Regulation of secretion of parathormone and secretory protein-I from separate intracellular pools by calcium, dibutyryl cyclic AMP, and (1)- isoproterenol

Dispersed porcine parathyroid cells were incubated at calcium concentrations between 0.5 and 3.0 mM in the presence of 3H- or 14C- amino acids to label newly synthesized parathormone. Up to four times more hormone was secreted at the lower calcium concentration but its specific radioactivity, from 30 to 50 times that of the intracellular pool, did not change. Dibutyrl cyclic AMP doubled immunoactive parathormone secretion at each calcium concentration, but there was no increase in secretion of radioactive hormone if labeled amino acids and secretagogue were added simultaneously. Similarly, when the intracellular pool of parathormone was prelabeled with 3H-amino acids and then the cells were incubated in 14C-amino acids and dibutyryl cyclic AMP, the entire increase in hormone secreted was derived from the prelabeled pool. (1)-isoproterenol increased intracellular cyclic AMP and acted on hormone secretion in a manner indistinguishable from dibutyryl cyclic AMP. In similar double-label experiments dibutyryl cyclic AMP preferentially enhanced secretion of secretory protein-I, a calcium-regulated protein of the parathyroid of unknown function. Calcium, alone, inhibited the intracellular level of cyclic AMP in a concentration-dependent fashion. These data are consistent with the existence in the parathyroid cell preparation of two hormone and secretory protein pools that may be individually recruitable--one consisting of most recently synthesized protein, the other consisting of older "storage" protein. The data do not allow one to decide whether the two pools coexist within individual cells or whether, instead, they exist in separate cells of the dispersed gland preparation.     

The secretion of parathormone and glycosylated proteins by parathyroid cells in culture

The secretion of radioactive peptides by dispersed porcine parathyroid cells incubated with [³H]- or [¹⁴C]amino acids, [³H]glucosamine and [³H]mannose was analyzed. After incubation, the culture medium contained radioactive parathormone, as expected, and two radioactive glycopeptides: SP I and SP II. SP I appears to be identical with $\text{parathyroid}\text{secretory}\text{protein}$, heretofore not recognized as a glycoprotein. SP II has not been previously identified. SP I, but not SP II or parathormone, was adsorbed by Concanavalin A possibly reflecting a high mannose content of this molecule. Raising the concentration of calcium in the medium suppressed the secretion of radioactive parathormone and SP I in a similar fashion but did not affect the secretion of SP II. Our results suggest that SP I may play a fundamental role in parathyroid synthetic or secretory processes.     

Formation and secretion of fragments of parathormone. Identification of cleavage sites

Monolayer cultures of bovine parathyroid cells or fresh gland slices were incubated with radioactive amino acids in order to study the formation and metabolism of parathormone (PTH). PTH, secretory protein I, and COOH-terminal fragments of PTH were all released into media within 30 min, most strongly in the first hour after synthesis. Peptides in tissue, cells, and media were separated using reverse-phase high performance liquid chromatography. In eluates of media, six radioactive peaks were prominent. The first four and the sixth were immunoreactive in a COOH-terminal specific PTH radioimmunoassay, but only the sixth was reactive in an NH2-terminal specific assay. Under conditions where recovery of PTH(1-34) was quantitative, gel filtration of media was used to show that no NH2-terminal fragments of PTH were secreted. Sequence analyses of secreted COOH-terminal peptides indicated that the NH2 termini of the first three peaks corresponded to residues 43, 37, and 34 of PTH. The fourth peak contained a mixture of two peptides with NH2 termini at residues 24 and 28 of PTH. The fifth could not be identified; the sixth was PTH. Cleavages at the 23-24 bond of PTH occurred within minutes of the formation of PTH itself, and the other peptides were formed more slowly. Mandatory cleavage of PTH at the 23-24 peptide bond would destroy the biological activity of the hormone on kidney and bone, a situation consistent with the possibility that intracellular PTH metabolism participates in secretory regulation. The results showed that different peptides were generated in parathyroid cells than were previously shown to be produced by cathepsin B or D. The results suggest that the proteolytic pathway which results in the secretion of PTH fragments is nonlysosomal in nature.     

Study of acute pharmacologic effects of prolactin on calcium and water transport in the rat colon by an in vivo perfusion technique

We investigated the acute effect of intraperitoneally administered prolactin on calcium and water transport in colon of sexually mature female Wistar rats using an in vivo perfusion technique. Test solution containing (in mM) NaCl, 100; KCl, 4.7; MgSO4, 1.2; CaCl2, 20; D-glucose, 11; sodium ferrocyanide (Na4Fe(CN)6), an index of net water transport, 20; and 0.7 (microCi 45CaCl2 (1 Ci = 37 GBq) was perfused througth the 8-cm colonic loop for 60 min at perfusion rates of 0.5 or 1.0 mL x min(-1). Calcium and water transport was also studied under a no flow condition to stimulate the condition often found in the colon by in vivo ligated colonic loop for 30 min. Control results showed no correlation between calcium transport and water flux. Flow of luminal solution at 0.5 and 1.0 mL x min(-1) was found to reverse net calcium absorption from 0.04+/-0.01 nmol x g(-1) dry weight x h(-1) to net calcium secretion of 0.04+/-0.04 and 0.9+/-0.02 nmol x g(-1) dry weight x h(-1), respectively. Neither 0.4, 0.6, nor 1.0 mg x kg(-1) prolactin had any effect on calcium fluxes in the colon. On the other hand, at a perfusion rate of 1 mL x min(-1), 0.4 mg x kg(-1) prolactin significantly decreased net water absorption from 3.86+/-0.90 to 0.88+/-0.64 mL x g(-1) dry weight x h(-1) (P < 0.001), and the higher doses of 0.6 and 1.0 mg x kg(-1) prolactin reversed net water absorption to net water secretion of 2.20+/-0.63 and 2.33+/-0.89 mL x g(-1) dry weight x h(-1), respectively (P < 0.001). The stimulatory effect of prolactin on water transport was completely abolished by reducing the perfusion rate from 1.0 mL x min(-1) to zero. The stimulatory effect of prolactin on water secretion at perfusion rate of 1.0 mL x min(-1) was also abolished when luminal [Na+] was reduced from 180 to 80 mM. We concluded that, unlike in the small intestine, calcium fluxes in the colon are not related to water transport and did not respond at all to prolactin. Water transport, on the other hand, was reversed from net absorption to secretion by prolactin. We propose that this prolactin-induced water secretion is probably mediated by recycling of luminal sodium in the vicinity of tight junctions.     

Glucose-stimulated insulin release by individual pancreatic beta cells: potentiation by glyburide.

To investigate the effect of glyburide on insulin secretion by individual beta cells from normal rats, we employed a reverse hemolytic plaque assay. Pancreata were harvested from female Wistar-Furth rats, the pancreatic islets isolated, and the latter dispersed into single cells. These cells were mixed with protein A-coated ox erythrocytes, the mixture was placed in a Cunningham chamber in the presence of insulin antiserum, and the cells were exposed to the various test substances. Having developed hemolytic plaques around the insulin-secreting cells with complement, the percentage of plaque-forming cells was determined and the plaque areas (reflecting the amount of insulin secreted) were quantitated. For the purpose of validation, we demonstrated that (i) plaque-forming (but not nonplaque-forming) cells could be identified as insulin secreting by an independent immunofluorescent technique, (ii), plaques did not form if insulin antiserum was deleted from the preparation, (iii) plaques failed to develop if insulin antiserum was preabsorbed with insulin, and (iv) incubation with non-protein A-coated RBC or omission of complement resulted in no plaque formation. In addition, both the percentage of plaque-forming cells and the mean plaque are increased upon exposure to glucose (0.75-20 mM) in a concentration-dependent manner at 5- and 60-min incubation times. Moreover, somatostatin suppressed the percentage of plaque-forming cells and diminished the mean plaque area of cells which continued to secrete insulin in response to glucose. Exposure of cells to 100 nM glyburide in the presence of 5 mM or 20 mM glucose had no effect on the percentage of plaque-forming cells present at 5 min or 60 min. Similarly, glyburide did not alter mean plaque area at 5 or 60 min when cells were co-incubated with 5 mM glucose. However, mean plaque area was markedly enhanced at 5 and 60 min in response to glyburide and 20 mM glucose. These results demonstrate that glyburide (i) does appear to enhance insulin secretion by an effect directly on the pancreatic beta cell; (ii) does not act by recruiting previously noninsulin-secreting cells into a secretory pool; (iii) does not potentiate the effect of glucose, at fed concentrations, on insulin secretion by individual cells; but (iv) does augment insulin secretion by beta cells stimulated with supraphysiologic concentrations of glucose.     

Characteristics of the response of dispersed guinea-pig adrenal cells to low doses (1-50 pg/ml) of alpha 1-24 adrenocorticotrophin

Isolated adrenal cells prepared by tryptic digestion of the guinea-pig adrenal gland are sensitive to low concentrations (less than 25 pg/ml) of adrenocorticotrophin (ACTH). Cell which have been pre-incubated for 2 h. centrifuged and resuspended in fresh culture medium prior to the introduction of 10 pg/ml ACTH for 60 min show a marked increase (328 +/- 109 nmol/l; mean +/- SD) in cortisol secretion over the control compared to freshly dispersed cells (75 +/- 45 nmol/l). Further potentiation of the ACTH effect was seen with the pre-incubated cells by suplementing the medium with calcium (8 mM) and ascorbate (2 mM) but not with theophylline (1 mM). Basal cortisol secretion was not affected by any of the additives. In the presence of 8 mM calcium and after 60 min incubation 10 pg/ml ACTH stimulated cortisol secretion from 328 nmol/l over the control to 839 +/- 382 nmol/l. The effect of ascorbate (2 mM) was to further increase the effect of ACTH at all dose levels tested (1-25 pg/ml). The concentration of ACTH required to provoke half maximal cortisol secretion decreased from 95 pg/ml with normal medium to 12 pg/ml with calcium -ascorbate supplemented medium. Using this supplemented medium the cells were sensitive to 1 pg/ml and cortisol secretion was stimulated 10-fold over the control with 50 pg/ml, a dose which saturated the system.     

Secretion of oxytocin and progesterone by ovine corpora lutea in vitro

The mechanisms involved in the control of oxytocin and progesterone secretion by the ovine corpus luteum have been investigated in vitro using luteal slice incubations. Oxytocin and progesterone were secreted at constant rates from luteal slices for 2 h of incubation (366 +/- 60 pg X mg X h and 18.9 +/- 0.18 ng X mg X h, respectively). Secretion of progesterone, but not of oxytocin, was significantly (p less than 0.02) stimulated in the presence of ovine luteinizing hormone. Incubation of luteal slices in medium containing 100 mM potassium, however, resulted in increased secretion of oxytocin and, to a lesser extent, of progesterone (294 +/- 59% and 142 +/- 15%, respectively, p less than 0.05). Basal oxytocin secretion was reduced during incubation in calcium-free medium, compared to secretion in the presence of calcium (70 +/- 15 and 175 +/- 25 pg X mg X 20 min, respectively, p less than 0.01), whereas progesterone secretion was not altered in the absence of calcium. Secretion of both hormones by luteal slices was stimulated by the addition of the calcium ionophore A23187 (p less than 0.05). Addition of prostaglandin F2 alpha (2.8 microM) had no effect on secretion of either oxytocin or progesterone. We have demonstrated that oxytocin and progesterone can be stimulated, independently, from corpus luteum slices incubated in vitro. The pattern of release is consistent with the proposal that oxytocin, but not progesterone, is associated with and actively released from luteal secretory granules. Our results also indicated that prostaglandin F2 alpha does not directly stimulate release of oxytocin or progesterone from luteal cells in vitro.     

Multiple episodes of induced secretion of human growth hormone from recombinant AtT-20 cells

Recombinant AtT-20 cells expressing human growth hormone (hGH) secreted the hormone at a constant, basal rate of 0.3–0.5 ng/10⁵ cells-hour when exposed to medium without secretagogues. When triggered with 8 bromo-cyclic AMP, cells secreted hGH at an initial rate of 1.7 ng/10⁵ cells-hour while intracellular hGH declined sharply. Upon extended exposure to secretagogue, secretion decreased gradually to the basal rate and intracellular hGH stabilized at a value 40% the initial. In cells switched from secretion to growth medium, the total rate of hGH accumulation intracellularly and in medium was 2.2 times that observed with cells never exposed to secretagogue; however, only a fraction of the hormone was stored intracellularly and the rest was secreted. When cells were exposed alternately to growth and secretion medium, induced cells secreted at rates at least two times higher than uninduced controls during the first five cycles. The induced response deteriorated with time, however, in parallel with outgrowth of attached cells by foci of round cells, and by the eighth cycle induced secretion did not occur. Operational modifications that may improve the performance of cycling schemes are discussed.     

Barium distinguishes separate calcium targets for synthesis and secretion of peptides in neuroendocrine cells

The effect of barium and potassium on the secretion and biosynthesis of enkephalin in bovine chromaffin cells, and prolactin and beta-endorphin in rat anterior pituitary cells, was examined to determine whether calcium-dependent secretion and biosynthesis are mediated by the same or by different calcium targets within the neuroendocrine cell. In the presence of 1.8 mM calcium, barium and potassium stimulated the secretion of all three peptides over 30 min, and increased the levels of proenkephalin and prolactin mRNA in 24 hr. These effects were inhibited by the calcium channel blocker D600. When the extracellular calcium concentration was lowered to 0.1 mM or less, secretion elicited by potassium was blocked, whereas secretion elicited by barium was enhanced, indicating that barium wholly substitutes for extracellular calcium in mediating peptide secretion. On the other hand, stimulation of proenkephalin and prolactin mRNA by both potassium and barium was inhibited when the extracellular calcium concentration was reduced. We conclude that calcium acts at two different intracellular targets to activate secretion versus biosynthesis of both enkephalin and prolactin. This appears to be the first report in which two different calcium-dependent processes in the intact cell are distinguished by a calcium ion agonist. Calcium-dependent processes such as protein phosphorylation, protein translocation, and enzyme activation may thus be related to events in the intact cell such as peptide synthesis and secretion on the basis of selective stimulation by barium.     

Secretion of proalbumin by canavanine-treated Hep-G2 cells

The two processing sites in the conversion of preproalbumin to albumin are marked by arginine residues. Therefore, to study the mechanisms of albumin processing and secretion, the arginine residues of nascent albumin were replaced with canavanine by the incubation of Hep-G2 cells with this arginine analog. During a 4-h interval, canavanine inhibited (67%) the secretion of nascent albumin and increased the intracellular transit time of albumin secretion from 24 to 39 min. At 1 h, canavanine inhibited total protein synthesis by 19% and albumin synthesis by about 40%. Both the intracellular and secreted albumin produced by canavanine-treated cells were analyzed by DEAE-cellulose chromatography and were found to be more acidic than normal proalbumin and albumin. Further analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the albumin produced and secreted by canavanine-treated cells appeared to have a larger molecular weight (by 4000) than serum albumin. The canavanine-treated cells were incubated with L-[3H]leucine and L-[3H]phenylalanine and the location of radioactive L-leucine and L-phenylalanine in the 30 NH2-terminal amino acid residues of secreted albumin was determined. The results indicated that canavanine-treated cells secreted proalbumin (79%) and also some fully processed albumin (21%). Preproalbumin was not secreted. Untreated Hep-G2 cells mostly secreted fully processed serum albumin (93%) with only traces of proalbumin (7%).     

Opioidergic control of gonadotropin secretion in the female rabbit: divergent effects of morphine on secretion of follicle-stimulating hormone and luteinizing hormone

The nature of secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was followed in female rabbits on a daily basis from age 36 to 60 days by sequential 5-min blood sampling over 1- to 2-h periods each day. Both LH and FSH were found to be secreted in a pulsatile manner. The mean LH pulse amplitude over the 25 days was 0.95 +/- 0.32 ng/mL and for FSH it was 10.15 +/- 1.11 ng/mL. Mean plasma LH levels were significantly increased from 1.46 +/- 0.08 ng/mL in 36 to 42-day-old rabbits to 1.89 +/- 0.12 ng/mL in 43 to 50-day-old rabbits and remained elevated from 50 to 60 days. FSH levels during the same periods also rose significantly from 14.93 +/- 0.79 to 19.57 +/- 2.05 ng/mL. To examine the influence of endogenous opioid peptides on the release of LH and FSH in 36 to 60-day-old female rabbits, morphine sulfate at 0.2, 0.5, 2.0, and 5.0 mg/kg was administered subcutaneously after 30 min baseline sampling, and blood was taken for another 60-120 min. Morphine at all doses and at all ages inhibited the amplitude and frequency of LH pulses but had no effect on FSH secretion. To determine whether the effects of morphine on LH secretion could be reversed with naloxone, females aged 82-114 days were used. Naloxone administered 1 h after morphine reversed the inhibitory effects of morphine, whereas the simultaneous administration of naloxone with morphine had variable effects but seemed to delay the LH increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium fluxes in isolated acinar cells from rat parotid. Effect of adrenergic and cholinergic stimulation.

Rat parotid acinar cells dispersed by a combination of enzymatic treatments remain sensitive to adrenergic and cholinergic agonists. Previous studies have implicated Ca2+ in both adrenergic and cholinergic responses. This paper describes the effects of adrenergic and cholinergic stimulation upon 45Ca2+ fluxes in isolated parotid acinar cells. Suspensions of dispersed cells took up 45Ca2+ from the medium. The net rate of isotope influx was increased by the adrenergic agonists epinephrine, norepinephrine, isoproterenol, and phenylephrine, and by the cholinergic agonists acetylcholine and carbamylcholine. In 1 mM Ca2+, epinephrine was capable of increasing the 45Ca2+ influx in 40 min to three times that of resting cells. Isoproterenol, a beta-adrenergic agonist, was only half as effective as epinephrine in stimulating maximal calcium uptake although it was equally effective in stimulating maximal amylase release in the same cells. Experiments with the alpha-adrenergic antagonist phentolamine, the beta-adrenergic antagonist propranolol, and the cholinergic antagonist atropine confirmed that alpha- and beta-adrenergic and cholinergic stimulation each had a direct stimulatory effect on 45Ca2+ uptake. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate also caused some stimulation of net calcium uptake. Direct measurement of Ca2+ efflux indicated that the increased calcium uptake in the presence of epinephrine was not the indirect result of a decrease in efflux. The rates of both basal and epinephrine-stimulated calcium uptake increased with increasing calcium concentration in the medium. Epinephrine had little effect on the rate of calcium uptake at 0.15 mM Ca2+. Although the energy poison NaCN had little effect on the basal rate of calcium uptake, the stimulable component of calcium uptake was inhibited by NaCN at all calcium concentrations tested (0.2 to 4.1 mM).     

Action of aldosterone on cultured renal collecting duct cells during the latent period.

The effects of aldosterone on protein synthesis in the latent period were investigated on cultured renal collecting duct cells from neonatal rabbit kidneys. Tissue was incubated with radioactively labelled uridine and amino acids and then precipitated with trichloroacetic acid in order to determine the intracellular precursor pool and identify new synthesis of RNA and protein. During the latent period, aldosterone increased the intracellular radioactive uridine pool and total radioactive RNA content already 20 and 60 min after its application; conversely 40 min after aldosterone introduction, no stimulation was found. Further experiments revealed that the intracellular radioactive amino acid pool was generally increased by aldosterone after 20, 40 and 60 min, while a distinct increased radioactive protein content was found to be induced by aldosterone only after 40 min. This indicates that aldosterone increases the uptake of RNA and protein precursors and the new synthesis of RNA and proteins. These events seem to to be regulated not continuously but intermittently. The induced proteins possibly take part in the mediation of the early hormone response. Experiments with the aldosterone antagonist, spironolactone, provide evidence for the specificity of the described hormone effects. The results after application of the Na+ channel blocker, amiloride, and the Na+/K(+)-ATPase inhibitor, G-strophanthin, indicate that the aldosterone effects are controlled by Na+ channels and Na+ pumps and therefore by the intracellular Na+ content. The inhibitory effect of cycloheximide on the aldosterone-induced protein synthesis indicates the role of these proteins on the hormone-stimulated Na+ transport.     

Methionine-enkephalin in a porcine endometrial cell line and its responsiveness to potassium depolarization.

Immunoreactive methionine-enkephalin (ir-MENK) has been identified in the porcine uterine fluid and endometrium. Previously, we have established a porcine endometrial cell line of epithelial origin (PE-1) by transfecting primary endometrial cells with temperature sensitive SV40 DNA. The current study was conducted to identify and characterize ir-MENK present in PE-1 cells, and to investigate the effect of KCl depolarization on the kinetics of ir-MENK secretion. PE-1 cells were cultured at 33C until confluency was reached (33C cells), after which they were incubated at 40C for 2 days (40C cells). Ir-MENK in PE-1 cells was analyzed by Sephadex G-15 gel filtration and reverse phase (RP)-HPLC. Analysis of 40C cell extract by Sephadex G-15 and RP-HPLC indicated that the major portion of ir-MENK present in PE-1 cells was eluted at a position similar to that of synthetic MENK. The effect of temperature on ir-MENK synthesis in PE-1 cells was examined by measuring ir-MENK content in 33C and 40C cells over a 14-day culture period. Compared to 33C cells, 40C cells maintained higher and steadier levels of ir-MENK, suggesting that synthesis of ir-MENK is temperature sensitive. KCl stimulated ir-MENK secretion at all concentrations tested (5-60 mM for 60 min), with 30 mM being the optimal concentration. Temporal analysis of ir-MENK secretion showed that incubation for 60 min with 30 mM KCl allowed maximal secretion. Secretion of ir-MENK from PE-1 cells resulted in depletion of ir-MENK in cell content. These results demonstrate that PE-1 cells contain ir-MENK which is biochemically similar to synthetic MENK, PE-1 cells synthesize ir-MENK in a temperature sensitive manner, and these cells secrete ir-MENK upon KCl stimulation.     

Influence of divalent cations upon complement-mediated enzyme release from human polymorphonuclear leukocytes.

The complement component, C5a provokes the selective release of granule-associated enzymes from the intact, viable cytochalasin B-treated human polymorphonuclear leukocytes (PMN) in the absence of phagocytosis or cellular adherence to surfaces. Consquently, in this experimental system the influence of divalent cations on these two processes can be disregarded and their effects on enzymes secretion can be studied directly. Cytochalasin B-treated PMN exposed to C5a in calcium and magnesium-free media consistently secreted significant amounts of the granule-associated enzymes, beta-glucuronidase and lysozyme. The basal secretory response was not diminished if cells were preincubated with 5.0 mM EDTA, nor was it influenced if 1.0 mm or 2.0 mM EDTA were present in the reaction mixtures. The addition of calcium (up to 1.5 to 2.0 mM) produced a concentration-dependent enhancement of beta-glucuronidase release, whereas increasing amounts of calcium (above 2.0 mM) inhibited secretion of this enzyme. Lysozyme release was similarly enhanced by the addition of calcium, but inhibition with high concentrations was not observed. Calcium per se, in the absence of C5a, provoked only the release of lysozyme from these cells. The effects of calcium upon enzyme release were not associated with alterations in the state of assembly of cytoplasmic microtubules. These findings provide another example of the role of calcium in "stimulus-secretion coupling" and provide evidence that exocytosis of various granules in human PMN is regulated by independent mechanisms involving calcium.     

Restoration of Catecholamine Content of Previously Depleted Adrenal Medulla In Vitro: Importance of Synthesis in Maintaining the Catecholamine Stores

The functional integrity of adrenal chromaffin storage vesicles was studied in the perfused rat adrenal gland subjected to intense exocytosis. Continuous perfusion with 55 mM K+-Krebs solution produced a large and uninterrupted secretion of catecholamines. Total amounts secreted within 45 min were 4.66 micrograms and represented almost 30% of the total tissue catecholamine content. If perfusion with excess K+ was extended to 90 min, the secretion increased further to 5.76 micrograms. Despite such a large secretory response, the catecholamine content of the K+-stimulated adrenal medulla was comparable to that of unstimulated control, suggesting an enhanced resynthesis to maintain the normal levels. Pretreatment of rats with alpha-methyl-p-tyrosine, and including this agent in the perfusion medium during stimulation with K+, caused a marked reduction in catecholamine content. The degree of depletion depended on the extent of stimulation with K+ (45% in 45 min and 60% in 90 min). Although depleted catecholamine stores did not show spontaneous recovery in 2 h, inclusion of tyrosine, L-3,4-dihydroxyphenylalanine or dopamine (but not epinephrine or norepinephrine) completely restored the catecholamine content of previously depleted adrenal medulla. Repletion achieved by tyrosine was time dependent (evident in 30 min and maximum in 2 h) and blocked by alpha-methyl-p-tyrosine but not by calcium deprivation. The ratio of epinephrine to norepinephrine remained constant during various stages of the experiment, suggesting both types of vesicles were equally affected by different treatments. The secretory response (10 Hz for 30 s) was unaffected even though tissue catecholamine stores were significantly depleted (50%). In summary, we have demonstrated that catecholamine content of the isolated perfused adrenal gland can be reduced by stimulation of exocytotic secretion in the presence of tyrosine hydroxylase inhibitor. Since the depleted stores can be fully refilled by synthesis of catecholamines from its precursors, it is suggested that chromaffin vesicles may be reutilized for the purpose of synthesis, storage, and secretion of adrenal medullary hormones.     

Insulin, glucagon, and somatostatin secretion by cultured rat islet cell tumor and its clones

Cells derived from rat islet tumor and grown in culture (parent cells-RIN-m) and two clones obtained from them were used to study the effect of various secretagogues on insulin, glucagon, and somatostatin secretion. Parent cells secreted all three hormones in various quantities, while clone 5F secreted predominantly insulin and clone 14B secreted predominantly somatostatin. The secretory behavior of these cells were compared to each other and to that of normal islets. In general, as in the case of normal islets, insulin secretion was stimulated by calcium, potassium, tolbutamide, theophylline, and glucagon. It was inhibited by somatostatin. Glucagon secretion was stimulated by calcium, arginine, and theophylline. Somatostatin secretion was stimulated in clone 14B by arginine, tolbutamide, theophylline, and insulin. These cells differ from normal islets, in that they do not respond to glucose or arginine with increased insulin secretion. Also somatostatin failed to inhibit glucagon secretion. The similarity in insulin secretory responses of parent cells and clone 5F suggests that local or paracrine islet hormone secretion plays only a negligible role in the control of other hormone secretion in these cells.     

Evidence for the production of a proteinaceous hemolytic exotoxin by wild-type strain of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 (Cyanobacteria)

A proteinaceous hemolysin produced by a wild-type strain of Synechocystis sp. PCC 6803 was purified from cell-free culture supernatants by successive column chromatography on DEAE-Sepharose Fast Flow and Sephacryl S-300 High Resolution. The molecular mass of the hemolysin, determined by SDS-PAGE, was approximately 81 kDa. The hemolysin was heat labile and showed potent hemolytic activity against rabbit and sheep erythrocytes. The hemolysin started to be secreted during the exponential growth phase and accumulated maximally at the stationary phase. The production of hemolysin varied with the amount of calcium present in modified BG-11 culture medium. Hemolysin production decreased in calcium-free medium, whereas it increased in medium containing 0.48 mM calcium. In contrast, the potency of hemolysin, as shown by hemolysis assay, was enhanced by deprivation of calcium (EDTA treatment) but decreased in the presence of calcium. Our results show that calcium stimulated production and secretion of hemolysin, but inhibited hemolytic potency.     

Evidence that potassium channels regulate prolactin secretion in GH4C1 cells by causing extracellular calcium influx

Tetraethylammonium (TEA), a K+ channel blocker, induced prolactin (PRL) secretion in GH4C1 cells in a dose-dependent manner when applied at a concentration from 1-20 mM. During continuous exposure to TEA, a significant increase in PRL secretion occurred by 20 min and the response was sustained until the end of a 60-min exposure. Blocking Ca2+ influx by employing a Ca(2+)-depleted medium or the Ca2+ channel blocker, nifedipine, prevented induction of PRL secretion by 20 mM TEA. Preincubation of the cells for 10 min with 20 mM TEA did not inhibit PRL secretion induced by thyrotropin-releasing hormone (TRH), phorbol 12-myristate 13-acetate (TPA) or by cell swelling produced by 30% medium hyposmolarity, but significantly depressed that induced by depolarizing 30 mM K+. BaCl2, another K+ channel blocker, had the same effect on PRL secretion as TEA. The data suggest that blocking K+ channels may cause membrane depolarization, thereby inducing Ca2+ influx which is a potent stimulus for PRL secretion in GH4C1 cells.     

Differential carbohydrate processing and secretion of thyrotropin and free alpha subunit. Effects of 1-deoxynojirimycin

In pulse-chase experiments we compared the kinetics of early carbohydrate processing and subsequent secretion of thyroid-stimulating hormone (TSH) and free alpha subunit under control conditions and after treatment with 1-deoxynojirimycin, an inhibitor of glucosidases I and II. Under control conditions TSH achieved resistance to endo-beta-N-acetylglucosaminidase H (endo H) more rapidly than free alpha (t1/2 0.3 h versus 0.9 h); however, free alpha was secreted more rapidly than TSH (t1/2 2.2 h versus 3.4 h). With 1-deoxynojirimycin, oligosaccharides co-migrating with G3Man9GlcNAc and G2Man9GlcNAc were demonstrated on TSH for the first time, suggesting that previous pulse-chase studies did not disclose these intermediates due to rapid removal of glucose residues from the common G3Man9GlcNAc2 precursor. 1-Deoxynojirimycin delayed the rate of attainment of endo H resistance for both TSH and free alpha, but there was no effect on subunit combination. With 5 mM 1-deoxynojirimycin the amount of secreted free alpha was reduced to 65% of control; secreted TSH was reduced markedly to 17% of control without intracellular accumulation, suggesting increased intracellular degradation. There was no significant toxicity from these doses of 1-deoxynojirimycin on the production or secretion of the two major nonglycosylated pituitary proteins, growth hormone and prolactin, or on at least 10 other secretory proteins. Basal differences in the relative rates of TSH and free alpha processing and secretion as well as differential sensitivity to 1-deoxynojirimycin suggest separate secretory pathways for these two closely related proteins.