Galanin can inhibit insulin release by a mechanism other than membrane hyperpolarization or inhibition of adenylate cyclase.

Studies on the mode of action of galanin to inhibit insulin release in RINm5F cells have shown that basal and glyceraldehyde-stimulated release were both inhibited. Galanin was inhibitory at concentrations in the low nanomolar range. Binding studies with 125I-labeled galanin indicated that the RINm5F cells exhibit a single set of sites estimated to be of the order of 30,000 sites/cell. Displacement of 125I-galanin by galanin from the receptor sites occurred over a similar concentration range to that which inhibited insulin release. Half-displacement was achieved with 2 nM galanin. Measurements of bis-(1,3-diethylthiobarbiturate) trimethineoxonol (bis-oxonol) fluorescence showed that galanin hyperpolarized the RINm5F cell plasma membrane. Measurements of intracellular free calcium, [Ca2+]i by means of the fluorescent indicator fura-2 showed that galanin decreased [Ca2+]i. As galanin did not inhibit either basal or glyceraldehyde-stimulated insulin release in the presence of the Ca2+ channel blocker nitrendipine, the hyperpolarization and reduction of Ca2+ entry appear to be a possible explanation for the galanin effects. However, quantitatively, the effects on membrane potential and [Ca2+]i appear to be insufficient to account for the potent inhibition of insulin release. Furthermore, evidence for an additional mechanism of action was obtained from experiments with 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester which stimulates insulin secretion by at least two mechanisms, one Ca2+ dependent and one Ca2+ independent. TPA-stimulated insulin release was inhibited by galanin over the same concentration range as for the inhibition of glyceraldehyde-stimulated release. Galanin inhibited TPA-stimulated release in the presence of maximally effective concentrations of nitrendipine and in the absence of extracellular Ca2+. These effects cannot be explained by hyperpolarization of the plasma membrane and consequent reduction of Ca2+ entry via the voltage-dependent Ca2+ channels. One suggested mechanism for the action of galanin is inhibition of adenylate cyclase. However, it was found that galanin inhibits insulin release even in the presence of 8-Br-cAMP, an agent which effectively bypasses adenylate cyclase. Therefore, an additional mechanism for the inhibitory effect of galanin must be present. All of the effects of galanin were sensitive to pertussis toxin. These data suggest two G-protein-dependent actions of galanin, one to hyperpolarize the plasma membrane and one at a distal point in stimulus-secretion coupling, close to the exocytotic event.

Characterization of calcium channels of a glucose-responsive rat insulinoma.

Voltage-dependent calcium channels are important in the control of calcium influx into excitable cells. With the use of cells isolated from a glucose-responsive rat insulinoma, the effect of the calcium-channel blocker nitrendipine on insulin release was examined. When 50 nM nitrendipine was added with a stimulatory glucose concentration (30 mM), first-phase insulin release was preserved, whereas the second phase was inhibited by 39.4 +/- 5.3%. When 50 nM nitrendipine was also present during basal and stimulated insulin release, the insulin release from fresh cells was significantly less during both phases compared with cells not exposed to nitrendipine (P less than 0.05). In cells cultured for 1 day in 30 mM glucose, a diminished insulin response was seen on stimulation with 30 mM glucose. Nitrendipine (50 nM) did not lead to a further decrease in insulin release. Saturable binding sites in homogenate, whole cells, and purified plasma membranes prepared from the insulinoma were characterized using [3H]nitrendipine. The Kd (in pM) for homogenate from uncultured cells was 225 +/- 34 (n = 11), whereas the Bmax (in fmol/mg protein) was 52 +/- 5. The number of apparent binding sites for [3H]nitrendipine was reduced by approximately 50% in cultured cells (Bmax = 30 +/- 5 fmol/mg protein). The reduction in insulin release from cultured cells correlated well with the reduction in nitrendipine binding. It is concluded that the decrease in the number of apparent binding sites for nitrendipine after culture represents a reduction in functional calcium channels associated with influx of calcium and insulin release.

Phorbol ester-stimulated insulin secretion by RINm5F insulinoma cells is linked with membrane depolarization and an increase in cytosolic free Ca2+ concentration.

In studying the regulation of insulin secretion by phorbol esters, we examined their effects on the cytosolic free Ca2+ concentration ([Ca2+]i), using the Ca2+ indicator fura-2 in the rat insulin-secreting beta-cell line RINm5F. [Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i. Ca2+-free conditions eliminated the increase in [Ca2+]i and resulted in a reduced stimulation of insulin release by TPA. The Ca2+ channel blocker nitrendipine (300 nM) inhibited both the increase in [Ca2+]i and the increased rate of insulin secretion. Another phorbol ester, 4 beta-phorbol 12,13-didecanoate, which activates protein kinase C, also induced an increase in [Ca2+]i and in the rate of insulin release, while 4 alpha-phorbol 12,13-didecanoate, which fails to stimulate protein kinase C, was without effect. Further studies with bis-oxonol as an indicator of membrane potential showed that TPA depolarized the beta-cell plasma membrane. From these results, it is concluded that TPA depolarizes the plasma membrane, induces the opening of Ca2+ channels in the RINm5F beta-cell plasma membrane, increases [Ca2+]i, and results in insulin secretion. The action of TPA was next compared with that of a depolarizing concentration of KC1 (25 mM), which stimulates insulin secretion simply by opening Ca2+ channels. TPA consistently elicited less depolarization, a smaller rise of [Ca2+]i, but a greater release of insulin than KC1. Therefore an additional action of TPA is suggested, which potentiates the action of the elevated [Ca2+]i on insulin secretion.

[3H]Ro 15-1788 binding to benzodiazepine receptors in mouse brain in vivo: marked enhancement by GABA agonists and other CNS drugs.

Administration of the benzodiazepine receptor antagonist, [3H]Ro 15-1788, to mice intravenously was found to label these receptors in brain. Binding of [3H]Ro 15-1788 in vivo was strongly blocked by pretreating mice with clonazepam or diazepam. Marked enhancement of [3H]Ro 15-1788 binding in vivo was induced by progabide or sodium valproate. This effect was greater than a similar enhancement of [3H]flunitrazepam binding. The increased membrane-bound [3H]Ro 15-1788 elicited by progabide was completely dissociated on subsequent incubation with Ro 15-1788, diazepam or clobazam, indicating that the enhanced binding occurred at benzodiazepine receptors. Compounds that exert diazepam-like actions and/or indirect GABAergic activity (cartazolate, pentobarbital, methaqualone, levonantradol, phenytoin) elicited enhancement of [3H]Ro 15-1788 in vivo. Other CNS agents (atypical neuroleptics, GABA antagonists, baclofen, some 5-HT1 agonists) also induced elevation of [3H]Ro 15-1788 binding in vivo, as did drugs exerting vasodilatatory effects (papaverine, nimodipine, verapamil, prazosin, N6-cyclohexyladenosine). Possible explanations for enhancement of [3H]Ro 15-1788 binding in vivo include increase in the number of benzodiazepine receptors induced by GABA or GABAergic drugs or effects of binding enhancers that elevate brain levels of [3H]Ro 15-1788, such as accelerating cerebral blood flow, competing for radioligand binding sites in plasma or increasing metabolic stability of the radioligand.

Sensitive enzyme immunoassay with beta-D-galactosidase-Fab conjugate for detection of type A influenza virus antigen in clinical specimens.

The most sensitive method for diagnosis of type A influenza virus infection is isolation of the agent in cell culture. However, detection and identification may require several days to complete. This delay in diagnosis prevents effective use of the antiviral agents available for treatment of type A influenza infection. As a rapid diagnostic method, enzyme immunoassay (EIA) is attaining increased usage for direct detection of viral antigen in clinical specimens. Standard EIA techniques, however, are usually not sensitive enough for reliable detection of viral antigen in respiratory secretions. We developed a conjugate consisting of the antigen-binding fragment of goat antirabbit immunoglobulin G coupled to beta-d-galactosidase, using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Other immunoreagents in our EIA consisted of guinea pig and rabbit antisera to influenza A/Brazil/11/78 (H1N1) for microtiter plate coating and primary antiserum, respectively. The sensitivity of this EIA was tested with 60 clinical specimens containing influenza A/England/333/80 (H1N1) which closely resembles A/Brazil. Of 31 initial specimens, collected within 24 h of the onset of symptoms, 27 (87%) were positive, using a fluorgenic substrate, and 18 of 29 (62%) specimens obtained 12 to 60 h after the initial specimens were positive, for a total of 75% (45 of 60). All positive reactions were specific, as shown in a confirmatory test with preimmune and hyperimmune guinea pig globulins. Clinical specimens negative for virus (n = 33) or containing heterologous respiratory viruses (n = 26) were negative in this system. These results indicate that EIA systems can be developed with a sensitivity approaching that required for clinical usefulness.