C. Michael Moriarty

Role of calcium in the regulation of adenohypophysial hormone release.

Abstract It is now accepted by most investigators that the initial action of most peptide hormones involves an interaction with a specific receptor on (or in) the plasma membrane of the target cell. A cascade of intracellular events results and culminates in the physiological response characteristic of the interaction of the particular hormone with its target cell. The regulation of hormone release from the adenohypophysis by the hypothalamic releasing hormones is presumed to occur via a similar process. The nature of the interaction at the cell surface as well as the details and sequence of the subsequent intracellular events are largely unknown. We do know, however, that two of the key factors regulating the intracellular secretory machinery in most cells are 1) the adenylate cyclase — cyclic AMP — protein kinase system and 2) the divalent cation, calcium. Since there have been several recent reviews (1–3) which have covered the role of the cyclic nucleotides in pituitary hormone secretion, this discussion will be restricted to a consideration of the regulatory role played by calcium. As was the case with tissues, the early work regarding calcium and the adenohypophysis followed the pattern of determining the ability of secretagogues to release pituitary hormones subsequent to various manipulations designed to remove what was often implicity considered to be extracellular calcium.

Involvement of intracellular calcium in hormone secretion from rat pituitary cells

Cells were dissociated from normal rat pituitaries by a combination of mechanical agitation and enzymatic action, seeded into culture flasks and grown in monolayer culture. When such cells were exposed to an extract of the hypothalamic stalk-median eminence area (HSME) a dose-dependent secretion of ACTH was observed. A 2-h exposure to Ca-free media significantly reduced the HSME-stimulated release of ACTH but the measured levels were still greater than the unstimulated controls. When the 45Ca2+ uptake into the cultured cells was measured both control and HSME-stimulated cells yielded identical results (60-80 nmol Ca/mg cell protein). Upon removal of the calcium associated with the surface coat it was found that HSME actually decreased the cellular uptake of calcium. Since variations in uptake can result from changes in influx or efflux as well as from variations in pool size or turnover times of calcium exchange with intracellular compartments, a series of isotope washout experiments were performed. Neither HSME nor theophyline affected the rate constant of calcium efflux from what is believed to be the cytosol pool to the extracellular media. Both agents, however, prompted a shift of intracellular calcium into a more tightly bound compartment. The data suggest that the calcium required for pituitary hormone secretion is derived primarily from an intracellular rather than extracellular origin. It may be that, via the action of cyclic AMP, such calcium can be mobilized from intracellular stores and shifted to a more tightly bound compartment where it can participate in the intracellular processes associated with secretion.

Blood histamine and solid malignant tumors

SummaryA clinical study was performed to determine whether patients with a newly diagnosed solid malignant tumor manifest an alteration in whole-blood histamine levels. Our results indicate that such patients have blood histamine nearly three times greater than either normal, healthy individuals or noncancerous disease controls. Following surgical removal of the tumor, blood histamine levels remained high for 2 months and then dropped close to the normal range 3 months after surgery. Basophil counts did not change significantly in the presence of a malignant tumor. Patients receiving either chemotherapy or radiation therapy, and terminal cancer patients who were no longer receiving any therapy except for pain control had blood histamine within or below the normal range. By analogy with animals studies, we suggest that nascent histamine synthesis is increased in the presence of a developing tumor. The clinical usefullness of this observation remains to be determined.

Cytochemical analysis of intracellular calcium distribution in the anterior pituitary of the rat

SummaryIn an attempt to assign morphologic identities to previously distinguished functional calcium compartments in the anterior pituitary of the rat, we employed the potassium pyroantimonate technique for cation localization. Tissues were incubated for In at 37°C in control medium; with 10mM theophylline; or with depolarizing amounts of potassium. Precipitate was quantified on photomicrographs of tissue prepared for electron microscopy with a Talos Systems Digitizer. The nature of the electron dense precipitate was dependent on the experimental state of the tissue. Treatment with 5 mM EGTA abolished the dense precipitate. Electron microprobe analysis also confirmed that calcium was the predominant cation in the observed precipitate. The most significant changes in precipitate deposition occurred along the plasma membrane, the limiting membrane of secretory granules and within mitochondria. Dense precipitate was present along the plasma membrane only in cells treated with potassium. Control tissue exhibited higher levels of precipitate associated with the limiting membrane of secretory granules than either theophylline-treated or potassium-treated tissue. Mitochondria contained more precipitate in potassium-treated tissue than in controls; the mitochondria of theophylline-treated tissue contained intermediate levels of precipitate. Addition of either theophylline or depolarizing amounts of potassium has been associated with hormone secretion in anterior pituitary tissue of normal rats. Kinetic studies in our laboratory indicate that intracellular calcium shifts occur. The pyroantimonate technique is useful in verifying morphologically the calcium compartments involved in shifts in intracellular calcium.

Variations in the response of enzyme-dissociated rat pituitary cells to thyrotropin releasing hormone.

Abstract While exploring the interaction between thyrotropin releasing hormone (TRH) and normal rat anterior pituitary cells in monolayer culture we observed that cells dissociated with the use of trypsin did not respond to TRH with an increase in either TSH or prolactin (PRL) release. The dissociated cells were cultured for 3 days, then washed to remove serum proteins and exposed to 10−6M TRH for 3 hours. TSH and PRL secretion from stimulated and unstimulated cultures was determined by radio-immunoassay and normalized using cell protein. When such trypsin-dissociated cells were exposed to 0.5 mM dibutyryl cyclic AMP the release of both TSH and PRL doubled indicating that the intracellular secretory machinery was functional and that the block to TRH was proximal to the formation of cyclic AMP and presumably at the level of a TRH surface receptor. Previous studies have shown that such trypsin-dissociated cells respond to LHRH and a crude hypothalamic extract with a dose dependent increase in LH, FSH and ACTH release. This rules out a non-specific effect of trypsin. When pituitary cells were dissociated with a non-trypsin technique, the unstimulated release of both TSH and PRL was comparable to that found with the trypsin-dissociated cultures. However, these cultures did respond to TRH with an increase in TSH release although again no effect was seen with PRL. The susceptibility of the cells to trypsin suggests the possibility that a protein moiety may be closely associated with the function of the receptor.