Dexamethasone-mediated inhibition of calcium transients and ACTH release in a pituitary cell line (AtT-20).

In the corticotroph-like murine pituitary tumor cell line, AtT-20, adrenocorticotropic hormone release is triggered by corticotropin-releasing hormone and is attenuated by the synthetic adrenal steroid dexamethasone. The precise mechanisms by which dexamethasone inhibits secretion are under investigation. We examined whether dexamethasone can modulate release via regulation of calcium homeostasis. More specifically, we have evaluated the effects of dexamethasone on calcium current, intracellular calcium concentration, and adrenocorticotropic hormone release. Using perforated patch-clamp and calcium imaging with fura PE3/AM, we found that dexamethasone decreases calcium current and intracellular calcium levels. The inhibition of current by dexamethasone is not, however, altered by the calcium channel antagonists nifedipine (L-type) or omega-agatoxin IVA (P/Q-type), despite the presence of these calcium channel subtypes in AtT-20 cells and the exclusive coupling of adrenocorticotropic hormone release to the L-type channel in these cells. We also evaluated the temporal relationship between dexamethasone-mediated inhibition of secretion and calcium influx. Whereas a prolonged (2 h) incubation with dexamethasone inhibits corticotropin-induced release by approximately 40%, a rapid (10 min) incubation (a time interval sufficient for dexamethasone-mediated inhibition of calcium transients) does not inhibit release. These data suggest, therefore, that dexamethasone does, indeed, modulate calcium homeostasis in AtT-20 cells, but that this effect is not responsible for its inhibition of secretion.

Genetic and clinical advances in Prader-Willi syndrome.

Prader-Willi syndrome is a developmental disorder with distinctive dysmorphic features, specific neurobehavioral attributes, and a characteristic learning profile. Advances continue to be made in understanding the factors associated with the loss of imprinted gene expression within chromosome 15q11-q13. These advances are helping providers make certain diagnoses early and are helping scientists uncover new genetic pathways. In addition, efforts to further understand the role of recombinant growth hormone therapy in Prader-Willi syndrome and the genetic information responsible for the neurobehavioral profile are additional targets for research.

Calcium currents in a pituitary cell line (AtT-20): differential roles in stimulus-secretion coupling.

The purpose of the present investigation was to identify voltage-dependent calcium channel subtypes that control the release of ACTH in AtT-20 cells, a clonal mouse pituitary cell line. Using the perforated patch-clamp technique, we identified dihydropyridine (nimodipine)-, omega-Agatoxin IVA-, and omega-Conotoxin MVIIC-sensitive calcium currents. No omega-Conotoxin GVIA-sensitive currents are present in these cells. There also existed a considerable resistant component to the recorded inward current that was inhibited by cadmium, a nonselective calcium channel antagonist. Using RIA, we examined the contributions of each of the pharmacologically distinct calcium channel populations to CRH- or potassium chloride (KCI)-stimulated release of ACTH at various time intervals (10 sec to 60 min). We found that nimodipine markedly inhibited ACTH release at all intervals tested, whereas omega-Agatoxin IVA, omega-Conotoxin MVIIC, and omega-Conotoxin GVIA had no significant effect. Moreover, the inhibition by nimodipine was comparable to that seen after cadmium application, and the effects of these two antagonists were not additive. These data suggest that although AtT-20 cells possess dihydropyridine-, omega-Agatoxin IVA-, and omega-Conotoxin MVIIC-sensitive calcium channels as well as a considerable toxin-resistant current, only the dihydropyridine-sensitive calcium channels appear to be coupled to CRH- or KCI-induced ACTH release.

Autoactive peptides act at three distinct receptors to depolarize the bag cell neurons of Aplysia.

1. In response to brief stimulation of an afferent input the bag cell neurons of Aplysia depolarize by 15-20 mV and generate an afterdischarge that, in vitro, lasts for approximately 30 min. During the discharge these neurons secrete three small peptides [bag cell peptides (BCPs)], Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu (alpha-BCP), Arg-Leu-Arg-Phe-His (beta-BCP), and Arg-Leu-Arg-Phe-Asp (gamma-BCP), that share a common core sequence and that have electrophysiological effects on the bag cell neurons themselves. We have now studied the action of these three peptides on bag cell neurons isolated in culture. All three peptides were found to be capable of producing a depolarization of these cells. 2. The ability of alpha-, beta-, and gamma-BCP to induce a depolarization in isolated bag cell neurons exhibits a seasonal variability. The response to the peptides is maximal from early summer through late fall and parallels the frequency of egg-laying in vivo. 3. The depolarization induced by alpha-, beta-, and gamma-BCP desensitizes with repeated application of peptide. Desensitization of the response to one peptide does not, however, prevent the response to application of one of the other two peptides. This suggests that separate autoreceptor populations exist for alpha-, beta-, and gamma-BCP. 4. As reported previously, desensitization of the depolarizing response to the peptides was also observed after preincubation of intact clusters of bag cell neurons with a high concentration of all three peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol induction of rhodamine 123 efflux and the multidrug resistance pump in rat pituitary tumor cells.

Rhodamine 123 is a fluorescent dye that localizes in mitochondria, is a substrate for the multidrug resistance pump, and is retained for long periods of time by carcinoma cells. 17 beta-Estradiol causes GH4C1 cells (rat pituitary tumor cells) to lose rhodamine 123 fluorescence faster than untreated cells. We found that estradiol induces accumulation of the mRNA for the multidrug resistance pump 3-5-fold, with maximum induction occurring within 1 day at 10(-9) M estradiol. Immunoblot analysis demonstrated that estradiol induces a protein of 150 kDa that reacts with an antibody to P-glycoprotein, the multidrug resistance pump. The reduced retention of rhodamine 123 caused by estradiol is prevented by verapamil and cyclosporin, inhibitors of the pump. A clone resistant to the effects of estradiol on rhodamine 123 has greatly reduced levels of mRNA for the pump. The effect of estradiol is more marked on rhodamine 123 retention than it is on that of rhodamine 110 or tetramethylrhodamine methyl ester. We conclude that estradiol enhances rhodamine 123 efflux by inducing the multidrug resistance gene. The specificity for rhodamine 123, compared with other analogs, may be caused by differences in accessibility to the pump.

Inhibition of peptide release from invertebrate neurons by the protein kinase inhibitor H-7.

The protein kinase inhibitor H-7 has been shown to prevent the potentiation of action potentials that normally accompanies an afterdischarge in the bag cell neurons of Aplysia. We have now shown that H-7 attenuates the release of ELH from these neurons during an afterdischarge without influencing the firing frequency or length of the afterdischarge.

Hyperosmotic media inhibit voltage-dependent calcium influx and peptide release in Aplysia neurons.

The bag cell neurons of Aplysia provide a model system in which to investigate the effects of hyperosmolality on the electrical and secretory properties of neurons. Brief stimulation of these neurons triggers an afterdischarge of action potentials that lasts approximately 20-30 min, during which time they release several neuroactive peptides. We have found that pre-incubation of intact clusters of bag cell neurons in hyperosmotic media prior to stimulation prevents the initiation of afterdischarges. Furthermore, an increase in osmolality of the external medium during an ongoing afterdischarge causes its premature termination. Hyperosmotic media attenuate the release of peptide evoked by both electrically stimulated afterdischarges and potassium-induced depolarization. The ability of high potassium to depolarize the bag cell neurons is, however, not impaired. Exposure of isolated bag cell neurons to hyperosmotic media also inhibits the amplitude of action potentials evoked by depolarizing current injection and attenuates the voltage-dependent calcium current. In isolated bag cell neurons loaded with the calcium indicator dye, fura-2, hyperosmotic media reduced the rise in intracellular calcium levels that normally occurs in response to depolarization. Our results suggest that the effects of hyperosmotic media on peptide secretion in bag cell neurons can largely be attributed to their effects on calcium entry.

Control of potassium currents and cyclic AMP levels by autoactive neuropeptides in Aplysia neurons.

The bag cell neurons of Aplysia are capable of generating an afterdischarge, which, in vivo, triggers egg-laying behavior. Pharmacologic elevation of cyclic AMP levels in isolated bag cell neurons has been shown to initiate repetitive firming similar to that seen during an afterdischarge, and to decrease outward currents measured under voltage-clamp. We have now examined the effects of three autoactive neuropeptides, alpha-, beta-, and gamma-bag cell peptide (BCP), on cyclic AMP levels and voltage-dependent potassium currents in these neurons. Previous work has shown that alpha-BCP lowers cyclic AMP levels in intact clusters of bag cell neurons. We have found that beta-BCP elevates cyclic AMP levels, whereas gamma-BCP, like alpha-BCP, lowers cyclic AMP levels. We used whole cell patch clamp technique to determine the effects of the peptides on the delayed voltage-dependent potassium currents in isolated bag cell neurons. As one would predict from their effects on cyclic AMP levels, beta-BCP decreased the amplitude of the delayed potassium currents whereas both alpha- and gamma-BCP increased the amplitude of these currents. In contrast, no consistent effects of these peptides on the transient voltage-dependent potassium current (A-current) were seen in these cells. Our results suggest that these three autoactive peptides may contribute to changes in second messengers and ionic currents during a bag cell afterdischarge.

Progressive potentiation of peptide release during a neuronal discharge.

1. In response to electrical stimulation, the bag cell neurons of Aplysia generate an afterdischarge that lasts 20-40 min. During this afterdischarge several neuroactive peptides are released. We have now studied the time course of release of two of these peptides, egg-laying hormone (ELH) and acidic peptide (AP). For the collection of released peptides, the artery to the bag cell clusters was perfused. The medium surrounding the clusters (artificial seawater, ASW) was completely exchanged at 5-min intervals before, during, and after stimulation of an afterdischarge. Peptides released into the external medium were analyzed with the use of high-pressure liquid chromatography. 2. Before stimulation, no detectable ELH and AP were found in the external medium. After the onset of an afterdischarge, the amount of ELH and AP released increased progressively until 15-20 min of firing. Toward the conclusion of an afterdischarge, the release of ELH and AP returned to control levels. 3. In contrast to the pattern of release of the peptides, the firing rate of the bag cell neurons is maximal within the first minute of afterdischarge and thereafter declines. 4. Release of the peptides from axonal varicosities occurs within the vascularized connective-tissue sheath that covers the clusters of bag cell neurons. Experiments were therefore carried out to establish whether the observed time course of release is affected by diffusion of the peptides through the vasculature into the external medium and, in particular, to determine whether the maximal rate of release at 15-20 min into the afterdischarge could be accounted for by a delay in transport of peptides from the neurites.(ABSTRACT TRUNCATED AT 250 WORDS)

Hippocampectomy and feature-positive discrimination.

The effects of hippocampal lesions on feature-positive discrimination were investigated using the nictitating membrane response preparation. During training, animals received a simultaneous reinforced compound as the conditioned stimulus (CS+) and a non-reinforced element as the CS-. The compound consisted of a tone and a light, with the tone being more salient than the light. The light and tone served as the CS- in Expts. 1 and 2, respectively. There were no significant differences between hippocampectomized animals and the controls (cortical and sham) when the CS- was the light; however, when the more salient tone stimulus was the CS-, hippocampectomized animals exhibited high levels of responding to both the CS+ and CS- and failed to acquire the discrimination. The results are discussed in terms of attentional and response inhibition theories of hippocampal function.