A nitric oxide/cyclic GMP-dependent protein kinase pathway alters transmitter release and inhibition by somatostatin at a site downstream of calcium entry.

We have examined the somatostatin-mediated modulation of acetylcholine release from intact chick embryo choroid tissue and compared these data with those obtained using acutely dissociated neuronal cell bodies from the chick ciliary ganglion. Acetylcholine release, evoked in a calcium-dependent manner by a high potassium (55 mM KCI) stimulation in both preparations, was inhibited almost completely by 100 nM somatostatin. Measurement of intracellular calcium in these neurons revealed that somatostatin blocked the large calcium transient that was observed in control neurons following KCI exposure. The modulatory effect of somatostatin on transmitter release was significantly attenuated by pre-treatment with pharmacologic agents that selectively block cyclic GMP (cGMP)-dependent protein kinase (PKG) or nitric oxide (NO) synthase. It is interesting that this prevention of somatostatin-mediated acetylcholine release inhibition occurred without reversal of the somatostatin-mediated block of the KCl-evoked calcium transient. Furthermore, a NO donor or cGMP analogue could block KCI-evoked acetylcholine release, but only cGMP could reduce the KCI-evoked calcium transient. Although cGMP could reduce the KCI-evoked calcium transient, a cGMP analogue was shown to reduce calcium ionophore-evoked transmitter release. Thus, somatostatin reduces acetylcholine release by modulating calcium influx, but the NO-PKG pathway can inhibit acetylcholine release, and alter somatostatin-mediated inhibition, by affecting transmitter release at some point after calcium entry.

Effect of cycloheximide and mRNA synthesis inhibition on death of trophically deprived ciliary ganglion neurons in culture.

1. The relationship between cycloheximide (CHX) and RNA synthesis inhibitors on trophic-deprived neuronal survival was studied with the use of primary cultures of stage (St) 34 chick ciliary ganglion (CG) neurons, to analyze the biological process of neuronal death caused by trophic factor withdrawal. Tissue culture conditions were refined by characterizing the additional medium components required to obtain 100% survival, for at least 1 wk, in the presence of an eye extract [choroid, ciliary body, iris, and pigment epithelium (CIPE)] as a trophic support for the neurons. Highly enriched neuronal cultures almost devoid of nonneuronal cells were used. 2. The time at which trophically deprived neurons cannot be rescued by the addition of trophic support, "commitment point," was established to be between 11 and 17 h after trophic deprivation. 3. CHX, an inhibitor of protein translation, reduced 3H-leucine incorporation by 90-95%, at a concentration of 10-100 micrograms/ml. The effect of the RNA transcription blockers actinomycin D (Act-D), alpha-amanitin, and 5.6 dichlorobenzimidazole riboside (DRB) on 3H-uridine incorporation into macromolecules was evaluated. Total RNA synthesis was inhibited by 10-25% by alpha-amanitin, whereas Act-D and DRB inhibited 80-97.5% of the 3H-uridine incorporation. 4. The effect of short- and long-term incubation with CHX on neuronal survival was analyzed. Continuous application of CHX promoted survival for 2-3 days, but thereafter neurons died regardless of whether CIPE was present or absent. Application of CHX for 6 h from the onset of the culture was enough to delay the commitment point up to 24 h after plating, and the addition of CIPE at this time maintained survival and promoted differentiation of CHX treated neurons. 5. The RNA transcription blockers Act-D, alpha-amanitin, and DRB were applied to both trophically deprived and trophically supported neurons, and the survival of each was evaluated. Neither drug was effective in supporting the survival of trophically deprived neurons in culture, and in most cases neurons even when cultured with CIPE died within 1-2 days in the presence of either drug. 6. Experiments using both CHX and mRNA synthesis blockers were performed to determine the effect of blocking mRNA transcription in trophically deprived neurons rescued by CHX. The addition of mRNA synthesis inhibitors precluded the effect of CHX on neuronal survival. 7. The effect of CHX (20 micrograms/ml) on RNA and protein synthesis was studied by measuring the incorporation of radiolabeled metabolic precursors (3H-leucine or 3H-uridine) into macromolecules. A 95% reduction in the protein synthesis was observed after 1 h of application of the drug, and by 24 h, 3H-leucine incorporation was reduced to 15-20% of the control values. Wash out of CHX after 6 h of incubation produced a recovery of protein synthesis up to 50% of control values 18 h later. CHX did not affect the synthesis of RNA for up to 12 h; however, it impaired the ability of the cell to take up metabolic precursors. 8. In conclusion, the present results support the hypothesis that the CHX effect on neuronal survival is due to its ability to induce the expression of survival or protective genes rather than to block the expression of killer proteins. This view is supported by 1) the 24-h delay of the commitment point following the short-term application of CHX, 2) the impaired ability of CHX to rescue trophic-deprived neurons by the addition of mRNA synthesis blockers, and 3) the fact that neuronal survival caused by trophic factors like CIPE, is blocked by blocking RNA transcription.

Somatostatin-induced inhibition of neuronal Ca2+ current modulated by cGMP-dependent protein kinase.

Neurotransmitter release is frequently regulated by peptides that modulate neuronal calcium channels. Whole-cell recordings show that the ion permeability and voltage dependence of these channels are controlled by a membrane-associated pathway involving GTP-binding proteins. Here we use perforated-patch recordings to show that, in addition to this pathway, the peptide somatostatin inhibits the calcium current in chick ciliary ganglion neurons by a second soluble pathway involving a cyclic GMP-dependent protein kinase (cGMP-PK). This somatostatin inhibition of Ca2+ current did not desensitize and was not characterized by the slowing of Ca(2+)-current activation (kinetic slowing) observed in whole-cell recordings. When cGMP-PK was inhibited, somatostatin inhibition of Ca2+ current resembled that observed with whole-cell recordings. cGMP agonists mimic the effect of somatostatin only in perforated patch recordings. An endogenous cGMP-PK therefore forms part of the mechanism by which somatostatin induces a sustained inhibition of neuronal calcium channels.

Developmental switch in the pharmacology of Ca2+ channels coupled to acetylcholine release.

The pharmacological specificity of Ca2+ channel-secretion coupling in acetylcholine (ACh) and somatostatin (SOM) release was studied in the chick eye choroid neuromuscular junctions and in dissociated ciliary ganglion (CG) neurons. ACh secretion changes in development from stage (St) 40, when release is dihydropyridine (DHP) and partially omega-conotoxin (omega-CgTX) sensitive, to posthatch, when release is insensitive to DHPs but sensitive to omega-CgTX. St 40 CG neurons cultured with striated muscle have release properties similar to those of St 40 iris and choroid but different from those of St 34 neurons, which are neither DHP nor omega-CgTX sensitive. SOM (also coreleased from posthatch choroid terminals) can inhibit ACh release in both posthatch and St 40 choroids, suggesting that the SOM receptor interacts with both DHP-sensitive and -insensitive channels.

Opiate and peptide inhibition of transmitter release in parasympathetic nerve terminals.

Somatostatin, morphine, and opioids inhibit transmitter release at intact neuromuscular junctions between ciliary ganglion neurons and the choroidal smooth muscle of the chick eye. Somatostatin and morphine, however, have no effect on release from terminals on the striated muscle target of the ciliary ganglion, the iris. In neuronal terminals of both the choroid and the iris, a high-affinity Na+-dependent choline uptake-mediated ACh synthesis is present at hatching. Both tissues exhibit a basal release of 3H-ACh which is potentiated severalfold during a 5 minute incubation in 55 mM K+ Tyrodes. Fifty percent of the basal release and 100% of the stimulated release are Ca2+ dependent and probably mediated through N-like voltage-dependent Ca2+ channels. Co-incubation of the choroid with 10 microM morphine sulfate blocks approximately 90% of the stimulated release. The same effect is seen with 100 nM somatostatin, 10 microM dynorphin, and 100 microM met-enkephalin arginine phenylalanine. Preincubation of the excised choroid with pertussis toxin (200 ng/ml) reverses the inhibitory effects of both morphine and somatostatin. In contrast, 3H-ACh release from terminals in the striated iris is not affected by either morphine or somatostatin at micromolar levels. These results suggest that both opiate and somatostatin receptors are present in the choroid target and that they may act through a final common pathway to modulate ACh release via G proteins. Second messengers such as cyclic AMP or diacylglycerol do not appear to mediate these effects; neither increasing cAMP levels in terminals nor activation of protein kinase C affects evoked release or its inhibition by morphine or other neuromodulators. It is unclear whether endogenous neuromodulation occurs in this system, although somatostatin-like immunoreactivity can be demonstrated in terminals of choroid neurons.