Specific amino acids moderate the effects on Ni2+ on the testosterone production of mouse leydig cells in vitro.

The purpose of this investigation was to study the effectiveness of two nickel-binding amino acids, histidine (His) and cysteine (Cys), to prevent the inhibitory action of Ni2+ on testosterone (T) production by mouse primary Leydig cell culture. The maximal human chorionic gonadotropin (hCG)-stimulated T response was measured by radioimmunoassay (RIA) in the culture media. Three types of experiments were performed. In a concentration-response study, Ni2+ (62.5 to 1,000 microM) was added to the cells simultaneously with equimolar or twice the equimolar concentrations of His or Cys and the cultures were maintained for 48 h. Nickel-induced reduction in T production was completely prevented by equimolar concentrations of His at Ni2+ concentrations of 125, 250, and 500 microM; equimolar or twice the equimolar concentrations of His were only partially effective at 1,000 microM Ni2+. Protective action of Cys was complete only at the lowest concentration of Ni2+ (125 microM). In a second series, the cells were incubated for various times (0.5 to 48 h) with 1,000 microM Ni2+ in the presence of 2,000 microM His or Cys. Increasing the time of incubation, the protective effect of both amino acids against Ni2+ was reduced. In a third series, attempts were made to reverse the action of 1,000 microM Ni2+ after incubation with cells for various times (0.5 to 24 h), followed by exposure to 2,000 microM His or Cys. Cell cultures were maintained for 48 h. A partial recovery of hCG-stimulated T production could be observed only if the amino acid was added not later than 4 h after the metal. This time was also required to elicit the T depression produced by Ni2+. Administration of either His or Cys at later times had no effect. Our results show that both His and Cys are able to moderate the effects of Ni2+ on Leydig cell T production, depending on the concentration of this metal ion, as well as on amino acid. However, at higher Ni2+ concentrations the complete protection by His or Cys is only temporary. Administration of these amino acids after the Ni2+-produced decrease in T production was not able to reverse the process.

Anaphylactoid reactions activate hypothalamo-pituitary-adrenocortical axis: comparison with endotoxic reactions.

Infectious and allergic diseases represent distinct aspects of immune response that can be experimentally modeled as endotoxic reactions following bacterial lipopolysaccharide (LPS) administration and anaphylactoid reactions following systemic injection of foreign proteins, respectively. Although it is well established that LPS stimulates the activity of the hypothalamo-pituitary-adrenocortical (HPA) axis, such effects of anaphylactoid reactions are completely unknown. To evaluate the impact of anaphylactoid reactions on HPA regulation, secretion of adrenocorticotropin hormone (ACTH) was followed and the pattern of c-Fos induction in the hypothalamic paraventricular nucleus (PVN) was revealed in rats that were challenged with egg white or compound 48/80. Male rats were intravenously injected with 0.1 ml/100g b.wt. 1:1 diluted egg white or 50 microg/100 g b.wt. compound 48/80, blood samples were taken before and various time intervals between 15-240 min after challenge for plasma ACTH measurement. Anaphylactoid reactions resulted in a rapid, significant activation of ACTH secretion and induced c-Fos immunoreactivity in the corticotropin-releasing hormone (CRH)-secreting subset of the parvocellular neurosecretory neurons. In addition, magnocellular neurosecretory neurons and autonomic-related projection neurons in the PVN became also c-Fos positive upon challenge. Changes in these parameters are compared to those seen in rats challenged with bacterial endotoxin, LPS.

Effect of cholinergic drugs on the concentration of intracellular free calcium of rat pituitary intermediate lobe cells.

We tested the effect of cholinergic drugs on the concentration of intracellular free calcium in rat melanotropes. Acetylcholine, muscarine, carbachol, and nicotine resulted in a significant rise in this parameter. Effect of acetylcholine was reduced by atropine (non-selective muscarinic antagonist), pirenzepine (M1 muscarinic antagonist), and 4-DAMP (M3 > M1 muscarinic antagonist), but exposure to the M1 muscarinic agonist McN-A 343 resulted in a significantly smaller calcium-response than that seen in response to acetylcholine or to muscarine. This suggests the involvement of both M1 and M3 muscarinic receptors in the acetylcholine-induced calcium-rise. On the other hand, in the presence of atropine the acetylcholine-induced calcium-rise was not eliminated: this fact indicates that nicotinic receptors are also involved in the acetylcholine-induced intracellular calcium-rise. The acetylcholine-, and nicotine-induced calcium-rise was significantly reduced in presence of the neuronal-type nicotinic antagonist, mecamylamine. This suggests the involvement of a neuronal-type nicotinic receptor in the acetylcholine-induced intracellular calcium-response. Moreover, because in a further experiment almost 80% of the cells investigated responded to muscarine as well as nicotine, we conclude that both functionally active muscarinic and nicotinic receptors are present on the same cell.

Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland.

Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]diphenyl-acetoxy-N-methyl-piperidine ([3H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25 degrees C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [3H]QNB and [3H]4-DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M1, M3 subtypes selective antagonist) > atropine (general) > pirenzepine (M1). Methoctramine (M2) didn't antagonise the [3H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca2+ level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M1 and M3 muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined.

Catecholaminergic control of intracellular free calcium and beta-endorphin secretion of rat pituitary intermediate lobe cells.

Individual melanotropes and intermediate lobes were tested to elucidate the role of alpha- and beta-adrenergic and D-2 dopamine receptors in the regulation of concentration of intracellular free calcium ([Ca2+]i) and release of beta-endorphin. Hormone secretion was studied in a superfusion system, while [Ca2+]i was measured microspectrofluorimetrically. Noradrenaline (1 microM) resulted in a slight decrease, then a marked increase in [Ca2+]i and secretion of beta-endorphin. The nonselective beta-adrenergic agonist isoproterenol (1 microM) increased [Ca2+]i and secretion of beta-endorphin; this effect was blocked by the beta-antagonist propranolol (10 microM). The alpha-adrenergic agonist phenylephrine (1 microM) increased [Ca2+]i and beta-endorphin secretion, but this effect was not blocked by terazosin or prazosin (alpha1-adrenergic antagonists, 1 microM). Administration of the alpha2-adrenergic agonist xylazine (1 microM) increased [Ca2+]i but did not affect secretion of the hormone. Biphasic effect of noradrenaline was tested in presence of adrenergic and dopaminergic antagonists. The noradrenaline-induced rise in [Ca2+]i and beta-endorphin secretion was decreased by propranolol, but this drug did not modify the inhibition. In the presence of 1 microM sulpiride (selective D-2 dopaminergic antagonist), the inhibitory phase of the curve was abolished, and the subsequent increase was reduced. This suggests that activation of dopamine D-2 receptors is involved not only in the inhibition, but also in the subsequent increase, which may originate from a rebound after the termination of the activation of these inhibitory receptors. Our data suggest the presence of several distinct types of catecholamine receptors in the rat intermediate lobe, and the dominant involvement of D-2 and beta-adrenergic receptors in the noradrenaline-induced regulation of melanotropes.