Isolation and characteristics of bovine pituitary secretory granules.

Secretory granules containing primarily growth hormone and prolactin were isolated from bovine anterior pituitaries. Marker enzyme analysis and electron microscopy indicated that the secretory granule fraction did not contain measureable amounts of other intracellular organelles. Such isolated granules were resistant to a variety of chemical and physical challenges including variations in osmolarity, ionic strength, EGTA, sonication, boiling, etc. The only treatments that were found to routinely result in granules lysis were alkaline pH and 0.5% SDS. Nonspecific leakage of both growth hormone and prolactin was less than 9% of total hormone pool even after a 60-min incubation. The release of prolactin but not growth hormone could be increased by lowering the free calcium concentration. Conversely, 10(-5) M ionophore A23187 caused a decrease in nonspecific hormone leakage. This raises the possibility that a nonexocytosis secretory pathway might be involved in pituitary hormone release. The initial secretory granule fraction was further purified using discontinuous sucrose gradient ultracentrifugation to yield a subfraction highly enriched in prolactin granules. These granules had the same stability characteristics as the original secretory granule fraction. The use of such granules should prove useful in our efforts to understand how calcium regulates cellular secretion.

The effect of calcium and cyclic AMP on amylase release in digitonin-permeabilized parotid gland cells.

Rat parotid cells were permeabilized with digitonin to examine their secretory dynamics. Cells were isolated by a modification of the method previously described by Hootman [1985). J. Biol. Chem. 260, 4186-4194) in which alpha-chymotrypsin was included. The final preparation consisted of approx. 40-60% single cells. The cells were 85-90% viable by trypan blue exclusion and secreted amylase when stimulated with isoproterenol. Digitonin (2 or 5 microM) was sufficient for permeabilization while 2 microM digitonin was somewhat more effective in maintaining cell integrity as indicated by lactate dehydrogenase release. Digitonin had minimal effects on intracellular granules in the whole cell and was, thus, relatively selective. The response of digitonin-permeabilized cells to calcium (without secretagogues) in the incubation medium was monitored by amylase release. For a wide range of applied free calcium concentrations (1 X 10(-7) M to 10(-4) M) a statistically significant increase in amylase secretion was observed. Control cells did not release amylase to a similar extent without secretagogue. Cyclic AMP (50 microM) significantly enhanced amylase secretion from digitonin-treated cells at all concentrations of free calcium tested. Neither calcium nor cyclic AMP alone was sufficient to stimulate maximal amylase release. Our results provide direct evidence for a model in which calcium and cyclic AMP work on separate pathways as interacting regulators of exocytosis.

Bioactive and immunoactive ACTH in the rat pituitary: influence of stress and adrenalectomy.

Tissue levels of bioactive and immunoactive ACTH were measured in both the anterior and neuro-intermediate lobes of the rat pituitary. Similar concentrations of bioactive (65 ng/mg) and immunoactive (83 ng/mg) ACTH were found in the anterior lobes control rats. A 2-min ether stress had no effect on either bioactive or immunoactive ACTH levels in the anterior lobe. Twenty-four h after adrenalectomy the anterior lobe content of both bioactive and immunoactive ACTH decreased only to return to supranormal levels 21 days after the operation. A 30-min neurogenic stress had no effect on anterior lobe bioactive ACTH content but reduced the immunoactive ACTH level to 50 ng/mg. Synthetic alphah-17-39 ACTH was used in our radio-immunoassay in order to measure the C-terminal ACTH activity of the neuro-intermediate lobe. The concentration of such C-terminal activity in control rats (890 ng alphah-17-39 ACTH/mg) considerably exceeded the amount of bioactive ACTH (15 ng/mg). This is presumably due primarily to the presence of the so-called corticotropin-like intermediate lobe-peptide (CLIP). The amounts of bioactive or C-terminal immunoactive ACTH in the neuro-intermediate lobe were not affected by ether stress nor short term (24-h) or long term (21-day) adrenalectomy. Neuro-intermediate lobe bioactive ACTH decreased (to 8 ng/mg) only with the introduction of a 30-min neurogenic stress. Neurogenic stress had no effect on the concentration of CLIP, but when the stress was imposed 24 h after adrenalectomy, a significant reduction was observed. The data support the presence of bioactive ACTH in the intermediate lobe of the rat pituitary and suggest that such ACTH is preferentially released by neurogenic stress and not appreciably regulated by circulating levels of glucocorticoids. Until the biological function and/or target organ of CLIP is identified, the significance of the changes in tissue levels of C-terminal immunoactive ACTH will remain unknown.

Control of bioactive corticotropin release from the neuro-intermediate lobe of the rat pituitary in vitro.

The purpose of this study was to identify agents capable of regulating the release of biologically active ACTH from the isolated neuro-intermediate lobe of the rat pituitary. Agents found to be potent secretagogues included acetylcholine (100 mug/ml), hypothalamic stalk-median eminence extract (0.33 eq), arginine antidiuretic hormone (100 mU/ml) and serotonin (100 mug/ml). Lower doses of arginine antidiuretic hormone (5.5 mU/ml) and serotonin (2 mug/ml) were ineffective. Dopamine 2 and 5 mug/ml) inhibited the release of biologically active ACTH whereas norepinephrine (5 mug/ml) did not. Dexamethasone (0.25 mug/ml) did not alter the basal or stimulated release of ACTH from the isolated neuro-intermediate lobe in contrast to its effect on ACTH release from the isolated anterior pituitary. Similarly, the tripeptide, prolyl-leucyl-glycinamide, which has been reported by some to inhibit MSH release, had no effect on either the basal or stimulated release of ACTH. The data suggest that regulation of ACTH release from the neuro-intermediate lobe in vivo may involve both stimulatory (acetylcholine) and inhibitory (dopamine) inputs.

Enriched populations of rat pituitary thyrotrophs in monolayer culture.

Rat anterior pituitary cells were dissociated and subjected to 4 h of unit gravity sedimentation. Eighty-five 8-ml fractions were collected, and nine pooled fractions were placed in monolayer culture for 1 week. The attached cells were immunocytochemically stained for TSH using an antiserum previously shown to be specific for the beta subunit of TSH and all culture media saved for subsequent assay of TSH. Thyrotrophs were localized both immunocytochemically and by radioimmunoassay to 1-2 fractions from the top of the sedimentation chamber. Typically, it was found that 60-80% of the cells in these fractions were immunocytochemically identified as thyrotrophs. Electron microscopic observations indicated that such cells displayed the classical morphological features associated with thyrotrophs. The enriched thyrotroph cultures responded to 1.0 mM dibutyryl cyclic AMP with an increased release of TSH indicating that the intracellular secretory machinery was not altered by the procedures employed. In all cases, the basal TSH secretion decreased with time in culture. In addition, the fractions containing the most TSH in the culture media were usually one fraction lighter (lower sedimentation rate) than the fractions which were found immunocytochemically to contain the highest percentage of thyrotrophs. The results suggest the possibility of two functionally distinct thyrotroph cell types as has been suggested for the pituitary somatotrophs.

Involvement of intracellular calcium in hormone secretion from 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.

A 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.

Effect of lead on TRH and GRF binding in rat anterior pituitary membranes.

The effect of lead on binding of the hypothalamic peptides thyroid releasing hormone (TRH) and growth hormone releasing factor (GRF) to rat anterior pituitary receptors was examined in this study. Concentrations of lead ranging from 0.01 to 1 microM did not alter [3H]TRH binding; concentrations above 1 microM increased TRH association with pituitary receptors. A previously uncharacterized ligand, [125I]GRF (human 1-44 amide), was used to examine the binding of GRF to anterior pituitary receptors. A high affinity site (GRFH = 18.1%, KH = 11.5 pM) was displaced by human growth hormone releasing factor (hGRF) (1-44)-NH2 or hGRF (1-29)-NH2 but not by rat growth hormone releasing factor (rGRF) (1-29)-NH2. Use of this ligand also revealed a class of low affinity binding sites (GRFL = 81.9%, KL = 0.39 microM) which has not been previously described. The low affinity site could be displaced by hGRF (1-44)-NH2, hGRF (1-29)-NH2 and rGRF (1-29)-NH2. A synthetic growth hormone releasing peptide (GHRP) also interacted with the low affinity GRF binding site. Lead dose-dependently displaced the binding of [125I]GRF to its pituitary receptors. The IC50 of lead for inhibiting [125I]GRF binding was 0.195 mM added lead or 52 pM free lead. These data suggest that one mechanism by which lead may affect pituitary function is through inhibition of receptor binding.

Effect of exposure to low level lead on growth and growth hormone release in rats.

This study was undertaken to examine the effect of exposure to low level lead on growth and growth hormone (GH) release. Female pups exposed to lead beginning in utero were smaller than controls on postnatal day 7 (P = 0.06). There was no corresponding effect in males. No overall differences in body weights were detected in either sex with respect to treatment effect. No differences in food or water intake were observed at any time. Pituitaries from 49-day-old lead-treated pups responded to in vitro incubation with growth hormone releasing factor (GRF) with a smaller increase in GH release than those from control pups (P = 0.08). In the case of the dams, lead did not affect body weight, body length, food consumption or pituitary responsiveness; however, water consumption was significantly increased in the lactating dam (P < 0.05). Interestingly, blood lead content in 5-day-old pups (43.3 +/- 2.7 micrograms/dl) exposed to lead in utero was more than twice that of their 49-day-old litter-mates (18.9 +/- 0.7 micrograms/dl). At 49 days blood lead levels in female pups (19.94 +/- 0.8 micrograms/dl) were significantly higher than those of male pups (17.00 +/- 1.1 micrograms/dl). Maternal blood lead levels on the same day averaged 22.7 +/- 2.5 micrograms/dl. This study suggests that exposure to a low level of lead can reduce pituitary responsiveness to a hypothalamic stimulus. In addition, the data reinforce the importance of considering age and sex when evaluating the toxic effects of lead.

Kinetic analysis of calcium distribution in rat anterior pituitary slices.

A kinetic analysis of exchangeable calcium associated with rat anterior pituitary slices was carried out. Slices were permitted to accumulate 45Ca for 1 h, and then a desaturation experiment was initiated. The resulting data was represented as a polyexponential function and analyzed with a weighted nonlinear least-squares method. The resulting coefficients and exponents were used to determine, at the steady state, the size of each calcium compartment, the unidirectional fluxes into and out of each compartment, and the corresponding rat constants. Three kinetically distinguishable compartments were defined. The first compartment exchanges with a half-time slower than calcium free in the extracellular space and can be removed by a 1.5-min exposure to Ca-free EGTA media suggesting that this calcium is bound in the extracellular matrix perhaps to the cell surfaces. The second compartment is intracellular and does not contain calcium accumulating structures such as mitochondria or microsomes. The third compartment has many of the characteristics associated with these organelles. Experiments were performed with two pituitary secretagogues, elevated K+ and ouabain, to assess changes in the study-state calcium distribution associated with enhanced secretion.

Role of calcium in acute stimulated release of prolactin from neoplastic GH3 cells.

Using a calcium-sensitive electrode to monitor calcium movements, we found that neoplastic GH3 cells experienced a net accumulation of calcium when exposed to elevated (50 mM) K+. Acute prolactin (PRL) release was also stimulated under these conditions. Both calcium uptake and PRL release could be blocked by the calcium antagonist methoxyverapamil (D-600). Thyrotropin-releasing hormone (TRH) also stimulated PRL release but had no effect on cellular calcium accumulation. Likewise, D-600 had no effect on TRH-induced PRL release. Such results indicate that enhanced secretory activity does not require an increase in intracellular calcium content. The observation that secretagogues do not stimulate PRL release in the absence of extracellular calcium was investigated. When GH3 cells were placed in a Ca-free medium, they underwent a prompt and sustained loss of cellular calcium. The loss of such intracellular calcium could be blocked with D-600. We conclude that the inability of TRH to stimulate the release of PRL in Ca-free medium is due to the loss of intracellular calcium and not to the absence of external calcium per se.

Calcium movements and intracellular calcium distribution in neoplastic GH3 cells.

Experiments were carried out to investigate the nature of the calcium homeostatic mechanisms in neoplastic GH3 rat pituitary cells. GH3 cells grown and maintained in Ham's F10 culture medium contained 35 nmoles calcium/mg cell protein. When stimulated by thyrotropin releasing hormone (TRH) or elevated K+ concentrations, only the latter caused cell calcium levels to rise although both resulted in hormone release. When exposed to EGTA, the GH3 cells lost calcium. When the temperature was lowered to 4 degrees C, the cells gained calcium and when rewarmed were able to extrude the previously accumulated calcium. The increased cell calcium following cold exposure could be blocked by prior treatment with rotenone. If rotenone was added subsequent to the cold exposure, it did not block the extrusion seen upon rewarming. In the absence of glucose in the medium, the GH3 cells took up more calcium upon exposure to 4 degrees C, and upon rewarming the cells could not return to their previous low levels. There are thus significant differences in calcium homeostasis between the neoplastic GH3 cells and their normal pituitary counterparts. When intracellular calcium was localized with the potassium pyroantimonate technique, there was calcium found in/on mitochondria, membrane bound vesicles and plasma membrane. Nuclear staining was sparse, and nucleolar staining was virtually absent. Upon stimulation with TRH, there was a decrease in mitochondrial calcium along with increases in both plasma membrane and nucleolar calcium levels. Since total calcium is unchanged, this indicates a significant calcium redistribution in response to TRH. The increased nucleolar calcium may reflect a calcium dependent increase in mRNA synthesis as has been reported. Since TRH presumably acts at a surface receptor, the increased plasma membrane calcium might be functionally related to receptor activation.

Effect of normal and cystic fibrotic serum on ion transport and mucus glycoprotein secretion from the isolated rabbit trachea.

It has been argued that individuals with cystic fibrosis (CF) might be expected to lead reasonably normal lives if the secondary changes associated with the disease could be eliminated or overcome. The purpose of this study was to characterize the electrical and transport properties of the isolated rabbit trachea in the presence and absence of control or CF serum. In addition, we sought to determine whether the presence of CF serum resulted in an alteration in either the amount or type of mucus glycoprotein secreted by the trachea. It was found that both control and CF sera inhibited the short-circuit current of the rabbit trachea due primarily to an inhibition of active sodium transport. The effect was irreversible and could be abolished by heating the serum prior to the experiment and was only observed when the serum was on the luminal surface. There was no differential effect between control or CF serum. Investigation also indicated that exposure of the isolated trachea to CF sera had no effect on the amount of mucus glycoproteins as indicated by the degree of sulfation in secretion samples eluted from DEAE cellulose following exposure to CF serum. It is possible that alterations in the type of mucus secreted subsequent to exposure to CF serum might relate to the turbidity of the mucus seen in such patients.

Childhood lead toxicity and impaired release of thyrotropin-stimulating hormone.

Decreased stature of children is epidemiologically associated with increased blood lead independent of multiple socioeconomic and nutritional variables. Since endocrine dysfunction occurs in adult lead workers, we studied two girls, 2 years of age, before and after calcium disodium edetate chelation for blood leads (PbB) of 19-72 micrograms/dl. The height of both children had crossed from the 50th to below the 10th percentile during the course of chronic lead toxicity. Basal free T4, T4, T3, cortisol, somatomedin C, and sex steroids were normal. A decrease in the growth hormone response and elevation of basal prolactin and gonadotropins were noted in one. Both children demonstrated blunted thyrotropin-stimulating hormone (TSH) responses to thyrotropin-releasing hormone (TRH) in six of seven challenges. This prompted in vitro studies of cultured cells from rat pituitaries. After incubation of pituitary cells with 0.1-10 microM Pb2+ for 2 hr, followed by the addition of TRH, there was a dose-dependent inhibition of TSH release. Lead did not interfere with the assay of TSH. To investigate the interaction of lead and calcium, 45Ca2+ kinetic analyses were done on rat pituitary slices after 1 hr incubation with 1.0 microM lead. The impaired late efflux was consistent with a decrease in the size and exchangeability of the tightly bound pool of intracellular microsomal or mitochondrial calcium. The rat pituitary cell model provides a model for the decreased TSH release of lead poisoning, supports the biological plausibility of a neuroendocrine effect on growth, and suggests that interference with calcium-mediated intracellular responses is a basic mechanism of lead toxicity.