Perifusion Medium Research Articles

Regulation by 1,4-diamines of the ornithine decarboxylase activity induced by ornithine in perifused tumor cells

Ornithine decarboxylase (ODC) activity of Ehrlich carcinoma cells was increased more than 36-fold after being maintained for 3.5 hr in vitro in a special chamber which allowed continuous perifusion with 0.5 mM ornithine; if incubated in vitro without perifusion the ODC activity was, of course, only 9-fold by the same concentration of ornithine. Ornithine withdrawal from the perifusion medium resulted in a decay of enzyme activity observed after 90 min; this decay was prevented by addition of 55 μM pyridoxal to the medium. The 1,4-diamines putrescine, spermidine, spermine, agmatine, histamine, serotonin, tryptamine, chlorpheniramine and harmaline at 55 μM strongly suppressed ODC induction by 0.5 mM ornithine in perifused Ehrlich ascites cells. Methyl derivatives also behave as strong inhibitors of ODC induction. On the contrary, N-acetylation paralleled with a decrease in the inhibition capacity: 55 μM N-acetyl putrescine, N-acetyl serotonin or N-ω-acetylhistamine suppressed ODC induction by ornithine in 66, 64 and 19%, respectively. The addition to the perifusion medium of the same concentrations of 1,3-diamines (1,3-diaminopropane, 1,3-diamino-2-propanol or the alkaloid gramine) as well as 1,5-diamines (1,5-diaminopentane and the antihistaminic doxylamine or cimetidine) failed to suppress the induction of ODC activity by ornithine. Interestingly, 1,4-benzenediamine, which strongly inhibits ODC activity when the induced enzyme is assayed in its presence, did not suppress the induction of the enzyme when both 0.5 mM ornithine and 55,u;M 1,4-benzenediamine were present in the perifusion medium. The inhibitory capacity in down-regulating ODC is not due to differences in the diamine uptake by the cells. The results suggest that the N—N distance (6 Å) and the charge of one amino group are important chemical characteristics for regulatory effects.

Insulin pulses less effective than continuous insulin in inhibiting PEPCK mRNA levels stimulated by cAMP and dexamethasone in perifused hepatoma cells.

Hepatic glucose production is stimulated in vitro twice as effectively by pulsatile as by continuous glucagon, given equivalent time-averaged doses. Efficacy studies of pulsatile insulin have yielded conflicting results. In the rat hepatoma cell line H-4-II-E-C3, insulin rapidly (t1/2 15 min) inhibits transcription of the gene and lowers mRNA levels for the gluconeogenic enzyme. PEPCK via a receptor-mediated process. We attached H-4-II-E-C3 cells to Cytodex-3 microcarriers and used a perifusion column system to test whether pulsatile insulin is more or less effective than equivalent time-averaged doses of continuous insulin. PEPCK transcription was induced by inclusion of cAMP analogue 8-(4-chlorophenyl-thio)-cAMP (0.1 mM) and dexamethasone (0.5 microM) in the perifusion medium. Three columns were exposed either to continuous, pulsatile, or no insulin. After 3 h, total nucleic acid was extracted, and mRNA(PEPCK) was measured with a sensitive-solution hybridization assay. Continuous insulin inhibited PEPCK expression in a dose-dependent fashion with EC50 1 x 10(-11) M. Equivalent time-averaged amounts of insulin delivered as pulses achieved significant inhibition but less effectively than continuous insulin. The apparent EC50 for pulsatile insulin increased from 2 x 10(-11) M to 5 x 10(-11) M as the oscillatory period was raised from 5 to 20 min, respectively. These observations suggest that insulin-mediated inhibition of PEPCK gene transcription is diminished by a pulsatile mode of administration in marked contrast to the pulse enhancement demonstrated for glucagon-mediated hepatic glucose production.

Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion

Free fatty acids in isolated pancreatic islets have been quantified by gas chromatography-mass spectrometry after stimulation with insulin secretagogues. The fuel secretagogue D-glucose has been found to induce little change in islet palmitate levels but does induce the accumulation of sufficient unesterified arachidonate by mass to achieve an increment in cellular levels of 38-75 microM. Little of this free arachidonate is released into the perifusion medium, and most remains associated with the islets. Glucose-induced hydrolysis of arachidonate from islet cell phospholipids is reflected by release of the arachidonate metabolite prostaglandin E2 (PGE2) from perifused islets. Both the depolarizing insulin secretagogue tolbutamide (which is thought to act by inducing closure of beta-cell ATP-sensitive K+ channels and the influx of extracellular Ca2+ through voltage-dependent channels) and the calcium ionophore A23187 have also been found to induce free arachidonate accumulation within and PGE2 release from islets. Surprisingly, a major fraction of glucose-induced eicosanoid release was found not to require Ca2+ influx and occurred even in Ca(2+)-free medium, in the presence of the Ca(2+)-chelating agent EGTA, and in the presence of the Ca2+ channel blockers verapamil and nifedipine. Exogenous arachidonic acid was found to amplify the insulin secretory response of perifused islets to submaximally depolarizing concentrations of KCl, and the maximally effective concentration of arachidonate was 30-40 microM. These observations suggest that glucose-induced phospholipid hydrolysis and free arachidonate accumulation in pancreatic islets are not simply epiphenomena associated with Ca2+ influx and that arachidonate accumulation may play a role in the signaling process which leads to insulin secretion.

Glyburide priming of beta cells: Possible involvement of phosphoinositide hydrolysis

In the simultaneous presence of 5.5 mM glucose, exposure of isolated perifused islets to the sulfonylurea glyburide (500 nM) acutely stimulated insulin release and amplified the subsequent insulin secretory responses to 10 mM glucose or 10 mM arginine. This sensitizing effect of glyburide developed within 10 min, was maintained for at least 40 min after glyburide removal from the perifusion medium, and was attenuated by the calcium channel blocker hitrendipine. In islets whose inositol-containing lipids were prelabeled during a 2-hr incubation period with myo[2- 3H]inositol, glyburide induced a concentration-dependent increase in labeled inositol phosphate accumulation. Nitrendipine abolished this stimulatory effect of glyburide. In perifused islets, the stimulatory effect of glyburide on phosphoinositide (PI) hydrolysis persisted after its removal from the medium and the duration of this effect paralleled the duration of sensitization. These findings suggest that glyburide-induced increases in PI hydrolysis account, at least in part, for its acute stimulatory effect on insulin output and its ability to sensitize islets to subsequent stimulation.

Effect of sodium and calcium on basal secretory activity of rat neurohypophysial peptidergic nerve terminals.

1. The release of arginine vasopressin (AVP) from a perifused preparation of peptidergic nerve terminals isolated from rat neurohypophyses was studied during manipulations of the external sodium and calcium concentrations. Intracellular concentrations of these two ions were manipulated by use of ouabain and a calcium ionophore, respectively. 2. Removal of extracellular Na+ caused, in a concentration-dependent manner, a significant decrease of secretory activity. Conversely, graded addition of Na+ to a Na(+)-free perifusion medium increased secretion. Half-maximal activation of secretory activity was attained at ca 75 mM [Na+]o. 3. Manipulations of extracellular Ca2+ did not affect the level of hormonal secretion in the absence of extracellular Na+. However, when Na+ was present in the perifusion medium, removal of extracellular Ca2+ induced an increase of secretory activity. 4. The effects of manipulations of [Na+]o were not dependent on the presence of Ca2+ in the perifusion medium nor on the nature of the Na+ replacement used (i.e. choline or mannitol). 5. Ouabain (0.1 mM) increased the basal secretory activity and potentiated the secretory response to removal of Ca2+ from the perifusion medium. 6. The Ca2+ ionophore A23187 stimulated, in a concentration-dependent fashion, the secretory activity of the peptidergic nerve terminals and this stimulation was strictly dependent on the presence of Ca2+ in the perifusion medium. 7. These results show that basal secretion is directly dependent on [Na]o and indicate that intracellular Na+ is an important factor in the control of secretory mechanisms. Evidence is presented in regard to a possible antagonistic effect of extracellular Ca2+ and Na+ on secretion.

Acute stimulation of pancreatic islets by inhibitors of lactic acid transport

The transport of L-lactate into rat pancreatic islets and RINm5F insulinoma cells was inhibited by α-cyano-4-hydroxycinnamate, α-fluorocinnamate, quercetin and by p-chloromercuribenzene-sulphonic acid. The addition of each of these compounds to perifused islets resulted in an immediate, marked stimulation of insulin release. Enhanced insulin secretion was accompanied by a similarly rapid and pronounced increase in the rate of 45Ca 2+ efflux from pre-loaded, perifused islets. In general, these stimulatory effects were most pronounced in the presence of a threshold concentration of glucose (5 mM) in the perifusion medium. In islets pre-loaded with 86Rb +, the addition of α-fluorocinnamate or quercetin caused a modest diminution in efflux rate whilst enhanced rates of 86Rb + outflow were apparent in the presence of 5 mM glucose. It is suggested that these inhibitors of lactic acid transport stimulate the β-cell, at least in part, by increasing the intracellular: extracellular lactate gradient, thereby promoting the electrogenic efflux of endogenous lactate from the cell.

Autonomous progesterone secretion from the bovine corpus luteum in vitro

While the regulation of progesterone secretion from the corpus luteum by LH has been convincingly demonstrated, the secretory patterns in the absence of any pituitary LH inputs are yet unclear. Consequently, we investigated the progesterone secretion by an in vitro perifusion system to characterize spontaneous progesterone release from the isolated bovine corpus luteum. Slices (120 mg) of midluteal corpora lutea were placed in perifusion chambers and continuously perifused by Medium-199 for 160-320 min. Progesterone was determined by radioimmunoassay in the effluent fractions collected at 2-min intervals. The spontaneous progesterone release from all bovine corpora lutea was pulsatile. Pulses were observed at mean (+/- SEM) intervals of 17.7 +/- 1.5 min with amplitudes of 6.7 +/- 0.5 ng and release rates of 29.5 +/- 2.4 ng.ml-1.(2 min)-1 (N = 5). Addition of 6.7 nmol/l hCG to the perifusion medium appeared to increase the pulse amplitudes and release rates (195 +/- 25% over unstimulated conditions), but did not change the pulse frequencies (N = 3). Perifusions with calcium-free medium containing 50 mumol/l verapamil and 20 mmol/l EGTA tended to suppress the pulse frequencies and amplitudes of this spontaneous progesterone release, whereas addition of hCG reversed this decrease again (N = 3). When prostaglandin activity was inhibited by 100 mumol/l indomethacin added to the perifusion medium, both pulse frequencies and amplitudes of this progesterone release were enhanced (N = 4). During perifusions with 100 mumol/l of the anti-progesterone ZK 96.734, both the pulse frequencies and amplitudes increased (N = 4). These studies demonstrate an episodic progesterone release from the bovine corpus luteum perifused in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative effects of linogliride fumarate and tolbutamide sodium on insulin secretion and glucose utilization in rat pancreatic islets

AbstractThe effects of linogliride fumarate (0.1 mmol/liter) and tolbutamide sodium (0.5 mmol/liter) on insulin secretion and glucose utilzation were directly compared in isolated perifused rat islets. Both compounds potentiated glucose‐primed (5.5 mmol/liter) insulin release. The insulin secretory response at the concentration chosen was qualitatively different for the two agents. In the presence of glucose, the insulin secretory effect of tolbutamide was biphasic, whereas linoglirde predominantly increased second‐phase insulin secretion. In the absence of exogenous glucos, tolbutamide stimulated first‐phase insulin release wherease linogliride was ineffective without glucose in the perifusion medium. Neither linogliride (0.1 mmol/liter) nor tolbutamide (0.5 mmol/liter) stimulated islet cell glucose usage as measured by conversion of [5−3H]‐glucose to 3H2O. The insulin secretagogue effect of linogliride was completely abolished when islet cell glucose usage was partially blocked (22% reduction) by mannoheptulose (MH) (10 mmol/liter). When glucose usage was significantly inhibited (55%) by 2‐deoxyglucose (2DG) (10 mmol/liter), linogliride‐stimulated insulin secretion was not significantly reduced. In contrast, tolbutamide‐stimulated first‐phase insulin release was not inhibited when glucose usage was reduced by either MH or 2‐DG, wherease secondphase insulin release was significantly inhibited. In summary, linogliride potentiates insulin secretion in isolated islets by a glucose‐dependent process, but with a kinetic profile and response to metabolic inhibitors different from that of tolbutamide.

Release of pro-thyrotropin-releasing hormone connecting peptides PS4 and PS5 from perifused rat hypothalamic slices

Thyrotropin-releasing hormone prohormone contains five copies of the thyrotropin-releasing hormone progenitor sequence Gln-His-Pro-Gly, each flanked by pairs of basic amino acids and separated by intervening sequences (connecting peptides). Using a perifusion system for rat hypothalamic slices, we have studied the ionic mechanisms underlying the release of two connecting peptides originating from the thyrotropin-releasing hormone precursor: prepro-thyrotropin-releasing hormone-(160–169) (Ps4) and prepro-thyrotropin-releasing hormone-(178–199) (Ps5). Quantification of these two peptides in the effluent fluid was performed using sensitive and highly specific radioimmunoassay procedures. Reverse phase high performance liquid chromatography analysis of the effluent perifusate showed that released peptides co-eluted with synthetic Ps4 and Ps5. The secretion of Ps4 and Ps5 was stimulated by depolarizing agents such as (i) high potassium concentrations, (ii) ouabain, an Na +/ K +-ATPase inhibitor, and (iii) veratridine, a stimulator of voltage-operated Na + channels. The response to potassium (70 mM) was not affected by the specific Na + channel blocker tetrodotoxin. The K + channel blocker tetraethylammonium did not modify K +-evoked release of Ps4 and Ps5. These data suggest that voltage-operated Na + channels are not involved in the stimulatory effect of high K + on the release of Ps4 and Ps5. The lack of effect of picrotoxin, a Cl − channel blocker, on the secretion of the connecting peptides indicates that chloride ions play a minor role in the release process.In contrast, deprivation of Ca 2+ in the perifusion medium suppressed K +-evoked release of the two peptides, indicating that voltage-operated Ca 2+ channels are implicated in the release process. Taken together, the present results show that non-thyrotropin-releasing hormone peptides originating from the thyrotropin-releasing hormone precursor are secreted by mediobasal hypothalamic fragments. The release of these peptides is stimulated by depolarization through a calcium-dependent process. These data indicate that Ps4 and Ps5 may be released at the level of the median eminence into the portal circulation, suggesting that these peptides may play a role in the control of anterior pituitary cells.

Inhibin secretion by individual porcine follicles perifused in vitro

To examine the relative contribution of dominant and nondominant follicles to ovarian inhibin secretion, individual porcine follicles (n = 69, diameter 4 to 9 mm) were perifused in vitro for 12 hours and then histologically examined via light microscopy. Follicle-stimulating hormone (FSH, 1 μ g/mL) was added during the second half of the perifusion period. Levels of 32 kD inhibin, estradiol (E 2 ), and progesterone (P) in perifusion media were determined by radioimmunoassay. Viable and atretic follicles of medium size (4 to 7 mm) secreted negligible amounts of inhibin. Significant levels of inhibin (2 to 5 ng/mL) were only found in media from large viable follicles. In contrast to media concentrations of E 2 and P, inhibin levels did not rise during FSH stimulation. Levels of inhibin correlated with those of P ( P < 0.01) but not with those of E 2 . In conclusion, mature dominant follicles may be the principal source of peripheral inhibin during the follicular phase.

Inhibition by the putative potassium channel opener pinacidil of the electrically-evoked release of endogenous dopamine and noradrenaline in the rat vas deferens

The effect of pinacidil on the release of endogenous noradrenaline and dopamine from the sympathetic innervation of the rat vas deferens was examined. Amine release was evoked by electrical stimulation (1, 2, 5 and 10 Hz) or by depolarization with high potassium (75 mmol/l) in the medium. Dopamine and noradrenaline were measured by means of high pressure liquid chromatography with electrochemical detection. Pinacidil (1, 5, 10 and 50 mumol/l) produced a concentration-dependent inhibition of the electrically stimulated (2 Hz) overflow of noradrenaline and dopamine. Only pinacidil 50 mumol/l increased the spontaneous loss of dopamine and noradrenaline. The inhibitory effects of pinacidil (5 mumol/l) on amine overflow were also observed at other frequencies of stimulation (1, 5 and 10 Hz). The magnitude of the inhibitory effect on noradrenaline release was approximately the same at all frequencies (63% to 56% reduction); for dopamine, the higher the frequency of stimulation, the greater the inhibitory effect of pinacidil (up to 73% reduction). When the preparations were continuously stimulated for 70 min at 2 Hz, pinacidil (5 mumol/l) reduced the overflow of dopamine and noradrenaline during the first 40 or 30 min of stimulation only. The addition of phentolamine (1 mumol/l) to the perifusion medium slightly reduced the inhibitory effect of pinacidil on amine overflow, but the inhibition by pinacidil remained statistically significant. Tetraethylammonium (10 mmol/l) completely abolished the inhibitory effect of pinacidil (10 mumol/l). Pinacidil (5 mumol/l) did not reduce the potassium-evoked release of the amines. The results demonstrate that pinacidil impairs transmitter release from the sympathetic innervation of the rat vas deferens, probably as a consequence of the opening of potassium channels.

Effect of Extended Exposure to Hypertonicity on Vasopressin Messenger Ribonucleic Acid Content in Hypothalamo-Neurohypophyseal Explants*

The feasibility of using organ-cultured explants of the rat hypothalamo-neurohypophyseal system (HNS) to study the mechanisms regulating the vasopressin (VP) mRNA content of the HNS was examined by evaluating the effect of exposure to hypertonicity on the VP mRNA content of these explants. Different effects were observed after a step increase in osmolality and a gradual increase in the same amount over 24 h. The VP mRNA content of control HNS explants determined from a RNA protection assay was 22 +/- 6 pg. It gradually decreased to 23% and 9% of the control value during 24 and 48 h in culture, respectively. Northern blot analysis revealed a single band of VP mRNA approximately 700 bases long in explants cultured for 36 h. Explants exposed to the step increase in osmolality were maintained in static culture. The control explants were placed directly into isotonic medium (299 mosmol/kg H2O). The explants exposed to the step increase were placed directly into hypertonic medium (greater than 304 mosmol/kg H2O). After 24 h in culture, basal VP release was measured, and all explants were then exposed to a further acute 15 mosm/kg H2O increase in osmolality. The highest basal release of VP was observed in the explants maintained under isotonic conditions (299 mosm/kg H2O). These explants significantly increased VP release in response to the acute increase in osmolality. Basal VP release was lower in explants maintained in hypertonic medium (greater than 304 mosmol/kg H2O), and these explants did not respond to the acute hypertonic pulse. VP mRNA content was significantly decreased in explants maintained for 24 or 48 h in hypertonic medium compared to that in explants maintained in isotonic medium (47 +/- 10% and 57 +/- 6%, respectively; P less than 0.01). No significant difference existed in the VP content of the posterior pituitary between the groups. To achieve a slow increase in osmolality, explants were perifused in individual chambers with medium at 2.1 ml/h. A gradual increase in osmolality (16 mosmol/kg H2O medium) was achieved by increasing the NaCl concentration in the perifusion medium. In response to this stimulus there was a significant increase in VP release, which was sustained for 9 h. VP mRNA content in the hypertonic group was 165 +/- 19% of that in control explants (P less than 0.001), but no difference existed in VP content in the posterior pituitary compared to that in time control explants.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of High Potassium Concentration and Dihydropyridine Ca2+-Channel Agonists on Cytoplasmic Ca2+and Aldosterone Production in Rat Adrenal Glomerulosa Cells

The aldosterone secretory response of isolated rat adrenal glomerulosa cells to potassium was studied in a cell-perifusion system. Increasing the potassium concentration from 3.6 to 5.4 mM in the perifusion medium caused a rapid 40-fold stimulation of aldosterone production which was maintained during the 2 h period of stimulation. A dose of 8.4 mM potassium elicited a 100-fold increase of hormone production with rapid onset and a slowly decreasing plateau. When the potassium concentration was further increased to 18 mM, there was a rapid stimulation of aldosterone production comparable to that evoked by 8.4 mM potassium; however, the response declined very rapidly to levels still above basal. The dihydropyridine-agonist BAY-K 8644 (100 nM) greatly enhanced the aldosterone response to 5.4 mM potassium but did not significantly modify the response evoked by 8.4 mM potassium. The effect of BAY-K 8644 on the aldosterone response was inhibitory at 18 mM potassium concentration, suggesting that the character of dihydropyridine modulation of the secretory response was voltage dependent, showing reversal at relatively negative potentials. When the cytoplasmic Ca2+ concentration was monitored in glomerulosa cells by the fluorescent Ca2(+)-probe Fura-2, potassium evoked a rapid dose-dependent increase in free Ca2+, with elevated steady-state Ca2(+)-levels throughout stimulation, even at potassium concentrations higher than 18 mM. Moreover, BAY-K 8644 (100 nM) enhanced the cytoplasmic Ca2+ response to all potassium concentrations tested up to 30 mM. The initial 30 sec 45Ca2+ uptake, an indicator of potassium-stimulated voltage-sensitive Ca2+ influx into these cells, showed a fast increase and only an initial inactivation in response to 18 mM potassium. This response was enhanced by 100 nM BAY-K 8644, with no sign of enhanced inactivation or inhibition caused by the dihydropyridine agonist. These results indicate that the correlation between Ca2+ influx, cytoplasmic Ca2+ levels, and the secretory response in adrenal glomerulosa cells is lost at potassium concentrations higher than 8 mM, especially in the presence of the dihydropyridine agonist, BAY-K 8644.

Normal and Adenomatous Human Pituitaries Secrete Thyrotropin-Releasing HormoneIn vitro: Modulation by Dopamine, Haloperidol, and Somatostatin

TRH is present in human normal pituitaries and in pituitary adenomas. In this study we demonstrated that the same tissues can release TRH in vitro. Fragments from seven normal pituitaries (10-15 mg/syringe) and dispersed cells from eight prolactinomas, four GH-secreting and two nonsecreting adenomas (1-3 x 10(6) cells/syringe) were perifused using a Krebs-Ringer culture medium. After 1 h of equilibration the perifusion medium was collected every 2 min (1 mL/fraction) for 3 h. TRH, PRL, and GH were measured by RIA under basal conditions and in the presence of 10(-10) to 10(-6) mol/L dopamine (DA), alone or concomitant with haloperidol, or in the presence of 10(-10) or 10(-6) mol/L somatostatin. Both normal pituitary fragments and pituitary adenomatous cells (from all types of adenomas studied) spontaneously released TRH in vitro. TRH was detected in the perifusion medium either immediately after the end of the equilibration period or 30-60 min later. The molecular identity of TRH was assessed by high pressure liquid chromatography. There was no difference in the profile and the rate of TRH secretion between normal and tumoral tissues, and no correlation was found between the level of TRH release and that of PRL or GH secretion. DA stimulated TRH release from normal pituitaries and from PRL- and GH-secreting adenomas at doses as low as 10(-10) mol/L. A concomitant decrease in PRL and GH release was observed from adenomatous cells and in one case of normal tissue. Haloperidol (10(-7) mol/L) antagonized the effect of 10(-8) mol/L DA on both TRH and PRL secretion in normal pituitary and in prolactinomas. DA had no effect on TRH release from two nonsecreting tumors. The amounts of TRH released during 1 h of perifusion were 60-1640 pg/2 mg wet wt tissue in normal pituitaries and 54-2174 pg/10(6) cells in adenomas; these values were very high compared to those precedently reported within the tissues. These results indicate that pituitary cells can release TRH in vitro and suggest that TRH might be synthesized in situ. We suggest that TRH could act on pituitary hormone secretion and/or cell proliferation via a paracrine and/or an autocrine mechanism.

Secretory patterns of 1α-hydroxycorticosterone in the isolated perifused interrenal gland of the dogfish, Scyliorhinus canicula

An isolated in-vitro perifused interrenal gland preparation from the dogfish Scyliorhinus canicula was used to study production of quantitatively the major corticosteroid 1 alpha-hydroxycorticosterone (1 alpha-OH-B), measured by radioimmunoassay. Basal secretory rates were 877.1 +/- 145 (S.E.M.) fmol/mg per 15 min (n = 14) and the preparation remained viable for up to 22 h, as reflected in a brisk response to 10 microM cyclic AMP (cAMP) after this time. Steroid production responded in a dose-dependent manner to porcine ACTH, with 10 microM producing a maximum stimulation of 225% above the basal secretory rate. cAMP (10 microM) produced an increase of 278% above basal, while 1 microM forskolin increased basal secretory rates by 127%. [Val5]- and [Ile5]-angiotensin II (0.1 microM) increased 1 alpha-OH-B production by 120 and 372% respectively over basal secretory rates. Increasing the concentration of K+ in the perfusate from 8 mM to 12, 18, 28 and 40 mM produced a significant rise only at 28 mM. Alterations in the concentration of Na+ and osmolarity of the perifusion medium had inconsistent effects on steroid production. Increased concentrations of urea (from 360 to 720 mM) increased the basal secretory rate by 121%, whilst reducing the concentration of urea (from 360 to 90 mM) had no effect.

Role of Protein Kinase-C in the Adrenocorticotropin Secretory Response to Arginine Vasopressin (AVP) and the Synergistic Response to AVP and Corticotropin- Releasing Factor by Perifused Rat Anterior Pituitary Cells*

Arginine vasopressin (AVP) stimulates biphasic release of ACTH from anterior pituitary corticotrophs. The response consists of an initial transient spike phase lasting less than 3 min and a subsequent sustained plateau phase that persists for as long as AVP is present. AVP also acts synergistically with CRF on ACTH release. We have previously shown that the initial spike phase of the response mainly requires release of intracellular Ca2+ and is independent of calmodulin, whereas the sustained plateau phase, like the monophasic sustained response elicited by CRF, involves the influx of extracellular Ca2+ via L-type voltage-sensitive Ca2+ channels and activation of calmodulin. We have also shown that the synergism between AVP and CRF does not require extracellular Ca2+ influx. In this study we examined the role of Ca2+/phospholipid-dependent protein kinase-C (PKC) in the two phases of the response to AVP and in the synergism between AVP and CRF. We exploited the observation that prolonged exposure to phorbol esters down-regulates PKC. Dispersed adult male rat anterior pituitary cells were incubated in static suspension culture for 4-5 days, 0.5 microM phorbol 12-myristate 13-acetate (PMA) or 0.0005% dimethylsulfoxide vehicle alone was added, and the incubation was continued for 24 h. The cells were preperifused with PMA-free perifusion medium for 3 h and then perifused with various agents for 10-20 min. Effluent fractions were collected every 30 sec or 1 min and subjected to ACTH RIA. Pretreatment with PMA inhibited the subsequent response to 100 nM PMA and 100 microM dioctanoylglycerol, but not to 5 microM forskolin or to depolarization with 56 mM KCl, demonstrating specific down-regulation of PKC. PMA pretreatment had no effect on the initial spike phase of the response to AVP, but inhibited the sustained plateau phase by 57% (P less than 0.005) and, consequently, the integrated total response by 33% (P less than 0.05). Pretreatment had no effect on the response to CRF. However, pretreatment with PMA completely blocked both phases of the synergistic response to the combination of AVP and CRF. These results indicate that activation of PKC is required for the sustained phase of the response to AVP and both phases of its synergistic interaction with the protein kinase-A pathway, but is not involved in the initial spike phase of the response to AVP, which presumably is mediated by inositol 1,4,5-trisphosphate-stimulated mobilization of intracellular Ca2+, or in the independent activation of the protein kinase-A pathway by CRF.

Comparative studies on the release of vasopressin and 7B2 from isolated hypothalami

A new protein, 7B2, has been found in the anterior, intermediate and posterior pituitary and the hypothalamus. It has been previously suggested that 7B2 is synthesized in the hypothalamus and is transported to the posterior lobe in a similar way to vasopressin (AVP). We examined the in vitro release of AVP and 7B2, using rat hypothalami in a perifused column system. Membrane depolarization with KCl (56 mM) caused a marked stimulation of AVP release (from 116% to 263% above basal values). Release of 7B2 was also stimulated by potassium from 106% to 212% above basal values. Potassium pulses in calcium-free medium failed to release AVP or 7B2. Osmotic and cholinergic stimulation increased AVP secretion by 205% and 282% above basal values, respectively, but had no significant effect on 7B2 secretion. Chromatographic profiles of perifusion media revealed one immunoreactive 7B2 peak eluting at a coefficient of 0.34 corresponding to that of rat hypothalamic 7B2. Similarly AVP coeluted with AVP standard. Thus AVP and 7B2 are differentially released by cholinergic and osmotic stimuli in vitro.

Direct effect of parathyroid hormone on insulin secretion from pancreatic islets

Indirect evidence indicates that parathyroid hormone (PTH) interacts with pancreatic islets and modulates their insulin secretion. This property of PTH has been implicated in the genesis of impaired insulin release in chronic renal failure. We examined the direct effect of PTH-(1-84) and PTH-(1-34) on insulin release using in vitro static incubation and dynamic perifusion of pancreatic islets from normal rats. Both moieties of the hormone stimulated in a dose-dependent manner glucose-induced insulin release but higher doses inhibited glucose-induced insulin release. This action of PTH was modulated by the calcium concentration in the media. The stimulatory effect of PTH was abolished by its inactivation and blocked by its antagonist [Tyr-34]bPTH-(7-34)NH2. PTH also augmented phorbol ester (TPA)-induced insulin release, stimulated adenosine 3',5'-cyclic monophosphate (cAMP) generation by pancreatic islets, and significantly increased (+50 +/- 2.7%, P less than 0.01) their cytosolic calcium. Verapamil inhibited the stimulatory effect of PTH on insulin release. The data show that 1) pancreatic islets are a PTH target and may have PTH receptors, 2) stimulation of glucose-induced insulin release by PTH is mediated by a rise in cytosolic calcium, 3) stimulation of cAMP production by PTH and a potential indirect activation of protein kinase C by PTH may also contribute to the stimulatory effect on glucose-induced insulin release, and 4) this action of PTH requires calcium in incubation or perifusion media.

Hyposmolar Stimulation of Secretion of Thyrotropin, Prolactin, and Luteinizing Hormone Does not Require Extracellular Calcium and Is not Inhibited by Colchicine, Cytochalasin B, Ouabain, or Tetrodotoxin

Hyposmolar stimulation of thyroid-stimulating hormone, prolactin, and luteinizing hormone secretion by dispersed perifused rat pituitary cells was not depressed by removal of Ca2+ from the perifusion medium or by 0.1 mM colchicine, 20 microM cytochalasin B, 0.1 mM ouabain, or 3 microM tetrodotoxin. The secretory response induced by medium hyposmolarity or by thyrotropin-releasing hormone was not appreciably different at 23, 37, or 43 degrees C, but was markedly reduced or abolished when the experiments were performed at 1 degree C. These data indicate that microtubules or microfilaments, transport of extracellular Ca2+ into the cytoplasm, and plasmalemma ion transport mechanisms sensitive to ouabain or tetrodotoxin are not essential components of the mechanism by which extracellular hyposmolarity induces secretion.

Non-Steroidal Follicular Factors Attenuate the Self-Priming Action of Gonadotropin-Releasing Hormone on the Pituitary Gonadotroph

Pituitary glands of pro-oestrous and oestrous rats were perifused in series with isolated ovarian follicles and the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responses to gonadotropin-releasing hormone (GnRH) were measured. Pituitary glands from pro-oestrous rats, perifused with the effluent medium from isolated preovulatory follicles, showed a significant reduction in the self-priming effect of GnRH as observed from the typical biphasic LH response to a continuous GnRH challenge. The initial FSH response to GnRH was also reduced. A similar trend was seen in the LH responses of pituitary glands from oestrous rats but results were not significant; there was no parallel reduction in FSH release. It is unlikely that the attenuation of GnRH self-priming was due to the steroids released by the isolated follicles. LH responses of pro-oestrous pituitaries, perifused with medium containing 200 pg oestradiol/ml, 5 ng progesterone/ml or 200 pg testosterone/ml were similar to paired controls as were the responses when all three steroids were added to the perifusing media at identical concentrations. Furthermore, when steroids were charcoal extracted from the effluent medium of perifused isolated follicles the biological activity of this medium in attenuating GnRH self-priming was not destroyed. Finally, the effects of purified bovine inhibin and steroid-free human follicular fluid (hFF) on LH responses were compared. Purified inhibin at a concentration of 20 or 50 ng/ml perifusing medium had no effect on GnRH-stimulated LH release although GnRH-stimulated FSH secretion was significantly reduced toward the end of an extended perifusing period.(ABSTRACT TRUNCATED AT 250 WORDS)