult and senescent rats. In conclusion, our results favor the idea that a Ca(2+)-dependent and a Ca(2+)-independent, insulin-sensitive pathway remain involved throughout the life span. We provided the evidence for an impaired regulatory role of protein kinase C and calcium in hepatocytes from the older age groups.

Influence of aging on the beta- and glucagon-receptor-mediated glycogenolysis in rat hepatocytes.

The influence of aging on beta-receptor and glucagon-receptor control of glycogenolysis was investigated in rat hepatocytes. The beta-receptor-induced glucose output was detectable only in senescent rats, was partly dependent on extracellular Ca2+, and was inhibited by 4 beta-phorbol 12-myristate 13-acetate (PMA), insulin, and the Ca(2+)-antagonists, verapamil and nifedipine. Chelation of extracellular Ca2+ potentiated the effect of nifedipine only. In contrast, glucagon-stimulated glycogenolysis, similar in mature and senescent rats, was independent on extracellular Ca2+ and was unaffected by PMA. Verapamil, in senescent rats only, and nifedipine, in mature and senescent rats, inhibited glucagon-stimulated glucose output only in the presence of Ca2+. Insulin inhibited glucagon-induced glucose output, irrespective of the age of the rat and the presence of Ca2+. We conclude that the beta-receptor component in the adrenergic regulation of glycogenolysis in senescent rats consists of a major Ca(2+)-independent and a minor Ca(2+)-dependent part, displaying different sensitivity towards protein kinase C (PKC), Ca(2+)-antagonists, and insulin. Aging does not change the capacity of glucagon to induce a full glycogenolytic response in the absence of extracellular Ca2+; Ca(2+)-influx, however, seems to be involved when extracellular Ca2+ is present, and this sensitivity is increased on aging.

Pharmacological characterization of a beta 3-receptor agonist (BRL 37,344) and a partial agonist (CGP 12,177A) in neonatal rat liver plasma membranes.

The pharmacological properties of BRL 37,344 (sodium-4-(2'-[2-hydroxy-2- (3-chloro-phenyl)ethylamino]-propyl)phenoxyacetatesesquihydrate), a beta 3-selective agonist, and CGP 12,177A) (-)-4-(3-t-butyl amino-2-hydroxypropoxy) benzimidazole-2-one], a non-selective beta-antagonist, recently characterized as a partial beta 3-agonist in rat adipose tissue, were studied in comparison with isoproterenol, a non-selective beta-agonist, in plasma membranes prepared from the livers of newborn rats. Competition binding curves obtained with [125I]iodocyanopindolol ([125I]CYP) as ligand and isoproterenol or BRL 37,344 as competitor were characterized by the presence of a high and a low affinity binding site; the high affinity binding site was no longer detectable when guanidylimidobisphosphate (GppNHp) was present in the incubation mixture. Competition curves with CGP 12,177A were monophasic and independent of GppNHp. In the presence of 10(-7) M of the beta 2-selective antagonist ICI 118,551 [erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylamino butan-2-ol], a concentration which blocks most of the beta 2-receptors, ligand binding was reduced to 32% of its maximum. Under these conditions, isoproterenol further displaced the ligand, and competition curves still displayed the high and the low affinity binding sites; BRL 37,344, however, caused no further displacement of ligand, except at the highest concentrations. This suggests that BRL 37,344 occupies only the ICI 118,551-sensitive binding sites, i.e. beta 2-receptors. Isoproterenol and BRL 37,344 both stimulated adenylate cyclase (EC 4.6.1.1) activity concentration dependently, although the stimulating effect of BRL 37,344 was about half of what was found for isoproterenol. Furthermore, BRL 37,344 inhibited concentration dependently the isoproterenol-induced stimulation of adenylate cyclase, and the inhibition was dependent on the concentration of isoproterenol. The stimulating effect of isoproterenol and BRL 37,344 on adenylate cyclase was blocked by ICI 118,551, whereas the beta 1-selective antagonist CGP 20,712A ((+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino)-3-[4-(1-methy l-4- trifluoromethyl-2-imidazolyl)-phenoxy]-2-propanolmethane sulphonate) was ineffective. CGP 12,177A failed to stimulate adenylate cyclase activity. From these results we suggest that BRL 37,344 acts as a beta 2-partial agonist in rat liver. The results obtained with CGP 12,177A are typical for a non-selective beta-antagonist. We therefore conclude that there is no pharmacological evidence for the presence of beta 3-receptors in livers from newborn rats.

Influence of age on the beta 1- and beta 2-adrenergic receptors in rat liver.

The influence of maturation and aging on beta receptors in rat liver was studied. Competition binding experiments with the nonselective beta-antagonist propranolol and the subtype selective antagonists ICI 118,551 (beta 2) ICI 89,406 (beta 1), and CGP 20,712A (beta 1) revealed the presence of a mixed beta 1 and beta 2 receptor population in crude plasma membrane preparations from livers of newborn, mature, and senescent rats. The percentage of beta 1 receptors was lowest in livers from newborn rats and was increased in livers from mature and senescent rats. This increase is caused by a decrease in beta 2 receptor density on maturation, although the beta 1 receptor density is nearly constant throughout the life span of the rat. Isoproterenol-stimulated adenylate cyclase activity was inhibited in livers from senescent rats by propranolol and ICI 118,551 and to a lesser extent by ICI 89,406 and CGP 20,712A. The isoproterenol-stimulated glucose output in hepatocytes from senescent rats was inhibited concentration dependently by propranolol, ICI 118,551, ICi 89,406, and CGP 20,712A. From these results we conclude that beta 1 and beta 2 receptors are present in livers from rats of the three age groups and that the beta 1 to beta 2 receptor ratio is increased in livers from mature and senescent rats compared with newborn rats. Both beta receptor subtypes are linked to the cAMP second messenger system in newborn and senescent rats; beta 1 and beta 2 receptors are equally involved in the regulation of glycogenolysis in hepatocytes from senescent rats.