Cyclic Nucleotides and the Release of Vasopressin from the Rat Posterior Pituitary Gland

Abstract: The effect of ATP, Mg²⁺, or MgATP on the release of luteinizing hormone-releasing hormone (LH-RH) from hypothalamic granules was examined under in vitro conditions. Granules, isolated from adult male hypothalami, were incubated at 37°C in a buffered (pH 7.8) medium containing 0.15 m -KCl. The addition of ATP to the incubation mixture did not stimulate the release of LH-RH. In contrast, the addition of MgATP stimulated the release of LH-RH, the release being 62% greater than control. The addition of Mg²⁺ to the incubated granules also stimulated the release of LH-RH. However, the magnitude of this Mg²⁺-stimulated release of LH–RH was significantly ( P < 0.01) lower than that of the MgATP-stimulated release, indicating that ATP stimulates LH-RH release in a Mg²⁺-dependent manner. As both MgATP and Mg²⁺ alone stimulated LH-RH release, we characterized further these two release processes by incubating the granules under one of the following conditions: incubation at 4°C in a buffered medium containing 0.15 m -KCl or incubation at 37°C in a medium that does not contain KCl. Under these two incubation conditions, the MgATP-stimulated release of LH-RH was not manifested, whereas the Mg²⁺-stimulated release of LH-RH was manifested. On the basis of these differences, we propose that two different processes can lead to the release of LH-RH from isolated hypothalamic granules: one process involves ATP and Mg²⁺ (MgATP) and another process involves Mg²⁺ alone.     

Factors affecting the reactivation of the mitochondrial adenosine 5'-triphosphatase and the release of ATPase inhibitor protein during and following the reenergization of mitochondria from ischemic cardiac muscle

In the present study we examined three factors affecting the reversal of the ischemia-induced inhibition of the mitochondrial ATPase described by us earlier (W. Rouslin (1983) J. Biol. Chem. 258, 9657-9661). These factors were the pH, the MgATP concentration, and the pCa of the medium in which mitochondria were sonicated following their reenergization in vitro. It was found that the extent of ATPase reactivation, on the one hand, and the extent of inhibitor protein release, on the other, following the reenergization in vitro and subsequent sonication of intact mitochondria isolated from 20-min-ischemic canine cardiac muscle were affected differently by each of the three factors studied. While raising the pH of the medium in which the mitochondria were sonicated subsequent to reenergization from approximately 7.0 to approximately 8.2 resulted in marked parallel increases in both ATPase reactivation and inhibitor protein release, lowering the pH of the medium to approximately 6.4 resulted in a marked decrease in ATPase reactivation but also in the apparent irreversible binding and/or denaturation of a portion of the ATPase inhibitor. Increasing the MgATP concentration of the sonication medium from zero to 2.0 mM resulted in approximately a one-third decrease in ATPase reactivation. The effect upon inhibitor release was more dramatic. MgATP at 2 mM decreased inhibitor release by approximately two-thirds. The pCa of the sonication medium was varied between 9.0 and 3.5 using Ca-ethylenebis(oxyethylenenitrilo)-tetraacetic acid (EGTA) buffers. Decreasing the pCa of the medium from 9.0 to 3.5 had a paradoxical effect. It resulted in increases both in ATPase reactivation and in the amount of inhibitor bound to the particles. Such a paradoxical effect may be explained if one assumes the existence of two kinds of inhibitor-enzyme interaction sites, namely, regulatory and nonregulatory binding sites. Thus, decreasing the pCa may decrease interaction at regulatory sites while enhancing interaction at nonregulatory inhibitor binding sites.     

Comparison between the biological effects of LH-RH and buserelin on the induction of LH release from hemi-pituitary glands of female rats in vitro

The biological effects of LH-RH and the agonist [D-Ser(But)6-des Gly10]-LH-RH(1-9)-ethylamide (buserelin) were compared during 8 h of incubation with female rat hemi-pituitary glands. Similar dose-response relationships were found for LH-RH and buserelin as concerns the release of luteinizing hormone (LH) by pituitary glands from intact and ovariectomized rats. Also the LH secretion patterns from glands of intact rats were similar: an initial low response was followed by a fast increase (priming effect) after which the response declined again (desensitization). In a subsequent experiment pituitary glands from ovariectomized rats were first exposed to LH-RH or buserelin for 4 h and then further incubated in medium only. After discontinuation of the stimuli the rate of LH release decreased in all cases, but this decrease was significantly greater when the glands had been exposed to LH-RH. Short-term (1/2, 1 or 2 h) exposures to LH-RH or buserelin followed by an intervening period (1 1/2, 1 or 0 h, respectively) of incubation in medium only resulted in an almost similar, significant increase in the subsequent protein synthesis-independent LH response to LH-RH (priming effect). Only preincubation with LH-RH for 2 h was significantly more effective. The results demonstrate equal intrinsic activities for LH-RH and buserelin. Differences in the biopotencies for LH-RH and buserelin in vivo and in vitro may occur only after discontinuation of the external stimuli.     

MgATP-Stimulated Release of Luteinizing Hormone Releasing Hormone from Isolated Hypothalamic Granules: Evidence for a Selective Requirement for Monovalent Cations

Abstract: The purpose of this study was to characterize the kinetics and monovalent ion requirements of the MgATP-stimulated release of Iuteinizing hormone releasing hormone (LHRH) from isolated hypothalamic granules. LHRH granules were obtained from homogenates of hypothalami of adult male rats by differential centrifugation and then incubated in buffered media containing KCl. Under these conditions, the pH optimum for the MgATP-stimulated release of LHRH was between 7.5 and 8.0; the rate of release was linear for the first 5 min of incubation; and the magnitude of the release was a saturable function of KCl concentration. When the potassium in the incubation mixture was replaced with various monovalent cations (Li⁺, Na⁺, Cs⁺, choline, or tetraethylammonium), the magnitude of the MgATP-stimulated release of LHRH was inversely related to the size of the cation; release in the presence of Li⁺ was 12%, whereas release in the presence of tetraethylammonium was 0% of the total LHRH. When the chloride in the incubation mixture was replaced with various monovalent anions (Br⁻, I⁻, or isethionate), the magnitude of the MgATP-stimulated release of LHRH was not related to the size of the anion. Moreover, inclusion of anion transport inhibitors in the incubation mixture did not affect LHRH release, suggesting that anion transport is not required for the MgATP-stimulated release of LHRH. In summary, we suggest that the MgATP-stimulated release of LHRH from isolated hypothalamic granules involves an enzymatic event and that this release process does not have a selective requirement for monovalent anions, but has a selective requirement for monovalent cations.     

Feedback control of FSH secretion in the male rat

Site of feedback control of FSH secretion in the male rat was studied by measuring changes in serum LH, FSH and hypothalamic LH-RH by radioimmunoassay in rats after castration and after 500 rad X-irradiation to the testis. The rise in serum LH and FSH in castrated animals was associated with a significant fall in hypothalamic LH-RH 16 and 24 days after castration. Serum FSH rose significantly after X-irradiation without a significant change in serum LH or hypothalamic LH-RH content up to 30 days after irradiation. When pituitary halves from X-irradiated animals were incubated in vitro in the presence or absence of synthetic LH-RH, there was a significant rise in FSH (but not LH) released in the incubation medium in the absence of added LH-RH. The response of the pituitaries to LH-RH was, however, not different between control and irradiated rats. It is concluded that the testicular FSH-inhibitory substance acts predominantly at the pituitary gland on the LH-RH independent release of FSH.     

A critical examination of the evidence for an MgATP-dependent proton pump in rat liver lysosomes

(1) When lysosomes isolated from the livers of Triton WR 1339-treated rats were incubated for 30 min in the presence of 100 mM KCl and 14CH3NH2, a stimulation by MgATP of the calculated accumulation of the base was observed, in agreement with previous results (Schneider, D.L. (1979) Biochem. Biophys. Res. Commun. 87, 559-565). A similar stimulation was seen with MgITP. Excess EDTA had very little effect on the stimulation by MgATP. (2) There was little effect of MgATP or MgITP on the calculated accumulation of 14CH3NH2 if the base was added to the incubation medium 1, 3, 4 or 5 min before terminating the incubation instead of being present for the total incubation period of 30 min. (3) The accumulation of the basic dye, acridine orange, by a crude lysosomal preparation isolated from the livers of untreated rats was found to be stimulated by MgATP, in agreement with earlier results (Dell'Antone, P. (1979) Biochem. Biophys. Res. Commun. 86, 180-189). Similar results were obtained with a crude lysosomal preparation isolated from the livers of Triton WR 1339-treated rats. In both cases, the stimulation was partly oligomycin-sensitive. (4) There was very little or no effect of MgATP on the accumulation of acridine orange by preparations of pure lysosomes isolated from the livers of Triton WR 1339-treated rats. (5) Our data do not acquire us to postulate the existence of an MgATP-dependent proton pump in lysosomes.     

Low density lipoprotein degradation by rat mast cells. Demonstration of extracellular proteolysis caused by mast cell granules

The interaction between rat serosal mast cells and low density lipoproteins (LDL) was studied in vitro. When rat 125I-LDL was incubated with mast cells, it was bound to a binding site on the mast cell surface but was not internalized by the cells. Even though 125I-LDL was not internalized, its protein component, apolipoprotein B, was rapidly degraded. The proteolytic activity responsible for the degradation of apolipoprotein B was present in the extracellular fluid of mast cells. It could be shown that the degradation was caused entirely by specific cell organelles of mast cells, the granules, which were spontaneously released into the extracellular fluid during preparation and incubation of the cells. In contrast to uncontrolled spontaneous degranulation, a controlled specific degranulation of mast cells can be induced by treating the cells with the compound 48/80. When increasing amounts of 48/80 were added to mast cell suspensions, a dose-dependent release of granules was observed and an increase in the rate of 125I-LDL degradation resulted. The increase in 125I-LDL degradation closely followed the increase in granule release. Thus, a quantitative relationship between the amount of granules present in the extracellular fluid and the amount of degradation of 125I-LDL could be established. The apolipoprotein part of LDL was extensively degraded by isolated mast cell granules. Analysis by polyacrylamide gel electrophoresis showed that upon incubation of LDL with isolated granules, the apolipoprotein B band rapidly disappeared with simultaneous appearance of several low molecular weight bands. The degradation of 125I-LDL by mast cell granules proceeded optimally at neutral pH and at physiological ionic strength. The results show that mast cell granules are able to efficiently degrade LDL in vitro, once released from mast cells into the extracellular fluid.     

Effect of inhibin-like activity on LH-RH-stimulated release of FSH by pituitary glands from female rats in vitro

During long-term incubation of pituitary glands from intact female rats in the presence of inhibin-like activity, LH-RH-stimulated release of FSH becomes inhibited after 4 h of incubation. However, at the same time inhibition of basal FSH release is included. Therefore, glands were at first incubated for 4 h in the presence of inhibin-like activity to block basal release completely and thereafter LH-RH was added to the medium. It was found that LH-RH still could stimulate FSH release, despite the continuous presence of inhibin-like activity. This means that LH-RH-stimulated release of FSH could be investigated separately from basal release. Using this way of incubation, it was found that part of the action of LH-RH on FSH release was independent of protein synthesis. Also part of LH-RH-stimulated FSH release was independent of the presence of extracellular Ca2+. Furthermore it was found that LH-RH, when added after 4 h of incubation did stimulate FSH synthesis, in the presence as well as absence of inhibin-like activity. The present results indicate that LH-RH-stimulated release of FSH is not affected by inhibin-like activity. Complete inhibition of basal release and synthesis of FSH does not prevent LH-RH from stimulating FSH release and synthesis. It is suggested that two separate releasing mechanisms for FSH could exist in the pituitary gland.     

Effect of LH-RH on the release of sulfated lutropin: a potential in vitro bioassay for LH-RH

Sheep pituitary cells prelabelled with radioactive [35S] sulfate (35SO4(2-)) were incubated with different concentrations of LH-RH and the release of LH (lutropin) into the medium was monitored in terms of immunoprecipitable [35S] sulfated LH radioactivity and estimation of LH in the same sample by radioimmunoassay. A dose dependent response was obtained with a maximum of a 16 fold increase in immunoprecipitable 35SO4(2-) -labelled LH radioactivity in the medium which was confirmed by radioimmunoassay. Similar results were also obtained for Buserelin, a well known superactive analogue of LH-RH. However, the half maximal response for Buserelin was obtained at 3-5 nM in comparison to 80.5 nM for LH-RH. After the maximal response to LH-RH as well as Buserelin, a further increase in the concentrations caused a decrease in the release of immunoprecipitable [35S]-sulfate labelled LH into the medium. Differential labelling of stored and newly synthesized LH with radioactive [35S] sulfate and [3H]-labelled leucine revealed that there was a dose dependent increase in the [35S] sulfate labelled LH into the medium whereas the release of [3H]-leucine labelled newly synthesized LH did not show a parallel increase either at different concentrations of LH-RH or at different time intervals. The above observations strongly suggest the possibility of sulfation of LH being the potential signal indicating the storage of LH in sheep pituitary cells. Another important observation in our study was that the dose dependent response of LH-RH in the form of release of [35S]-sulfate labelled LH, which was monitored by immunoprecipitation with specific LH antiserum, can be used in an in vitro bioassay for LH-RH. We believe that a new cheap and sensitive in vitro bioassay could be developed on the basis of this observation.     

Inhibitory effect of MgATP on the release of regulatory light chain from scallop myosin and light chain composition of scallop myosin hybridized with abalone light chain 2 at 30 degrees C

The experimental conditions for release of the regulatory light chain (RLC) of scallop myosin at 30 degrees C were studied. Substantially all RLC was released from myosin by incubation for 5 min in medium containing buffer and KCl. This release of RLC was inhibited strongly by Ca2+, while the effect of Mg2+ was about 10,000 times weaker than that of Ca2+. Even in the absence of Ca2+, MgATP and MgADP inhibited the release of RLC, while the protective effect of AMPPNP was negligible. Other Mg nucleotides also showed some protective effect, though appreciably less than MgATP. The incubation of scallop myosin with abalone regulatory light chain (LC2) at 30 degrees C for 5 min produced a hybrid myosin. In the presence of 5 mM MgCl2, 1 of the 2 mol of RLC per mol of scallop myosin was exchanged with 1 mol of LC2. In the presence of Ca2+ or MgATP, myosin bound 1 extra mole of LC2 besides the 2 mol each of SH-LC and RLC.     

MgATPase activity of myosin subfragment 1. The dimer is more active than the monomer

The MgATPase activity of the rabbit skeletal myosin subfragment 1 (S1), in the steady state, was measured by means of the intrinsic fluorescence of tryptophan. This technique gave results similar to those obtained by other methods (linked or radioactive assays). The activity was measured under conditions that effect the monomer/dimer ratio. It is shown that there is a close correlation between MgATPase activity and the proportion of dimer. At 20 degrees C, for pH 6.9 to 8.1 and for [KCl] less than or equal to 1 M, the observed activity (kobs) can be linearly related to the proportion of dimer (Ed/Eo) by: kobs(s-1) = 0.016-7 X 10(-3)[KCl] + 0.031(Ed/Eo), where [KCl] is expressed in M. We deduce that, at 20 degrees C and for [KCl] = 0 M, the activity of the monomer is kmobs = 0.016 s-1 (Ed/Eo = 0) and that of the dimer kdobs = 0.047 s-1 (Ed/Eo = 1), i.e. a ratio kdobs/kmobs approximately equal to 3. Beyond pH approximately equal to 8.3, the activities of both the monomer and the dimer increased steeply with increasing pH value. In the standard conditions (pH 8.0, [KCl] = 0 to 100 mM), S1 is mainly in the form of a dimer, and such conditions are not appropriate for study of the S1 monomer. For studying the pure monomer, the conditions required at 20 degrees C and in bis-Tris-propane are: S1 concentration approximately equal to 0.2 mg/ml, pH 6.9 to 7.8, [KCl] approximately equal to 300 mM. For studying the pure dimer, the conditions required are: S1 concentration greater than or equal to 0.2 mg/ml, pH 7.8 to 8.1 and [KCl] approximately equal to 0. In both cases the MgATP concentration is about 50 microM. Finally, if great care is taken concerning the age of the S1 solutions and the evaluation of the proportion of dimer, the values of kobs are extremely precise: the uncertainty regarding the values of kobs, as determined by means of intrinsic fluorescence, does not exceed +/- 0.001 s-1. Beyond this error bar conditions are uncontrolled.     

Release of DEFB126 from macaque sperm and completion of capacitation are triggered by conditions that simulate periovulatory oviductal fluid

Capacitation of macaque sperm in vitro has been achieved efficiently only with the addition of both cyclic nucleotides and methylxanthines. The use of these exogenous sperm activators clouds an understanding of the normal mechanisms underlying capacitation and may slow early embryo development following in vitro fertilization (IVF). We demonstrate that culture medium which simulates periovulatory oviductal fluid with respect to bicarbonate (HCO(3)(-)) and glucose concentration induces capacitation in a high percentage of macaque sperm as determined by the ability of sperm to undergo both the release of coating protein DEFB126 and the zona pellucida-induced acrosome reaction (AR). Few sperm were able to undergo the AR following 6 hr incubation in medium containing either 35 mM HCO(3)(-) (approximately 7.2 pH) or 90 mM HCO(3)(-) (approximately pH 7.8) with 5 mM glucose. When glucose concentration was lowered to 0.5 mM to match levels reported for women at midcycle, the AR rate increased significantly in sperm incubated in both levels of HCO(3)(-), indicating that glucose interferes with sperm responsiveness to increasing HCO(3)(-) concentration observed in the primate oviduct during ovulation. Even greater synchronization of capacitation could be achieved with nonphysiologic extremes of alkalinity or energy substrate deprivation. In the latter case, sperm achieved high rates of IVF. A shift in pH from 7.2 to 7.8 in a HEPES-buffered medium was sufficient to remove DEFB126 from the surface of most sperm after only 3 hr. The loss of DEFB126 from sperm under periovulaory fluid conditions has implications for the timing of release of sperm from the oviductal reservoir.     

Mechanisms of loss of latency of lysosomal enzymes. Effects of incubation on the properties of lysosomal membranes.

1. The effects of sucrose and KCl on the loss of latency of lysosomal enzymes caused by incubation at 37 degrees C, pH 7.4, were examined by using Triton-filled lysosomes from rat liver and two fractions from livers of rats not injected with Triton. 2. After incubation, the percentage free activity of lysosomal enzymes was measured before and after cooling to 0 degrees C in order to determine the amount of latency lost at 37 degrees C without cooling and the additional amount lost on cooling the incubated lysosomes to 0 degrees C. 3. The latency that is lost without cooling is first decreased and then increased by increasing the osmotic strength of the incubation medium with KCl, or with sucrose in the presence of KCl. However, if the osmotic strength is increased with sucrose alone, loss of latency is decreased up to 0.25M-sucrose, but is increased only slightly at higher sucrose concentrations. Apparently the lysosome is permeated by hyperosmolar KCl but not by sucrose during incubation. 4. If the osmotic strength of the assay medium is increased with KCl, the loss of latency caused by incubation for 60 min in hyperosmolar KCl is repressed. Thus it appears that a KCl-permeated lysosome can be obtained which is relatively stable until exposure to lower osmolarities. 5. The loss of latency caused by cooling incubated lysosomes to 0 degrees C is largely eliminated if the osmotic strength of the medium in which the lysosomes are cooled is raised sufficiently with either sucrose or KCl. 6. Osmotic-fragility curves were obtained after incubation for 1 and 60 min at iso-osmoticity (0.2M-KCl or 0.25 M-sucrose). Although little loss of latency occurs at iso-osmoticity, lysosomes incubated for 60 min display greatly increased fragility on exposure to hypo-osmolar KCl, hypo-osmolar sucrose or hyperosmolar KCl. 7. It is suggested that permeability to KCl at 37 degrees C and the increase in fragility on exposure to hypo-osmolar conditions are both consequences of injury, probably from enzymic action, sustained by the lysosomal membrane during incubation at 37 degrees C.     

Effect of pH on lipolysis, cAMP and cAMP-dependent protein kinase activity in isolated rat fat cells

The effect of acidosis and alkalosis on lipolysis, cAMP production and cAMP-dependent protein kinase activity in isolated rat fat cells incubated in the presence of norepinephrine and norepinephrine plus theophylline has been investigated. The pH of the incubation medium was adjusted to 6.8, 7.4 and 7.8 respectively. Acidosis inhibited both norepinephrine- and norepinephrine plus theophylline-induced release of glycerol whereas alkalosis led to slight stimulation. Norepinephrine produced an increase in cAMP and cAMP-dependent protein kinase activity. However, comparison of both parameters in acidosis and alkalosis with those at pH 7.4 indicates that they were higher at pH 7.8 and lower at pH 6.8. Addition of theophylline in combination with norepinephrine increases cAMP production within 5 min, under acidosis to values similar to those obtained at pH 7.4 with norepinephrine. The same effect on protein kinase activity was obtained. In spite of this increment in cAMP and protein kinase activity produced by addition of norepinephrine plus theophylline, lipolysis remains inhibited by acidosis. Addition of theophylline at pH 7.4 and 7.8 induced a much higher cAMP production and cAMP-dependent protein kinase activity although at pH 7.8 there was a statistically significant increase in protein kinase activity at 10 min it did not induce a significant increase in lipolysis. This is discussed and possible mechanisms are suggested to explain the effect of acidosis and alkalosis on the lipolysis induced by norepinephrine in rat fat cells.     

Regulation of Ca2+ transport by isolated organelles of a rat insulinoma. Studies with endoplasmic reticulum and secretory granules

The regulation of extramicrosomal Ca2+ concentration maintained by suspensions of rat insulinoma microsomes was studied using Ca2+-selective minielectrodes. The Ca2+-transporting activity was MgATP dependent and correlated with the endoplasmic reticulum marker NADPH-cytochrome c reductase. When incubated in a high KCl medium containing Mg2+ and phosphate, the microsomes lowered [Ca2+] within less than 10 min to around 0.2 microM. They had a high Ca2+-sequestering activity since they were able to take up and retain several small Ca2+ additions. No evidence for a Na+/Ca2+ countertransport was obtained. The accumulated Ca2+ was released by the Ca2+ ionophore A23187 or upon transforming ATP into ADP using glucose plus hexokinase. The addition of ADP, at concentrations present in cells, resulted in a dose-dependent and reversible net Ca2+ efflux from the microsomes until a higher [Ca2+] steady state was reached. This was specific for ADP since GDP, UDP, CDP, IDP, and the nonhydrolyzable analogue methylene-ADP as well as AMP and cAMP did not reproduce the effect. Insulin secretory granules were unable to lower medium [Ca2+] or to take up a pulse addition of Ca2+. However, most of the large granular calcium content was released by A23187. The addition of Na+ and lowering or increasing medium pH by 0.2 pH unit did not induce Ca2+ uptake or efflux from the secretory granules. The results indicate that insulinoma endoplasmic reticulum but not insulin secretory granules may play a critical role in the regulation of cytosolic Ca2+. A variation in cellular ADP content following secretagogue addition might modulate Ca2+ fluxes across the endoplasmic reticulum and contribute in raising cytosolic Ca2+.     

Does the membrane potential control incorporation of tubulovesicles into the secreting apical membrane of the rat parietal cell?

The present studies were designed to examine the effect of changes in membrane potential by means of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) and variations in the pH of the medium on the secretory response of parietal cells. Studies were performed in vitro using isolated cells from rat stomachs and acid production was indirectly determined by 14C-aminopyrine (AP) accumulation. CCCP affected both basal and histamine-stimulated AP accumulation in a concentration-dependent manner. The AP accumulation ratios depended on pH of the incubation medium; the ratio was lowest at pH 6.6, and increased progressively as the pH of the medium increased to 7.8. Moreover, the decreases in AP accumulation ratios caused by simultaneous addition of CCCP and AP to cell suspensions compared to those in which CCCP was added to incubated cells after achieving the steady-state of AP accumulation were quantitatively similar. These findings suggest that the decrease in AP accumulation due to CCCP treatment is a consequence of an activation of acid secretion rather than an inhibitor of acid production. From the present and previously published data, we propose a working hypothesis: membrane recycling is dependent on changes in apical membrane potential.     

Evidence for calcium inactivation during hormone release in the rat neurohypophysis.

1. A study has been made of the relationship between 45Ca uptake into and hormone release from isolated rat neurohypophyses incubated in vitro. 2. Hormone secretion is triggered by high-K (56 mM) but long exposure to the stimulus does not generate a maintained release of hormone. 3. When hormone release began to wane, addition of Ba of La increased hormone output which suggests that the decline in output did not result from depletion of the neurosecretory granules at the nerve terminals. 4. 45Ca uptake is enhanced in the presence of high-K concentration, but the initial high rate declines during long exposure to the potassium stimulus with a time constant similar to that of the decline in hormone release. 5. After a period of incubation in a K-rich, calcium-free medium, addition of calcium to the medium induced hormone release. The magnitude of this release was dependent on the time of exposure to excess potassium. 6. After inactivation of secretion, mobilization of internal calcium by means of a calcium ionophore increased hormone release.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland.

The possibility that prostaglandin E2 (PGE2) may play a role in luteinizing hormone (LH) release was examined using an in vitro model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood. It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

A method for studying synaptosomal release of neurotransmitter candidates, as evaluated by studies on cortical cholecystokinin octapeptide release

A method for perfusing rat cortical synaptosomes for studying the regulation of cholecystokinin octapeptide (CCK-8) release has been developed and was found to have advantages over the static incubation system. Synaptosomes isolated from rat cortex were suspended in Biogel P2 columns and perfused with Krebs Ringer Bicarbonate buffer. One hundred mM KCl and 75 microM veratine stimulated CCK-8 release, which was Ca++-dependent. The synaptosomes were functionally viable for at least 135 min of incubation as indicated by multiple 100 mM KCl depolarizations and uptake of (3H)-norepinephrine and (14C)-choline. Dopamine and acetylcholine (10(-6)M) stimulated CCK-8 release while serotonin and norepinephrine were without effect. Approximately 20% of total occluded CCK-8 was released from synaptosomes by 100 mM KCl and degradation of CCK-8 was less than 10%. Perfusion of synaptosomes has several advantages over static incubation systems and allows systematic studies on the role of neurotransmitter in the regulation of neuropeptide secretion.