Effects of antimicrotubule agents,potassium and inhibitors of energy production on hCG secretion

Summary The BeWo trophoblastic cell line was employed to assess the requirement for microtubules and cellular energy in human chorionic gonadotropin (hCG) secretion. In contrast to the general inhibitory effect of colchicine and vincristine on hormone secretion in systems involving exocytosis, wide concentration ranges of these antimicrotubule agents caused enhancement of hCG secretion. Similarly, cytochalasin B, an agent that interferes with microfilament function, doubled both basal hCG secretion, and secretion of hCG stimulated by 1mm dibutyryl cyclic AMP plus 1mm theophylline (dbT). Inhibitors of cellular energy production (2,4-dinitrophenol, malonate, azide) decreased both secreted and cellular levels of hormone. High concentrations of K⁺ gave no enhancement of basal or dbT-stimulated hCG secretion, nor any reduction of cellular hCG levels. These findings contrasted with observations of others in secretory systems involving exocytosis, in which high K⁺ potentiated basal or stimulated hormone release and depleted cellular stores of hormone. It was concluded that the process of hCG secretion in the malignant trophoblast is fundamentally different from the mechanism of protein hormone secretion in other tissues. This investigation was supported by Grant No. CA 23357 awarded by the National Cancer Institute, DHEW.     

Cell swelling induced secretion of TRH by posterior pituitary,hypothalamic paraventricular nucleus and pancreatic islets: effect of L-canavanine

The aims of this study were to test if ethanol induces thyrotropin-releasing hormone (TRH) secretion in vitro from the posterior pituitary and hypothalamic explants by a mechanism involving cell swelling, and to characterize the pathway of stimulated secretion. Ethanol, at a concentration of 80 mM, stimulated the release of TRH from the posterior pituitary, the hypothalamic paraventricular nucleus, the median eminence, and the brain septum, when administered only in isosmolar but not in hyperosmolar medium. This indicates the involvement of a cell swelling-inducing mechanism. L-canavanine in a concentration of 3 mM, increased the basal and hyposmosis-induced TRH secretion from the posterior pituitary and the paraventricular nucleus, and both basal and ethanol-induced TRH secretion from isolated pancreatic islets. This indicates the presence of both constitutive and regulatory secretory pathways. Our results suggest that cell swelling induces exocytosis from clathrin coated granules.     

K+ channel inhibition modulates the biochemical and morphological differentiation of human placental cytotrophoblast cells in vitro

Maintaining placental syncytiotrophoblast, a specialized multinucleated transport epithelium, is essential for normal human pregnancy. Syncytiotrophoblast continuously renews through differentiation and fusion of cytotrophoblast cells, under paracrine control by syncytiotrophoblast production of human chorionic gonadotropin (hCG). We hypothesized that K(+) channels participate in trophoblast syncytialization and hCG secretion in vitro. Two models of normal-term placenta were used: 1) isolated cytotrophoblast cells and 2) villous tissue in explant culture. Cells and explants were treated with K(+) channel modulators from 18 h, and day 3, onward, respectively. Culture medium was analyzed for hCG, to assess secretion, as well as for lactate dehydrogenase (LDH), to indicate cell/tissue integrity. hCG was also measured in cytotrophoblast cell lysates, indicating cellular production. Syncytialization of cytotrophoblast cells was assessed by immunofluorescent staining of desmosomes and nuclei. Over 18-66 h, mononucleate cells fused to form multinucleated syncytia, accompanied by a 28-fold rise in hCG secretion. 1 mM Ba(2+) stimulated cytotrophoblast cell hCG secretion at 66 h compared with control, whereas 5 mM tetraethylammonium (TEA) inhibited hCG secretion by >90%. 0.1-1 mM 4-aminopyridine (4-AP) reduced cytotrophoblast cell hCG secretion and elevated cellular hCG; without altering cellular integrity or syncytialization. In villous explants, hCG secretion was not altered by 1 mM Ba(2+) but inhibited by 5 mM 4-AP and 5/10 mM TEA, without affecting LDH release. Anandamide, pinacidil, and cromakalim were without effect in either model. In conclusion, 4-AP- and TEA-sensitive K(+) channels (e.g., voltage-gated and Ca(2+)-activated) regulate trophoblast hCG secretion in culture. If these K(+) channels participate in hCG secretion in situ, they may regulate trophoblast turnover in health and disease.     

Increase in cellular glutamate levels stimulates exocytosis in pancreatic beta-cells

Glutamate has been implicated as an intracellular messenger in the regulation of insulin secretion in response to glucose. Here we demonstrate by measurements of cell capacitance in rat pancreatic beta-cells that glutamate (1 mM) enhanced Ca2+-dependent exocytosis. Glutamate (1 mM) also stimulated insulin secretion from permeabilized rat beta-cells. The effect was dose-dependent (half-maximum at 5.1 mM) and maximal at 10 mM glutamate. Glutamate-induced exocytosis was stronger in rat beta-cells and clonal INS-1E cells compared to beta-cells isolated from mice and in parental INS-1 cells, which correlated with the expressed levels of glutamate dehydrogenase. Glutamate-induced exocytosis was inhibited by the protonophores FCCP and SF6847, by the vacuolar-type H+-ATPase inhibitor bafilomycin A(1) and by the glutamate transport inhibitor Evans Blue. Our data provide evidence that exocytosis in beta-cells can be modulated by physiological increases in cellular glutamate levels. The results suggest that stimulation of exocytosis is associated with accumulation of glutamate in the secretory granules, a process that is dependent on the transgranular proton gradient.     

Actin Involvement in Exocytosis from PC12 Cells: Studies on the Influence of Botulinum C2 Toxin on Stimulated Noradrenaline Release

Botulinum C2 toxin is known to ADP-ribosylate actin. The toxin effect was studied on [3H]noradrenaline secretion of PC12 cells. [3H]Noradrenaline release was stimulated five- to 15-fold by carbachol (100 microM) or K+ (50 mM) and 10-30-fold by the ionophore A23187 (5 microM). Pretreatment of PC12 cells with botulinum C2 toxin for 4-8 h at 20 degrees C, increased carbachol-, K+-, and A23187-induced, but not basal, [3H]noradrenaline release maximally 1.5-to three-fold, whereas approximately 75% of the cellular actin pool was ADP-ribosylated. Treatment of PC12 cells with botulinum C2 toxin for up to 1 h at 37 degrees C also increased stimulated [3H]noradrenaline secretion, whereas toxin treatment for greater than 1 h decreased the enhanced [3H]noradrenaline release stimulated by carbachol and K+ but not by A23187. Concomitantly with toxin-induced stimulation of secretion, 20-50% of the cellular actin was ADP-ribosylated, whereas greater than 60% of actin was modified when exocytosis was attenuated. The data indicate that ADP-ribosylation of actin by botulinum C2 toxin largely modulates stimulation of [3H]noradrenaline release. Moreover, the biphasic toxin effects suggest that distinct mechanisms are involved in the role of actin in secretion.     

Differential steroidogenic responses of ovine luteal cells to ovine luteinizing hormone and human chorionic gonadotropin

Experiments were conducted in vitro on ovine small luteal cells to evaluate their steroidogenic response to ovine luteinizing hormone (oLH) and human chorionic gonadotropin (hCG) administered continuously throughout the experimental period or as a 15-min pulse. Both oLH and hCG stimulated a significant increase in progesterone secretion (P less than 0.001) by small luteal cells. Human chorionic gonadotropin administered continuously or as a pulse maintained progesterone secretion at 40-55% of experimental maximum at least 6 hr while oLH-stimulated progesterone secretion declined to basal levels by 4 hr after a 15-min pulse or declined to 25% of the experimental maximum within 6 hr under constant stimulation. The responses of small luteal cells to oLH and hCG were found to differ (P less than 0.001). The sustained progesterone secretion of luteal cells in response to a pulse of hCG may be due to longer residence of occupied receptor complex on the cell membrane. In contrast, the decline in oLH stimulated progesterone secretion, even when hormone is continuously present in the medium, may be related to a rapid internalization of receptor-hormone complexes and down-regulation of receptors.     

Dual effects of manganese on prolactin secretion

The effect of Mn2+ (a commonly used Ca2+ antagonist) on prolactin secretion from pituitary cells was investigated. In the presence of normal extracellular Ca2+ levels (2.5mM), Mn2+ inhibited basal, TRH- and K+- stimulated prolactin secretion. The Ca2+ ionophore, A23187, partially overcame the inhibitory effect of Mn2+. However, in the presence of low extracellular Ca2+ (less than 100 microM), which decreased basal prolactin secretion and abolished any stimulatory effects of TRH or K+, a paradoxical stimulatory effect was observed with Mn2+ in the presence of A23187. In the presence of Ca2+, Mn2+ appeared to be inhibitory due to its Ca2+ antagonistic effects, but at low Ca2+ levels, intracellular stimulatory effects of Mn2+ became apparent.     

Calcium influx in a rat mast cell (RBL-2H3) line. Use of multivalent metal ions to define its characteristics and role in exocytosis

An increase in concentration of cytosolic Ca2+ ([Ca2+]i) is associated with an accelerated influx of 45Ca2+ when cultured RBL-2H3 cells are stimulated with either antigen or analogs of adenosine although these agents act via different receptors and coupling proteins (Ali, H., Cunha-Melo, J.R., Saul, W.F., and Beaven, M.A. (1990) J. Biol. Chem. 265, 745-753). The same mechanism probably operates for basal Ca2+ influx in unstimulated cells and for the accelerated influx in stimulated cells. This influx had the following characteristics. 1) It was decreased when cells were depolarized with high external K+; 2) it was blocked by other cations (La3+ greater than Zn2+ greater than Cd2+ greater than Mn2 = Co2+ greater than Ba2+ greater than Ni2+ greater than Sr2+) either by competing with Ca2+ at external sites (e.g. La3+ or Zn2+) or by co-passage into the cell (e.g. Mn2+ or Sr2+); and 3) the inhibition of influx by K+ and the metal ions had exactly the same characteristics whether cells were stimulated or unstimulated even though influx rates were different. The dependence of various cellular responses on influx of Ca2+ was demonstrated as follows. The stimulated influx of Ca2+, rise in [Ca2+]i, and secretion, could be blocked in a concentration-dependent manner by increasing the concentration of La3+, but concentrations of La3+ (greater than 20 microM) that suppressed influx to below basal rates of influx markedly suppressed the hydrolysis of inositol phospholipids (levels of inositol 1,4,5-trisphosphate were unaffected). Some metal ions, e.g. Mn2+ and Sr2+, however, supported the stimulated hydrolysis of inositol phospholipid and some secretion in the absence of Ca2+. Thus a basal rate of influx of Ca2+ was required for the full activation of inositol phospholipid hydrolysis, but in addition an accelerated influx was necessary for exocytosis.     

Mechanisms of luteinizing-hormone exocytosis in Staphylococcus aureus-alpha-toxin-permeabilized sheep gonadotropes.

We have used primary gonadotropes permeabilized with the pore-forming protein Staphylococcus aureus alpha-toxin to investigate luteinizing hormone (lutropin, LH) exocytosis. The diameter of the alpha-toxin pores (2-3 nm) allows the exchange of small molecules, whereas larger cytosolic proteins are retained. Because of the slow exchange of small molecules through the pores, we have developed a protocol which combines prolonged pre-equilibration of the permeabilized cells at 0 degrees C before stimulation with strong Ca2+ buffering. Under these conditions, increasing the free Ca2+ concentration from 0.1 microM to 10 microM [EC50 (concentration effecting half-maximal response) 2-3 microM] resulted in a 15-20-fold increase in LH exocytosis. LH exocytosis was maximal in the first 3 min and completed by 12 min. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca2(+)-stimulated LH secretion gradually declined (greater than 90% decrease by 60 min). Addition of MgATP (5 mM) rapidly restored full Ca2(+)-stimulated LH secretion. MgATP supported Ca2(+)-stimulated LH secretion at a half-maximal concentration of 1.5 mM. UTP and adenosine 5'-[gamma-thio]triphosphate were 40 and 31% as effective as MgATP, whereas other nucleotide triphosphates were ineffective. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 50 nM) stimulated LH exocytosis at free Ca2+ concentrations as low as 1 nM and was additive with Ca2+ at higher free Ca2+ concentrations. PMA-stimulated exocytosis required MgATP at concentrations similar to those required for Ca2(+)-stimulated LH exocytosis. These results demonstrate that LH exocytosis can be triggered both by micromolar Ca2+ concentrations or, in the virtual absence of Ca2+, by PKC activation. Both mechanisms of stimulated exocytosis have an absolute requirement for millimolar ATP. Because they retain cytosolic proteins, alpha-toxin-permeabilized cells may have advantages over alternative permeabilization methods provided that conditions are used that compensate for slow diffusion through alpha-toxin pores.     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

Osmotic regulation of prolactin secretion. Possible role of chloride

The role of osmotic pressure in the exocytosis of prolactin from rat pituitary tumor (GH) cells in culture was investigated. Reducing the osmotic strength of the medium from 300 mosm to 150 mosm by removal of NaCl did not alter basal secretion of prolactin but inhibited secretion stimulated by thyrotropin-releasing hormone (TRH) and forskolin. Both basal and stimulated secretion of prolactin were inhibited by increasing the osmotic strength of the medium with NaCl (IC50 at approximately 500 mosm). The stimulated release of hormone from GH-cells was independent of sodium and unaffected by replacement of sodium ion with tetramethylammonium or choline, or by addition of 500 nM tetrodotoxin. Secretagogue-stimulated release was, however, dependent upon chloride. Exchange of medium chloride with benzoate or isethionate significantly inhibited the stimulated release of prolactin (IC50 at approximately 60 mM exchange) regardless of the secretagogue utilized (phorbol ester, forskolin, depolarization plus BAY K8644, or TRH). Exchange of medium chloride with either isethionate or benzoate reduced cell volume by 10% compared to 60% for sucrose and mannitol, suggesting that inhibition of secretion by isethionate exchange was not a result of increased intracellular osmotic pressure. Complete exchange of medium chloride with isethionate did not alter equilibrium [3H]methyl-TRH binding, resting internal [Ca2+], or the [Ca2+]i response to depolarization and TRH as measured with intracellularly trapped Fura 2. Chloride removal did not change resting internal pH and recovery from an acid load as measured by the intracellular pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The stimulated secretion of prolactin was also inhibited by exchange of chloride with isethionate in normal pituitary cells in primary culture and the ability of normal cells to respond to the dopamine agonist bromocryptine was not affected by the exchange. These results suggest that exocytosis of prolactin from GH-cells and normal pituitary cells in culture is an osmotically driven process that is chloride-dependent. Stimulated release is more chloride-dependent than constitutive release. The inhibitory effect of isethionate substitution occurs after signal transduction and is distinct from the site of dopamine inhibition of prolactin release.     

Pertussis toxin blocks both cyclic AMP-mediated and cyclic AMP-independent actions of somatostatin. Evidence for coupling of Ni to decreases in intracellular free calcium

The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

ATP dependence of H+ secretion

Cells in isolated rabbit gastric gland were made permeable to ATP by high voltage discharge across a gland suspension. In both normal (5.4 mM K+) and high K+ (108 mM) medium, this electrical shock resulted in a marked reduction in the ability of the parietal cell to produce and accumulate acid. Acid production was monitored both microscopically by acridine orange accumulation in the secretory canaliculus and by accumulation of the weak base [14C]aminopyrine. In 108 mM K+ solutions but not in 5.4 mM K+ solutions 5, mM ATP was able to restore the accumulation of these probes to control (unshocked) levels. When shocked glands had been previously stimulated by secretagogues, the aminopyrine accumulation ratio was only partly restored by ATP. Inhibition of mitochondrial respiration by cyanide, azide, or Amytal abolished acid secretion; the subsequent addition of ATP to shocked glands increased the aminopyrine accumulation ratio to 47 and resulted in an acridine orange fluorescence indistinguishable from that of histamine-stimulated, unshocked glands. We conclude that ATP can act as a substrate for H+ secretion in the parietal cell, and that perhaps no additional energy source is necessary.     

A comparative study on the effects of different bile salts on mucosal ATPase and transport in the rat jejunum in vivo.

The effects of deoxycholate, taurocholate and cholate on transport and mucosal ATPase activity have been investigated in the rat jejunum in vivo using closed-loop and perfusion techniques. In the closed-loops, 5 mM deoxycholate selectively inactivated (Na+ + K+)-ATPase, and net secretion of Na+ induced by 2.5 mM deoxycholate was due to reduced lumen to plasma flux of the ion; deoxycholate (2.5 mM) produced marked inhibition of 3-0-methylglucose transport. Luminal disappearance rates of deoxycholate (60.5 plus or minus 2.9% per g wet st of gut) greatly exceeded those of taurocholate (4.3 plus or minus 1.0). In the perfusion studies 1 mM deoxycholate induced net secretion of water, Na+ and C1-, and inhibited active glucose transport; concomitantly "total" ATPase, (Na+ + K+)-ATPase, and Mg-2+-ATPase were inhibited. At higher concentrations (5 mM) deoxycholate stimulated Mg-2+-ATPase activity. Taurocholate and cholate at 1mM had no effect on transport of (Na+ + K+)-ATPase. Mucosal lactase, sucrase and maltase activities were not affected by 1 mM deoxycholate, taurocholate or cholate. These results suggest that deoxycholate inhibits sodium-coupled glucose transport by inhibition of (Na+ + K+)-ATPase at the lateral and basal membranes of the epithelial cell, rather than from an effect at the brush-border membrane level.     

Muscarinic receptor enhancement of nicotine-induced catecholamine secretion may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

Bovine adrenal chromaffin cells possess both nicotinic and muscarinic cholinergic receptors, but only nicotinic receptors have heretofore appeared to mediate Ca2+-dependent exocytosis. We have now found that muscarinic receptor stimulation in bovine adrenal chromaffin cells leads to enhanced inositol phospholipid metabolism as evidenced by the rapid (less than 1 min) formation of inositol trisphosphate (IP3) and inositol bisphosphate (IP2). Muscarinic receptor-mediated accumulation of IP3 and IP2 continues beyond 1 min in the presence of LiCl and is accompanied by large increases in inositol monophosphate. Muscarinic receptor stimulation was also found to enhance nicotine-induced catecholamine secretion by 1.7-fold if muscarine was added 30 s before nicotine addition. Moreover, since the muscarinic antagonist atropine reduces acetylcholine-induced secretion, we conclude that muscarinic receptor stimulation somehow primes these cells for nicotinic receptor-mediated secretion, perhaps by causing small nonstimulatory increases in cytosolic free Ca2+ mediated by IP3. Furthermore, we show that small depolarizations of these cells with 10 mM K+, which themselves do not affect basal secretion, also enhance nicotine-induced secretion. Thus, small increases in cytosolic free Ca2+ produced either by physiologic muscarinic receptor stimulation or by small experimental depolarizations with K+ may prime the chromaffin cells for nicotinic receptor-mediated secretion.     

Regulation of leptin secretion from white adipocytes by insulin, glycolytic substrates, and amino acids

The aim of the present study was to determine the respective roles of energy substrates and insulin on leptin secretion from white adipocytes. Cells secreted leptin in the absence of glucose or other substrates, and addition of glucose (5 mM) increased this secretion. Insulin doubled leptin secretion in the presence of glucose (5 mM), but not in its absence. High concentrations of glucose (up to 25 mM) did not significantly enhance leptin secretion over that elicited by 5 mM glucose. Similar results were obtained when glucose was replaced by pyruvate or fructose (both 5 mM). L-Glycine or L-alanine mimicked the effect of glucose on basal leptin secretion but completely prevented stimulation by insulin. On the other hand, insulin stimulated leptin secretion when glucose was replaced by L-aspartate, L-valine, L-methionine, or L-phenylalanine, but not by L-leucine (all 5 mM). Interestingly, these five amino acids potently increased basal and insulin-stimulated leptin secretion in the presence of glucose. Unexpectedly, L-glutamate acutely stimulated leptin secretion in the absence of glucose or insulin. Finally, nonmetabolizable analogs of glucose or amino acids were without effects on leptin secretion. These results suggest that 1) energy substrates are necessary to maintain basal leptin secretion constant, 2) high availability of glycolysis substrates is not sufficient to enhance leptin secretion but is necessary for its stimulation by insulin, 3) amino acid precursors of tricarboxylic acid cycle intermediates potently stimulate basal leptin secretion per se, with insulin having an additive effect, and 4) substrates need to be metabolized to increase leptin secretion.     

The use of drugs to dissect the pathway for secretion of the glycoprotein hormone chorionic gonadotropin by cultured human trophoblastic cells

Agents that affect intracellular cation and pH gradients and inhibit energy production have been tested for their ability to modulate the processing and secretion of the free alpha subunit and the alpha beta dimer of human chorionic gonadotropin (hCG) by cultured human trophoblastic cells (JAR). Incubation of JAR cells with monensin or nigericin, monovalent cation ionophores that produce equilibration of Na+ and K+ across cellular membranes, dicyclohexylcarbodiimide, an agent that inhibits intracellular membrane ATPases, and methylamine, which neutralizes intracellular pH gradients, produced similar effects on hCG processing and secretion. All these agents inhibited the processing of the asparagine-linked oligosaccharide chains of free alpha subunit and the alpha and beta subunits contained in the hCG dimer. Moreover, after treatment of JAR cells with these agents, there was an intracellular accumulation of precursor forms and an inhibition of secretion of "mature" forms of hCG. Monensin affected the processing and secretion of hCG subunits differently at different concentrations. At 5 X 10(-7) M, monensin inhibited the processing of the asparagine-linked oligosaccharides of hCG without altering the rate-limiting step in the secretory pathway or blocking hCG secretion. The intracellular hCG subunit precursors in both control and monensin-treated cells contained a similar array of high mannose oligosaccharides, predominantly of the Man8GlcNAc2 and Man9GlcNAc2 types. However, monensin-treated cells secreted hCG subunits that contained endo H-sensitive oligosaccharides of the high mannose (mostly Man5GlcNAc2) and hybrid types rather than the endo H-resistant complex chains synthesized by control cells. Nevertheless, a full complement of serine-linked oligosaccharides was added to the hCG-beta subunit in monensin-treated cells. These results indicate that the intracellular movement of hCG from the rough endoplasmic reticulum to the cell surface was not inhibited by monensin at a concentration that impaired Golgi-localized steps in the processing of asparagine-linked oligosaccharides. At 5 X 10(-6) M, monensin significantly inhibited secretion of hCG and created a new rate-limiting step in the processing pathway. hCG subunits bearing Man5GlcNAc2 units accumulated intracellularly, suggesting that the equilibration of intracellular Na+/K+ pools blocked oligosaccharide processing at an intra-Golgi point, perhaps by inhibiting movement of the glycoprotein hormone from the "cis" to the "trans" Golgi compartment. Since the other drugs mentioned above produced similar effects on hCG processing and secretion, it appears that maintenance of intracellular cation and pH gradients is necessary for the intra-Golgi transport of glycoprotein hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.