Rapid responses to aldosterone in human distal colon.

Aldosterone at normal physiological levels induces rapid increases in intracellular calcium and pH in human distal colon. The end target of these rapid signaling responses are basolateral K+ channels. Using spectrofluorescence microscopy and Ussing chamber techniques, we have shown that aldosterone activates basolateral Na/H exchange via a protein kinase C and calcium-dependent signaling pathway. The resultant intracellular alkalinization up-regulates an adenosine triphosphate (ATP)-dependent K+ channel (K(ATP)) and inhibits a Ca2+ -dependent K+ channel (K(Ca)). In Ussing chamber experiments, we have shown that the K(ATP) channel is required to drive sodium absorption, whereas the K(Ca) channel is necessary for both cyclic adenosine monophosphate and calcium-dependent chloride secretion. The rapid effects of aldosterone on intracellular calcium, pH, protein kinase C and K(ATP), K(Ca) channels are insensitive to cycloheximide, actinomycin D, and spironalactone, indicating a nongenomic mechanism of action. We propose that the physiological role for the rapid nongenomic effect of aldosterone is to prime pluripotential epithelia for absorption by simultaneously up-regulating K(ATP) channels to drive absorption through surface cells and down-regulating the secretory capacity by inhibiting K(Ca) channels involved in secretion through crypt cells.     

Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells

Permeabilized cells treated with the adenosine triphosphate analog, [35S]adenosine-5'-0-(3-thiotriphosphate) ([gamma-35S]ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated 35S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of 35S from the cells over a 20 min period. Treatment of permeabilized cells with ATP gamma S inhibited secretion and 35S incorporation into the cells. Pretreatment with ATP gamma S resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate 35S from [gamma-35S]ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells.     

A reassessment of guanine nucleotide effects on catecholamine secretion from permeabilized adrenal chromaffin cells

The role of guanine nucleotides in catecholamine secretion was investigated in alpha-toxin-permeabilized chromaffin cells. The stable GTP analogues, GTP-gamma-S (guanosine 5'-(gamma-thio)triphosphate) and GMP-PNP (guanosine 5'-(beta,gamma-imido)triphosphate), potentiated calcium-evoked catecholamine release in a dose-dependent manner. This effect was reversed by GDP-beta-S (guanosine 5'-(beta-thio)diphosphate) indicating that a GTP-binding protein plays a modulatory role in the calcium-dependent secretory process in chromaffin cells. Calcium and the phosphorylating nucleotide ATP were both necessary for secretion, even in the presence of GTP analogues, suggesting that the activation of a GTP-regulatory protein alone does not trigger exocytosis in these cells. TPA (12-O-tetradecanoylphorbol-13-acetate), a direct activator of protein kinase C, was found to mimic the effects of the GTP analogues, inducing a dose-dependent potentiation of the calcium-evoked release in alpha-toxin-permeabilized cells. Treatment of the permeabilized cells with sphingosine, a potent inhibitor of protein kinase C, completely abolished the stimulatory effects of both TPA and GTP-gamma-S. Moreover, long term incubation of chromaffin cells with TPA, a treatment which depletes cells of protein kinase C activity, suppressed the stimulatory effects of GTP-gamma-S. Protein kinase C is activated when it becomes membrane-bound in the presence of calcium and diacylglycerol; here, GTP-gamma-S was found to enhance the calcium-induced translocation of protein kinase C to membranes in alpha-toxin-permeabilized cells. These results suggest that guanine nucleotides modulate secretion by activating protein kinase C-linked events in chromaffin cells. Furthermore, the potentiation of calcium-induced secretion in alpha-toxin-permeabilized cells following activation of protein kinase C either directly with TPA or indirectly with GTP analogues provides additional support for the concept that protein kinase C may exert a positive control directly on the intracellular exocytotic machinery.     

Effect of chronic and acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration on the function and structure of cultured chromaffin cells

Cultured bovine adrenal chromaffin cells were treated chronically with various concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the culture medium for 2–8 days or acutely for 10–15 min. Culture of cells with MPTP for periods of 2–8 days resulted in a marked loss of total cellular catecholamines and a parallel reduction in secretory response, but not the ratio of stimulated to unstimulated secretion. By the eighth day in culture, at the highest MPTP concentration (1000 μM), cell catecholamine content and secretion were only about 10% that of untreated cells. The proportion of total cellular catecholamines secreted was not altered by MPTP, suggesting that the secretory process was unaffected by the drug. The loss of secretory output was not prevented by inhibitors of monoamine oxidase or catecholamine uptake, drugs known to prevent MPTP-induced damage to central dopaminergic neurons. The subcellular organelles of MPTP-treated cells appeared relatively normal except for extensive depletion of the vesicle contents, in agreement with the biochemical data. The severity of the depletion appeared to be lessened in cells treated with monoamine oxidase inhibitors.Short term exposure to MPTP at concentrations less than 100 μM had little effect on secretion induced by carbachol. Higher concentrations of MPTP increased unstimulated release and reduced stimulated release. Pretreatment of the cells with MPTP resulted in a lasting reduction in their subsequent secretory responsiveness. MPTP alone, at concentrations greater than 100 μM induced catecholamine release that was unaffected by pretreatment of the cells with monoamine oxidase inhibitors or the catecholamine uptake inhibitor desipramine. MPTP-induced secretion by intact cells was calcium-dependent, while the small increase by permeabilized cells was not.     

Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2alpha (PI3K-C2alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2alpha is required for acquisition of fusion competence in neurosecretion.     

On the mechanism of action of dexamethasone in a rat mast cell line (RBL-2H3 cells). Evidence for altered coupling of receptors and G-proteins

Prolonged exposure of rat basophilic leukemia (RBL-2H3) cells, a cultured analog of rat mast cells, to 0.1 microM dexamethasone resulted in global suppression of various stimulatory events in response to Ag and a global enhancement of the same stimulatory events to the adenosine analog, N-(ethylcarboxamide)adenosine (NECA). We had previously shown that Ag and NECA both activate phospholipase C but by different mechanisms; cells that had been treated with cholera or pertussis toxin, for example, responded to Ag but not to NECA with the release of inositol phosphates, increase in levels of cytosolic Ca2+, and secretion. Because the toxins still inhibited the responses to NECA in dexamethasone-treated cells, the effects of dexamethasone may have been exerted at the level of receptor/G-protein coupling rather than at the level of effector systems. Additional evidence for this was the following: 1) NECA-induced hydrolysis of the inositol phospholipids was still enhanced after permeabilizing (with streptolysin O or Staphylococcus alpha-toxin) and washing the cells; 2) the response to the G-protein stimulant, guanosine 5'-(3-O-thio)triphosphate was also enhanced in permeabilized, dexamethasone-treated cells and 3) binding and kinetic studies suggested that the enhanced responsiveness to NECA was attributable in part to an increase in receptor number. The suppressive action of dexamethasone on Ag-induced hydrolysis of inositol phospholipids, however, was readily lost by permeabilizing RBL-2H3 cells. The results indicate, therefore, that treatment with dexamethasone leads to changes in receptor-coupling mechanisms that are either resistant to (i.e., NECA-mediated responses) or reversed by (i.e., Ag-mediated responses) cell permeabilization.     

The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl-imidodi-phosphate (AMP-PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production. In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products. A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.     

Tumor Necrosis Factor Modulates the Inactivation of Catecholamine Secretion in Cultured Sympathetic Neurons

Cytokines exert multiple effects on cellular functions. We studied the effects of cytokines on the calcium-dependent release of catecholamines in cultured neurons from neonatal rat superior cervical ganglia. Incubation of sympathetic neurons with recombinant human interleukin-1 beta (0.14-0.7 nM) or recombinant human tumor necrosis factor-alpha (1 nM) for 24-48 h had no effect on the baseline spontaneous release and the initial K(+)-evoked [3H]norepinephrine release, compared with untreated cells. A repeat K(+)-induced depolarization after 6 min resulted in a decrease of [3H]norepinephrine secretion to 69 +/- 5.8% (n = 11) of the initial secretion in recombinant human tumor necrosis factor-treated cells, but not in control cells. The secretory response was restored when the interval between the two K+ challenges was increased to 10 min. We conclude that the diminished secretory response to a repeat stimulus in recombinant human tumor necrosis factor-treated superior cervical ganglia neurons is due to a prolonged recovery from inactivation of secretion in these cells.     

Dual regulation of ACTH secretion by guanine nucleotides in permeabilized AtT-20 cells

1. We have examined the effects of guanine nucleotides on ACTH secretion from digitonin-permeabilized AtT-20 cells, with the aim of analyzing the involvement of GTP-binding proteins (G proteins) in the secretory process. 2. AtT-20 cells permeabilized with 20 microM digitonin displayed calcium-dependent secretion. The EC50 of calcium was approximately 2 microM and the maximal stimulation was 350% of basal release. 3. Nonhydrolyzable guanine nucleotides also stimulated ACTH release, in a virtually Ca2+-free medium. The EC50 of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) was approximately 15 microM and the maximal stimulation was approximately 230% of basal release. The effects of calcium and guanine nucleotides were not additive. 4. In the presence of the inhibitory hormone, somatostatin guanine nucleotides inhibited the calcium-stimulated secretion. 5. Both the stimulatory and the inhibitory effects on secretion of guanine nucleotides were independent of changes in cyclic AMP (cAMP) and calcium. It is suggested that G proteins influence an unknown step in the secretion process, which would be near or at the exocytotic site. 6. The results can be explained by assuming the existence of two types of G proteins, one with stimulatory effects on exocytotic release (GeS) and another with inhibitory effects (GeI).     

ATP-dependent and ATP-independent pathways of exocytosis revealed by interchanging glutamate and chloride as the major anion in permeabilized mast cells.

Most investigations of the mechanism of regulated exocytosis have involved the use of secretory cells permeabilized in glutamate-based electrolyte solutions. In our previous work we have used NaCl-based electrolyte solutions. For secretion to occur from rat mast cells under these latter conditions, a dual effector system comprising Ca2+ and a guanine nucleotide are required; together they are sufficient. Here we compare the secretion from mast cells permeabilized in solutions of different electrolytes. Replacement of Na+ by K+ had little effect. Replacement of Cl- by Br-, SO4-, gluconate, isethionate, acetate, tartrate, succinate, etc. affected the maximal extent of secretion elicited by the dual effectors Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (Ca2(+)-plus-GTP-gamma-S) but had little influence on the effective affinity for Ca2+. The dicarboxylic amino acids (L- and D-glutamate, and L-aspartate) permitted exocytosis to be elicited by Ca2+ or GTP-gamma-S alone. Secretion stimulated by GTP-gamma-S is strongly inhibited by Cl- (50% inhibition by 20 mM Cl-), whereas the extent of Ca2(+)-induced secretion is proportional to the concentration of glutamate in mixed electrolyte buffers. Unlike dual-effector stimulation, secretion due to the single effectors requires adenosine triphosphate (ATP) and is prevented by inhibitors of protein kinase C. These results point to the existence of two parallel pathways for control of exocytosis in permeabilized cells, one ATP dependent, the other ATP independent.     

Desensitization of pituitary gonadotropin secretion by agonist-induced inactivation of voltage-sensitive calcium channels

Gonadotropin-releasing hormone (GnRH) stimulates calcium mobilization and influx in pituitary gonadotrophs, and agonist-induced calcium entry through voltage-sensitive channels (VSCC) is required for the maintenance of gonadotropin secretion. However, prolonged or frequent exposure to GnRH attenuates the extracellular Ca2+-dependent cytosolic Ca2+ signal and diminishes hormone secretion. Measurements of membrane Ca2+ currents revealed significant impairment of VSCC activity in gonadotrophs during desensitization by GnRH. VSSC were also inactivated in a calcium-dependent manner during exposure to high K+. Prolonged inactivation of such Ca2+ channels by high K+ reduced the calcium and secretory responses to GnRH and vice versa. The calcium-dependent inactivation of VSCC during GnRH action appears to be a primary factor in the onset of desensitization in pituitary gonadotrophs. This mechanism could also account for the development of agonist-induced refractoriness in other calcium-regulated target cells.     

GTP-dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein

Guanosine triphosphate (GTP) has been implicated in the regulation of Ca(2+)-mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 microM free Ca(2+) causes maximum secretion-equivalent to that achieved with 100 microM free Ca(2+)-whereas GTPgammaS inhibits Ca(2+)-stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP-regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca(2+). The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP-dependent secretory apparatus requires at least one cytosolic protein. GTP-dependent secretion can be reconstituted with crude HL-60 and bovine liver cytosol. The reconstituting activity binds to GTP-agarose, suggesting that the cytosolic factor is a GTP-binding protein or forms a complex with a GTP-binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion-reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high-molecular-weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate-ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP-dependent secretion, and that activity from a BFA-insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion.     

Effects of the juvenile hormone analog pyriproxyfen on German cockroach, Blattella germanica (L.), tergal gland development and production of tergal gland secretion proteins

Male German cockroaches possess secretory glands that secrete fluid into a pair of transverse depressions on the seventh and eighth abdominal tergites. We investigated the effects of altered juvenoid titer during the first part of the last instar on tergal gland secretory tissue development and the production of tergal gland secretion proteins. Male fifth (last) instar nymphs (1-3 days post-emergent) were topically treated with the JH analog pyriproxyfen. Light and transmission electron microscopy demonstrated that treatment with pyriproxyfen produced a visible decrease in the amount of tergal gland tissue present, a deformation of the overall shape of the gland located on tergite seven, and a less orderly arrangement of the secretory cells in the tissue. The protein fraction of tergal gland secretion was examined in pyriproxyfen-treated and control insects 1, 5, and 15 days after the insects molted to the adult stage. Amounts of all tergal secretion proteins were reduced in treated insects.     

Induction of tissue plasminogen activator secretion from rat heart microvascular cells by fM 1,25(OH)(2)D(3)

We investigated the effects of 1,25-dihydroxyvitamin D(3) [25(OH)(2)D(3)] on tissue plasminogen activator (tPA) secretion from primary cultures of rat heart microvascular cells. After an initial 5-day culture period, cells were treated for 24 h with 1,25(OH)(2)D(3) and several of its analogs. The results showed that 1,25(OH)(2)D(3) induced tPA secretion at 10(-10) to 10(-16) M. A less calcemic analog, Ro-25-8272, and an analog that binds the vitamin D receptor but is ineffective at perturbing Ca(2+) channels, Ro-24-5531, were approximately 10% as active as 1,25(OH)(2)D(3). An analog that binds the vitamin D receptor poorly but is an effective Ca(2+) channel agonist, Ro-24-2287, required approximately 10(-13) M to induce tPA secretion. Combinations of Ro-24-5531 and Ro-24-2287 were approximately as potent as 1,25(OH)(2)D(3). Treatment of the cells with BAY K 8644 or thapsigargin also increased tPA secretion, suggesting that increased cytosolic calcium concentration ([Ca(2+)]) induces tPA secretion. The results suggested that the sensitivity of the tPA secretory response of microvascular cells to 1,25(OH)(2)D(3) was due in part to generation of a vitamin D-depleted state in vitro and in part to synergistic effects of 1,25(OH)(2)D(3) on two different induction pathways of tPA release.     

Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities

Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, when conventional methods of nucleotide extraction were employed. Under the reaction conditions specified in this report, the initial rate of hydrolysis of adenosine triphosphate was directly proportional to the activity of APase in the sample extracts and consequently can be used as a sensitive measure of APase activity. A method was devised for obtaining reliable nucleotide measurements in naturally occurring microbial populations containing elevated levels of APase activity. The metabolic significance of APase activity in microbial cells is discussed, and it is concluded that the occurrence and regulation of APase in nature is dependent upon microscale inorganic phosphate limitation of the autochthonous microbial communities.     

Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells

The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitude and duration, and release of ACTH and beta- endorphin immunoactivity. Isoproterenol increased both action potential frequency and hormone secretion. Raising the external calcium concentration increased the frequency and amplitude of the action potentials and stimulated secretion of ACTH and beta-endorphin immunoactivity. Thus, stimulation of secretory activity in AtT-20 cells was closely correlated with increased electrical activity. However, a complete blockade of action potential activity had no effect on basal hormone secretion in these cells. These results suggest that the mechanisms underlying stimulated hormone secretion are different from those responsible for basal secretory activity. It is proposed that the increased influx of calcium due to the increased action potential frequency initiates the stimulated release of hormone from these cells.     

Motions of the SecA protein motor bound to signal peptide: Insights from molecular dynamics simulations

SecA is an essential part of the Sec pathway for protein secretion in bacteria. In this pathway, SecA interacts with the N-terminal fragment of the secretory protein – the signal peptide, and couples binding and hydrolysis of adenosine triphosphate with movement of the secretory protein across the SecY protein translocon. How interactions with the signal peptide alter the conformational dynamics and long-distance conformational couplings of SecA is a key open question that we address here with molecular dynamics techniques. Analyses of protein motions indicate that the signal peptide alters SecA dynamics not only at the site where this peptide binds, but also at a nucleotide-binding domain. Hydrogen bond clusters contribute to the long-distance propagation of changes in SecA dynamics.     

Cell surface complexes (''cortices'') isolated from Paramecium tetraurelia cells as a model system for analysing exocytosis in vitro in conjunction with microinjection studies.

Cortex preparations isolated from Paramecium tetraurelia cells consist of surface with secretory organelles (trichocysts) still attached. In the absence of nucleotides, in media with a pCa of 5-5.5 and a pH of greater than or equal to 6.5, maximal exocytosis occurred when the Mg2+ concentration was lowered from 10 to 0.5 mM. ATP, as well as its non-hydrolysable analogues adenosine 5'-[gamma-thio]triphosphate (ATP[S]) and adenosine 5'[beta gamma-imido]triphosphate (App[NH]p), inhibited exocytosis at a concentration equivalent to that occurring in vivo (as determined by h.p.l.c.), but preincubation with ATP augmented the exocytotic response. GTP and its analogues only slightly stimulated exocytosis in vitro, but sensitivity to Ca2+ was increased significantly, in particular with GTP. These effects of nucleotides were rapidly reversible. Intracellular GTP concentrations (0.35 mM) would suffice for full activation with the pCai values assumed to occur in these cells during activation. On microinjection, ATP inhibited the secretagogue response in intact cells. Whereas microinjected GTP stimulated exocytosis (membrane fusion) without a secretagogue added, Gpp[NH]p remained without any effect; GTP[S] permanently abolished any triggered secretory response. Concomitantly, h.p.l.c. analysis of triggered and untriggered cells showed that GTP hydrolysis occurs immediately after synchronous (1 s) exocytosis in vivo. The precise site(s) of action of GTP during signal transduction in Paramecium cells remain to be determined.     

Pharmacological Evidence for the Modulation of Monoamine Release by Adenosine in the Invertebrate Nervous System

An in vitro preparation from the pedal ganglia of the marine bivalve, Mytilus edulis, was used to examine the modulation of transmitter release by adenosine and its analogs from invertebrate nervous tissue. The ganglia of this organism contain the monoamines dopamine (DA), serotonin (5-HT), and norepinephrine (NE), and the presynaptic release of these substances is known to be calcium-dependent. This organism also contains a DA-sensitive adenylate cyclase system which resembles that seen in mammals. Neural tissue from the pedal ganglia was incubated with labeled monoamines, and release studies were then conducted in superfusion chambers; release of monoamines was evoked by the addition of 50 mM KCl. Addition to the superfusion medium of the adenosine analog, 5'-N-ethylcarboxamidoadenosine (NECA; 10 nM), inhibited the release of 5-HT and DA, and to a lesser extent NE, whereas 100-fold higher concentrations of adenosine itself and the adenosine analog, R-N6-phenylisopropyladenosine, were required to achieve comparable levels of inhibition. The inhibitory effects of NECA on neurotransmitter release were blocked by the adenosine receptor antagonist, theophylline (IC50 = 10-14 microM). The results from this study indicate for the first time the possible role of adenosine as a modulator of neurotransmitter release in the invertebrate nervous system.