ATP stimulates calcium-dependent glutamate release from cultured astrocytes

ATP caused a dose-dependent, receptor-mediated increase in the release of glutamate and aspartate from cultured astrocytes. Using calcium imaging in combination HPLC we found that the increase in intracellular calcium coincided with an increase in glutamate and aspartate release. Competitive antagonists of P(2) receptors blocked the response to ATP. The increase in intracellular calcium and release of glutamate evoked by ATP were not abolished in low Ca(2+)-EGTA saline, suggesting the involvement of intracellular calcium stores. Pre-treatment of glial cultures with an intracellular Ca(2+) chelator abolished the stimulatory effects of ATP. Thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase from the Ca(2+) pump of internal stores, significantly reduced the calcium transients and the release of aspartate and glutamate evoked by ATP. U73122 (10 microM, a phospholipase C inhibitor, attenuated the ATP-stimulatory effect on calcium transients and blocked ATP-evoked glutamate release in astrocytes. Replacement of extracellular sodium with choline failed to influence ATP-induced glutamate release. Furthermore, inhibition of the glutamate transporters p-chloromercuri-phenylsulfonic acid and Ltrans-pyrolidine-2,4-dicarboxylate failed to impair the ability of ATP to stimulate glutamate release from astrocytes. However, an anion transport inhibitor, furosemide, and a potent Cl(-) channel blocker, 5-nitro-2(3-phenylpropylamino)-benzoate, reduced ATP-induced glutamate release. These results suggest that ATP stimulates excitatory amino acid release from astrocytes via a calcium-dependent anion-transport sensitive mechanism.     

Thymosin fraction 5 stimulates the release of prolactin from cultured GH3 cells

Thymosin fraction 5, a bovine thymus preparation, has recently been implicated in the regulation of neuroendocrine function. The purpose of the present study was to investigate the effects of thymosin fraction 5 treatment upon the GH3 rat pituitary cell line. Thymosin fraction 5 stimulated prolactin (PRL) release from these cells in a dose and time dependent manner. These results suggest that a product of the endocrine thymus may regulate the release of PRL.     

Albumin stimulates the release of lysophosphatidylcholine from cultured rat hepatocytes.

The effect of albumin on the release of [3H]lysophosphatidylcholine from cultured rat hepatocytes prelabelled with [Me-3H]choline was studied. In the absence of serum and albumin from the medium, the cells released essentially no [3H]lysophosphatidylcholine. Albumin stimulated this process dramatically, and it reached a plateau at 2 mg/ml. After an initial lag of 30 min, the release of [3H]lysophosphatidylcholine was linear for at least 4 h. At low concentrations, albumin slightly stimulated [3H]phosphatidylcholine release. The albumin had no measurable effect on the metabolism of cellular [3H]phosphatidylcholine, [3H]lysophosphatidylcholine or [3H]glycerophosphocholine. In addition, albumin did not alter the release of 3H-labelled water-soluble compounds, including [3H]glycerophosphocholine, into the medium. The possibility that the [3H]lysophosphatidylcholine was arising from catabolism of [3H]phosphatidylcholine in the medium by secreted enzymes was excluded. The effect on [3H]lysophosphatidylcholine secretion was also observed when the cells were incubated with alpha-cyclodextrin, a cyclic polysaccharide that has the ability to bind lysophosphatidylcholine. The albumin-released lysophosphatidylcholine was enriched in unsaturated fatty acids. Alteration of the fatty acid composition of cellular phosphatidylcholine gave rise to parallel changes in phosphatidylcholine and lysophosphatidylcholine in the medium. It is concluded that phosphatidylcholine is constantly being degraded in the rat hepatocyte to lysophosphatidylcholine which is released into the medium only when a suitable acceptor is present.     

An inhibitor of arachidonic acid metabolism stimulates luteinizing hormone (LH) release from cultured pituitary cells

5, 8, 11, 14 eicosatetraynoic acid ("ETYA", Roche 3-1428) is a competitive inhibitor of arachidonic acid metabolism. It effectively inhibits the action of both the lipoxygenases and the fatty acid cyclooxygenases both of which utilize arachidonic acid as a substrate. In the present work, we have shown that ETYA stimulates luteinizing hormone (LH) release from cultured pituitary cells (ED50 = 10 micrograms/ml). Stimulation is not due to contaminants present in the preparation, since highly purified ETYA (characterized by GC-MS) stimulates release, while contaminants removed by silicic acid chromatography do not. In addition, neither oxidized solutions of ETYA nor arachidonic acid itself stimulate LH release. ETYA stimulated release is dose dependent and is inhibited by ions which antagonize Ca2+ action. The observation that neither indomethecin (10, 100 micrograms/ml) nor meclofenamate (1.0, 10 micrograms/ml) stimulate LH release suggests that the effect of ETYA cannot be explained by an action on cyclooxygenase. The action of ETYA may be mediated either via an effect on lipoxygenase or through some nonspecific action (such as altered membrane fluidity).     

Endothelin-1 stimulates the release of preloaded [3H]D-aspartate from cultured cerebellar granule cells

We have recently reported that endothelin-1 (ET) induces phosphoinositide hydrolysis in primary cultures of rat cerebellar granule cells. Here we found that ET in a dose-dependent manner (1-30 nM) stimulated the release of preloaded [3H]D-aspartate from granule cells. The ET-induced aspartate release was completely blocked in the absence of extracellular Ca2+, but was unaffected by 1 mM Co2+ or 1 microM dihydropyridine derivatives (nisoldipine and nimodipine). At higher concentration (10 microM) of nisoldipine and nimodipine, the release was partially inhibited. Short-term pretreatment of cells with phorbol 12,13-dibutyrate (PDBu) potentiated the ET-induced aspartate release, while long-term pretreatment with PDBu attenuated the release. Long-term exposure of cells to pertussis toxin (PTX), on the other hand, potentiated the ET-induced effects. Our results suggest that ET has a neuromodulatory function in the central nervous system.     

An inhibitor of arachidonic acid metabolism stimulates luteinizing hormone (LH) release from cultured pituitary cells

5, 8, 11, 14 eicosatetraynoic acid (“ETYA”, Roche 3-1428) is a competitive inhibitor of arachidonic acid metabolism. It effectively inhibits the action of both the lipoxygenases and the fatty acid cyclooxygenases both of which utilize arachidonic acid as a substrate. In the present work, we have shown that ETYA stimulates luteinizing hormone (LH) release from cultured pituitary cells (ED₅₀ = 10 μg/ml). Stimulation is not due to contaminants present in the preparation, since highly purified ETYA (characterized by GC-MS) stimulates release, while contaminants removed by silicic acid chromatography do not. In addition, neither oxidized solutions of ETYA nor arachidonic acid itself stimulate LH release. ETYA stimulated release is dose dependent and is inhibited by ions which antagonize Ca²⁺ action. The observation that neither indomethecin (10, 100 μg/ml) nor meclofenamate (1.0, 10 μg/ml) stimulate LH release suggests that the effect of ETYA cannot be explained by an action on cyclooxygenase. The action of ETYA may be mediated either via an effect on lipoxygenase or through some nonspecific action (such as altered membrane fluidity).     

Angiotensin II increases cytosolic calcium and stimulates catecholamine release in cultured bovine adrenomedullary cells

In bovine adrenomedullary cells in primary culture, angiotensin II (AII) elicited virtually immediate, dose-related increments in cytosolic calcium [( Ca++]i) measured by the Quin 2 technique and stimulated approximately proportional secretion of norepinephrine, epinephrine, and dopamine measured by liquid chromatography with electrochemical detection. Peak responses of [Ca++]i to AII were similar to peak responses to nicotine or KCl. Pre-treatment with verapamil or washing the cells in calcium-free medium attenuated the stimulatory effect of AII on [Ca++]i. Pre-treatment with nicotine, which temporarily inactivates cholinergic receptor-activated calcium channels, did not affect [Ca++]i responses to AII. The results indicate functional effects of AII on cultured chromaffin cells. The mechanism of cellular activation by AII appears to include increases in [Ca++]i due to opening of membrane calcium channels which may be unrelated to cholinergic receptor-operated calcium channels.     

Control of phosphatase release from cultured tobacco cells

The activity of cultured tobacco XD-6 cells to release phosphataseinto the medium was enhanced after a time lag of 2 to 3 daysfollowing a marked increase in intracellular level of the enzymeduring Pi-omitted culture. The enhanced release of phosphatasehad a Q₁₀ value of about 2.3, and was suppressed by 2,4-dinitrophenoland azide. Cycloheximide did not inhibit the enzyme release,but Pi caused a rapid and drastic decrease in the rate of release.The release is suppressed even by 1 µM Pi. These results indicate that the rate of enzyme release is notdirectly limited by synthesis of the enzyme, but limited bythe transport, which may be suppressed by Pi, of the enzymeto the outside of the cell membrane. ¹ Present address: Laboratory of Applied Microbiology, Facultyof Agriculture, Yamagata University, Tsuruoka, Yamagata-ken997, Japan. (Received June 28, 1977; )     

The effects of dihydropyridines on neurotransmitter release from cultured neuronal cells

Depolarizing stimuli increase the release of transmitter substances from cultured PC12 pheochromocytoma cells and reaggregate cultures of mouse mesencephalic dopamine neurones. We measured the stimulated release of (3H) norepinephrine and (3H) dopamine from these systems respectively. In the cultured mouse dopaminergic neurones, several organic calcium channel blockers including nitrendipine, D-600, verapamil and diltiazem were unable to inhibit potassium-evoked transmitter release. However, release was blocked by 3 mM cobalt. The novel dihydropyridine calcium channel agonist BAY K8644 also had no effect on basal or evoked dopamine release. In contrast, BAY K8644 greatly stimulated the potassium-evoked release of (3H) norepinephrine from PC12 cells. The BAY K8644 enhanced release could be blocked by the dihydropyridine antagonist nitrendipine. These results indicate that while stimulus-secretion coupling in the PC12 cell line involves dihydropyridine sensitive calcium channels, this is not the case in primary cultured neurones.     

Leptin stimulates gonadotropin releasing hormone release from cultured intact hemihypothalami and enzymatically dispersed neurons

Leptin is a peptide released by adipocytes that has profound effects on central regulation of body metabolism. The present study represents an investigation into leptin effects on hypothalamic control of reproductive function, specifically on GnRH release. Adult male rats (gonadectomized or sham-operated) were used as donors of hypothalamic tissue that was used as intact hemihypothalami or as enzymatically dispersed hemihypothalami in a perifusion culture system. Continuous samples were collected at 10-min intervals for 8 to 10 hr and were assayed to measure temporal changes in GnRH release in response to various doses of leptin infused into the perifusion chambers. Leptin at the highest dose (10(-8) M) resulted in consistent and significant stimulation of GnRH release. There were no effects of treatment for surgical preparation (gonadectomy versus sham) or tissue preparation (intact versus dispersed hemihypothalami). The results of this study support the hypothesis that leptin plays a direct stimulatory role in the regulation of GnRH release. This study describes an important step in our understanding of the mechanism that connects changes in basal metabolism with reproductive function. These results indicate an intact interneuronal network is unnecessary for these leptin effects, but does not exclude a role for interneuronal networks in this regulatory pathway.     

Selective isoactin release from cultured embryonic skeletal muscle cells

The culture medium of embryonic quail myoblasts, labeled for 24 h with [35S]L-methionine, was analyzed by two-dimensional gel autoradiography. The major polypeptide observed had a 43,000 molecular weight and an isoelectric point of 5.4. This polypeptide could be specifically adsorbed to DNAse-I Sepharose. A tryptic peptide map of the [35S]methionine-labeled peptides of intracellular actin and the extracellular major polypeptide were virtually identical. These findings identify the released polypeptide as actin. A comparison of two-dimensional gel patterns of intracellular and extracellular labeled polypeptides showed a large number of differences indicating the actin release did not result from general cellular breakdown. The released actin was not filamentous as judged by its behavior during Bio-Gel A-5m chromatography (Bio-Rad Laboratories, Richmond, Calif.) The released actin did not originate solely from contaminating fibroblasts in the culture because actin was also observed in the medium in clonal myoblast cultures and in purified myotube preparations. Finally, the nonmuscle isoactins, as opposed to muscle alpha-isoactin, were released preferentially. These results indicate that within the developing muscle cell where both muscle and nonmuscle specific isoactins are simultaneously present, the different isoactins may be physically or functionally compartmentalized with the nonmuscle isoactins existing primarily at or near the cell surface.     

Lysophosphatidic acid stimulates fas ligand microvesicle release from ovarian cancer cells

Previous reports support that lysophosphatidic acid (LPA) upregulates Fas ligand (FasL) cell surface presentation on the ovarian cancer cells. In this study, we aim to investigate soluble FasL (sFasL) secretion associated with the small membrane microvesicles upon LPA stimulation, and to analyze the roles of cytoskeletal reorganization in FasL transport induced by LPA. Ovarian cancer cells were stimulated with LPA and spent media were harvested, concentrated, and ultracentrifugated to collect the supernatant and pellet. Western blot suggested that sFasL released from ovarian cancer cells were the mature form, and these sFasL are released with the small membrane microvesicles. Flow cytometry showed that the majority of microvesicles secreted contained FasL on their membrane, and these small membrane microvesicles are bioactive against activated human T lymphocytes. The microtubule-disrupting reagent nocodazole, not the actin-filament-disrupting reagent cytochalasin D pretreatment blocked FasL-expressing small membrane microvesicle release stimulated by LPA, suggesting that microtubules play an essential role in FasL microvesicle transport and exocytosis. LPA may promote ovarian cancer metastasis by counterattacking peritoneal cavity anti-tumor immunity.This work was supported by NCI UO1CA85133, NCI P50 CA83639, NIH R01 CA89503, NIH-RO1CA82562, and NIH RO1 CA01015.     

Porcine pancreatic phospholipase A2 stimulates secretin release from secretin-producing cells

We have isolated, from canine pancreatic juice, two 14-kDa proteins with secretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A2 (PLA2). In this study we have obtained evidence that secretin-releasing activity is an intrinsic property of pancreatic PLA2. Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid chromatography. They were tested for stimulation of secretin release from murine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enriched mucosal cell preparations isolated from rat upper small intestine. Each enzyme preparation was found to contain several components of secretin-releasing activity. Each bioactive fraction was purified to homogeneity by rechromatography and then subjected to mass spectral analysis and assays of PLA2 and secretin-releasing activities. It was found that the fraction with highest enzymatic activity also had the highest secretin-releasing activity and the same Mr as porcine pancreatic PLA2. Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA2, suggesting that it was identical to the enzyme. Purified porcine pancreatic PLA2 also stimulated secretin release concentration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum. Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory effect of purified pancreatic PLA2 on secretin secretion from STC-1 cells was inhibited by an L-type Ca2+ channel blocker, by down-regulation of protein kinase C or by pretreatment of the cell with pertussis toxin. It is concluded that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through the L-type channel and activation of protein kinase C.     

Electron donation to the plasma membrane redox system of cultured carrot cells stimulates proton release.

Membrane-permeable electron donors, duroquinol, diphenylcarbazide, pyrocatechol and tert-octylcatechol, promoted both reduction of an impermeant electron acceptor and proton transport with cultured carrot cells. These cells were preloaded with electron donors for 15, 30, 45 and 60 min. Aliquots of cells were removed at various times, washed free of excess electron donors and assayed for their effect on transplasma membrane redox with impermeable hexacyanoferrate (HCF III) as the electron acceptor and for simultaneous H+ excretion in the presence of hexacyanoferrate. All four electron donors stimulated HCF III reduction and associated H+ excretion. Below a rate of hexacyanoferrate reduction of 6 mumol/g dry wt. per min, the ratios of H+/e- were between 0.3 and 1 with low concentrations (0.1 mM) of the added electron donors. When hexacyanoferrate reduction exceeded 6 mumol/g dry wt. per min, proton release began to cascade to give ratios of 1 to 3, suggesting activation of an H(+)-ATPase or a proton transporter. This behavior by cultured carrot cells indicates that a certain threshold of proton concentration in a limited membrane domain must be reached in order for the proton channel to be opened.     

Transferrin stimulates iron absorption, exocytosis, and secretion in cultured intestinal cells

The cellularmechanism by which basolateral transferrin (Tf) produces an increase inapical-to-basolateral Fe flux in Caco-2 cells was analyzed. After apulse of ⁵⁹Fe from the apicalmedium, three types of basolateral⁵⁹Fe efflux were found: a⁵⁹Fe efflux that was independentof the presence of Tf in the basolateral medium, a⁵⁹Fe efflux in which⁵⁹Fe left the cell bound to Tf,and a Tf-dependent ⁵⁹Fe efflux inwhich ⁵⁹Fe came off the cell notbound to Tf. Furthermore, addition of Tf to the basolateral mediumdoubled the exocytosis rate of Tf and increased the secretion ofapolipoprotein A, a basolateral secretion marker. Both apotransferrinand Fe-containing Tf produced similar increases in⁵⁹Fe efflux, Tf exocytosis, andapolipoprotein A secretion. The Ca²⁺ channel inhibitor SKF-96365inhibited both the Tf-mediated increase in transepithelial Fe transportand the secretion of apolipoprotein A. Thus the activation oftransepithelial Fe transport by Tf seems to be mediated byCa²⁺ entry into the cells.