Neuronal Adenylate Cyclase Research Articles

A Gain-of-function Mutation in Adenylate Cyclase Confers Isoflurane Resistance in Caenorhabditis elegans

Volatile general anesthetics inhibit neurotransmitter release by a mechanism not fully understood. Genetic evidence in Caenorhabditis elegans has shown that a major mechanism of action of volatile anesthetics acting at clinical concentrations in this animal is presynaptic inhibition of neurotransmission. To define additional components of this presynaptic volatile anesthetic mechanism, C. elegans mutants isolated as phenotypic suppressors of a mutation in syntaxin, an essential component of the neurotransmitter release machinery, were screened for anesthetic sensitivity phenotypes. Sensitivity to isoflurane concentrations was measured in locomotion assays on adult C. elegans. Sensitivity to the acetylcholinesterase inhibitor aldicarb was used as an assay for the global level of C. elegans acetylcholine release. Comparisons of isoflurane sensitivity (measured by the EC₅₀) were made by simultaneous curve-fitting and F test. Among the syntaxin suppressor mutants, js127 was the most isoflurane resistant, with an EC₅₀ more than 3-fold that of wild type. Genetic mapping, sequencing, and transformation phenocopy showed that js127 was an allele of acy-1, which encodes an adenylate cyclase expressed throughout the C. elegans nervous system and in muscle. js127 behaved as a gain-of-function mutation in acy-1 and had increased concentrations of cyclic adenosine monophosphate. Testing of single and double mutants along with selective tissue expression of the js127 mutation revealed that acy-1 acts in neurons within a Gαs-PKA-UNC-13-dependent pathway to regulate behavior and isoflurane sensitivity. Activation of neuronal adenylate cyclase antagonizes isoflurane inhibition of locomotion in C. elegans.

Dopamine: Biphasic Dose Responses

The present article indicates that dopamine and/or its agonists induce biphasic dose-response relationships for numerous endpoints. These include locomotion, pain sensitivity, blood pressure, prolactin secretion, oxytocin release, heart rate, memory, and neuronal adenylate cyclase activity. Biphasic responses were reported predominantly with male Sprague-Dawley rats, but also with mice, dogs, monkeys, and humans. Regardless of the model or endpoint the maximum changes from the control were always modest being within the 10 to 80% range. The range of stimulatory responses was quite variable, extending from slightly greater than a factor of 10 for the endpoints such as memory, pain-vocalization, and diastolic blood pressure to the 106 range for prolactin release and the 108 range for oxytocin release. Mechanistic studies suggested that the stimulatory and inhibitory effects of dopamine are mediated by different receptors or receptor subtypes having opposite actions and different ligand affinities.

AMPA receptor-mediated regulation of a Gi-protein in cortical neurons.

Excitatory synaptic transmission in the central nervous system is mediated primarily by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. The myriad intracellular responses arising from the activation of the NMDA and AMPA receptors have previously been attributed to the flow of Ca2+ and/or Na+ through these ion channels. Here we report that the binding of the agonist AMPA to its receptor can generate intracellular signals that are independent of Ca2+ and Na+ in rat cortical neurons. In the absence of intracellular Ca2+ and Na+, AMPA, but not NMDA, brought about changes in a guanine-nucleotide-binding protein (Galpha[il]) that inhibited pertussis toxin-mediated ADP-ribosylation of the protein in an in vitro assay. This effect was observed in intact neurons treated with AMPA as well as in isolated membranes exposed to AMPA, and was also found in MIN6 cells, which express functional AMPA receptors but have no metabotropic glutamate receptors. AMPA also inhibited forskolin-stimulated activity of adenylate cyclase in neurons, demonstrating that Gi proteins were activated. Moreover, both Gbetagamma blockage and co-precipitation experiments demonstrated that the modulation of the Gi protein arose from the association of Galpha(il) with the glutamate receptor-1 (GluR1) subunit. These results suggest that, as well as acting as an ion channel, the AMPA receptor can exhibit metabotropic activity.

CAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (IH) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately -60 mV in nominally zero-Ca2+ Krebs solution containing tetrodotoxin (1 microM), tetraethylammonium (40 mM), Cd2+ (500 microM) and 4-aminopyridine (1 mM). IH developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time. IH was selectively blocked by extracellular Cs+ (1 mM). The activation of the H-channel conductance (GH) ranged between -55 and -120 mV. The GH was 80-150 pS (n = 4) at the half-activation voltage of -84 +/- 7 mV (n = 4). The reversal potential of IH obtained by instantaneous current voltage (I/V) relations was -41 +/- 6 mV (n = 4); it shifted to -51 +/- 8 mV (n = 3) in low-Na+ (20 mM) solution and to -24 +/- 4 mV (n = 4) in high-K+ (20 mM) solution. Forskolin (1-10 microM) produced an inward current and increased the amplitude of IH. Forskolin did not change the half-activation voltage of GH. 8-Bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP, 0.1-1 mM) and dibutyryl-cAMP (0.1-1 mM) enhanced IH. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhanced IH. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.

Mu-Opioid receptor-mediated reduction of neuronal calcium current occurs via a G(o)-type GTP-binding protein

It has recently been shown that the activation of mu-opioid receptors inhibits several components of calcium channel current in rat DRG sensory neurons. mu-Opioid receptors, acting through the pertussis toxin (PTX)-sensitive substrate Gi, also reduce the activity of neuronal adenylate cyclase, but the relationship of this effect to changes in calcium channel activity has yet to be determined. Using whole-cell recordings from acutely isolated rat DRG neurons, we examined the ability of the mu-opioid-selective agonist Tyr-Pro-NMe-Phe-D-Pro-NH2 (PLO17) to reduce calcium current after treatment with PTX and in the presence of the nonhydrolyzable GTP analog guanosine 5'-[-thio]triphosphate (GTP gamma S), to assess the role of G-proteins in the coupling of mu-opioid receptors to calcium channels. Inhibition of current by PLO17 was mimicked or rendered irreversible by intracellular administration of GTP gamma S, an activator of G-proteins, and was blocked by pretreatment of neurons with PTX. In contrast, when the catalytic subunit of cAMP-dependent protein kinase was included in the recording pipette, calcium currents increased in magnitude throughout the recording without attenuation of responses to PLO17. Thus, the mu-opioid-induced inhibition of calcium current occurs through activation of a Gi- or G(o)-type G-protein, but independent of changes in adenylate cyclase activity. As a first step in identifying this G-protein, we compared the ability of several antisera directed against specific regions of Gi and G(o)alpha subunits to block the inhibition in current by PLO17. Intracellular dialysis with an antiserum specific for G(o) (GC/2) attenuated calcium current inhibition by PLO17 in five of six neurons by an average of 75%. In contrast, there was no attenuation in the response to PLO17 when neurons were dialyzed with an anti-Gi1 alpha/Gi2 alpha antiserum (AS/7) or antibodies specific for alpha subunits of Gi proteins (Gi1/Gi2 or Gi3) in an identical manner. These results suggest that in rat DRG neurons mu-opioid receptors couple to calcium channels via the PTX-sensitive G(o) subclass of GTP-binding proteins.

Differential effects of chronic agonist administration on μ-opioid receptor- and muscarinic receptor-regulated adenylate cyclase in rat striatal neurons

In cultured rat striatal neurons exposed to 10 μM morphine or oxotremorine for 24 hours, we observed an increased (about 30 %) dopamine D 1 receptor-stimulated cyclic AMP production, whereas no desensitization of μ-opioid receptor or muscarinic cholinergic receptor was found. However, whereas upregulation of dopamine D 1 receptor-stimulated adenylate cyclase activity upon 7 days morphine exposure was at least as pronounced as observed after 24 hours of exposure to the opioid, this adaptive phenomenon was virtually absent following one week of oxotremorine treatment. This reduced adenylate cyclase sensitization upon 7 days oxotremorine exposure appeared to coincide with desensitization of muscarinic cholinergic receptors. A possible role of the resistance of μ receptors to desensitization and the (resulting) upregulation of the neuronal adenylate cyclase system upon chronic receptor activation in the development of opiate tolerance and dependence is suggested.

Dynorphin A and cAMP-dependent protein kinase independently regulate neuronal calcium currents.

The kappa-selective opioid peptide dynorphin A (DYN) inhibits neuronal adenylate cyclase activity and reduces neuronal voltage-dependent calcium currents. It is not yet known, however, whether the regulation of calcium channel activity is dependent on or independent of the adenylate cyclase/cAMP system. We used the whole-cell variation of the patch clamp technique to show that DYN reversibly reduced, in a naloxone-sensitive manner, calcium currents in acutely dissociated rat nodose ganglion neurons. DYN slowed the rate of current activation and had a greater effect on currents evoked from relatively negative holding potentials. These actions were mimicked by guanosine 5'-[gamma-thio]triphosphate, which activates GTP-binding proteins (G proteins), and were blocked by pretreatment with pertussis toxin, which inactivates Gi- and Go-type G proteins. In contrast, calcium currents recorded in the presence of the catalytic subunit of the cAMP-dependent protein kinase (AK-C), included in the recording pipette, increased in magnitude throughout the recording. DYN was applied to neurons before and after the effect of AK-C became apparent; the reduction of calcium currents by DYN was greater in the presence of AK-C than in its absence. We conclude that the acute reduction of neuronal calcium currents by DYN occurred by means of activation of pertussis toxin-sensitive Gi- or Go-type G proteins. The persistence of the action of DYN in the presence of AK-C indicates, however, that this effect was independent of a reduction of the activity of the adenylate cyclase/cAMP system and suggests in addition that phosphorylated channels may be preferentially inhibited by DYN.

Induction, blockade and restoration of a persistent hypersensitive state

A new model of chronic hypersensitivity was developed in the rat by daily intraplantar administration of either prostaglandin E 2 dopamine or isoprenaline, for a period of 2 weeks. Like other hyperalgesic mediators, dibutyryl-cAMP, when applied to the paws, caused an acute effect but did not produce persistent hypersensitivity. The persistent hypersensitive state was not affected by a typical non-steroidal anti-inflammatory drug (indomethacin), was temporarily inhibited by a centrally acting analgesic (morphine), was partially inhibited by a protein synthesis inhibitor (cycloheximide) and abolished by a single dose of peripherally acting analgesics such as dipyrone or N-methyl morphine. Once the residual hypersensitivity had been abolished with dipyrone or N-methyl morphine, a small dose of prostaglandin E 2, dopamine or Interleukin-1β, which in normal animals causes a mild and short lived effect, restored the persistent hypersensitive state. This ability to restore the persistent effect was not observed with intraplantar administration of dibutyryl-cAMP. Our results suggest the existence of a peripheral trace of inflammatory pain, a phenomenon which may be associated with stimulation of neuronal adenylate cyclase and protein synthesis. This concept may explain part of the puzzle of chronic inflammatory pain and lead to the development of new analgesics.

The gastrointestinal prokinetic benzamide derivatives are agonists at the non-classical 5-HT receptor (5-HT4) positively coupled to adenylate cyclase in neurons

We have previusly shown that a non-classical 5-hydroxytryptamine (5-HT4) receptor mediates the stimulation of adenylate cyclase activity in mouse embryo colliculi neurons in primary culture. The pharmacological characteristics of this receptor exclude the possibility that it belongs to the known 5-HT1, 5-HT2 or 5-HT3 receptor types. Here we report that this 5-HT receptor can be stimulated by 4-amino-5-chloro-2-methoxy substituted benzamide derivatives. All these compounds have been reported to be potent stimulants of gastrointestinal motility and some of them are 5-HT3 receptor antagonists. The rank order of potency of these substituted benzamide derivatives in stimulating cAMP formation was: cisapride greater than BRL 24924 greater than 5-HT greater than zacopride greater than BRL 20627 greater than metoclopramide. The non-additivity of benzamide and 5-HT activities suggests that 5-HT and the substituted benzamide derivatives act on the same receptor. Only ICS 205930, a recognized 5-HT3 receptor antagonist, competitively antagonized the stimulatory effect of cisapride, zacopride and BRL 24924. However, its pKi (6-6.3) for this new receptor was very different from its pKi for 5-HT3 receptors (pKi = 8-10). Other selective 5-HT3 receptor antagonists with an indole group (BRL 43694 and GR 38032F), with a benzoate group (cocaïne, MDL 72222) or with a piperazine group (quipazine) were ineffective in reversing the stimulatory effect of benzamide derivatives. Exposure of neuronal cells to potent agonists at this receptor such as BRL 24924 rapidly reduces its capacity to stimulate cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of Gs‐Stimulated Adenylate Cyclase in Brain Membranes by Low pH Pretreatment: Correlation with Altered Guanine Nucleotide Exchange

Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog [32P]P3 (4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate [Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of [32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of [32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.

Exchange of guanine nucleotides between tubulin and GTP-binding proteins that regulate adenylate cyclase: cytoskeletal modification of neuronal signal transduction.

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.

Calmodulin plays a dominant role in determining neurotransmitter regulation of neuronal adenylate cyclase.

Ca2+, through the mediation of calmodulin, stimulates the activity of brain adenylate cyclase. The growing awareness that fluctuating Ca2+ concentrations play a major role in intracellular signalling prompted the present study, which aimed to investigate the implications for neurotransmitter (receptor) regulation of enzymatic activity of this calmodulin regulation. The role of Ca2+/calmodulin in regulating neurotransmitter-mediated inhibition and stimulation was assessed in a number of rat brain areas. Ca2+/calmodulin stimulated adenylate cyclase activity in EGTA-washed plasma preparations from each region studied--from 1.3-fold (in striatum) to 3.4-fold (in cerebral cortex). The fold-stimulation produced by Ca2+/calmodulin was decreased in the presence of GTP, forskolin, or Mn2+. In EGTA-washed membranes, receptor-mediated inhibition of adenylate cyclase was strictly dependent upon Ca2+/calmodulin stimulation in all regions, except striatum. A requirement for Mg2+ in combination with Ca2+/calmodulin to observe neurotransmitter-mediated inhibition was also observed. In contrast, receptor-mediated stimulation of activity was much greater in the absence of Ca2+/calmodulin. The findings demonstrate that ambient Ca2+ concentrations, in concert with endogenous calmodulin, may play a central role in dictating whether inhibition or stimulation of adenylate cyclase by neurotransmitters may proceed.

Interactions between neurotransmitters that regulate cAMP and intracellular Ca2+ levels in the CNS.

Two major second messenger signalling systems play prominent roles in the regulation of neuronal activity — that which modulates intracellular cAMP levels and that which results in an elevation in intracellular Ca2+ concentrations. Intracellular Ca2+ is now known to be mobilized as a consequence of receptor modulation of inositol phosphate metabolism.1 It is widely recognised that both of these pathways can lead to changes in the level of intracellular protein phosphorylation through the mediation of distinct protein kinases -sometimes converging on a single target protein, as is the situation in the case of tyrosine hydroxylase.2 However, less widely recognized is the fact that these signalling systems also interact more immediately at the level of the generation of their rimary signals. Recent studies — for instance those by Jakobs et al3 — indicate that, in some cells, the Ca2+-activated, protein kinase C, eliminates hormone inhibition of adenylate cyclase which is mediated by inhibitory GTP regulatory elements. It has also been recognised for some time that Ca2+/calmodulin regulates a specific cyclic AMP phosphodiesterase in neuronal tissue; this interaction provides a mechanism whereby Ca2+ signals can decrease cAMP levels.4,5 Thus, the potential for interaction between systems that utilize Ca2+ and cAMP has already been established. In order to explore other potential mechanisms by which these two systems interact at the level of signal generation, recent studies from these laboratories have focussed on two major issues: 1) additional functions that may be served by putative “cyclase-inhibitory” receptors in the CNS. The physiological significance of adenylate cyclase inhibition in neuronal tissue has often been questioned, since the magnitude of the effect is so modest -rarely exceeding 25%. In the present studies additional functions supported by “cyclase inhibitory” receptors are investigated; and, 2) the impact of Ca2+-signals on the regulation of adenylate cyclase by cyclase-linked receptors. Over the past few years, a focus in one of these laboratories ha been the fact — first reported by Brostrom et al.6 — that Ca2+/calmodulin can regulate neuronal adenylate cyclase — particularly in terms of its implications for neurotransmitter regulation of adenylate cyclase. Initial studies on the hippocampal system had indicated that adenosine-Al receptor-mediated inhibition of adenylate cyclase could occur only upon Ca2+/calmodulin stimulation of activity.7 Current studies have attempted to determine how widespread this phenomenon is within the nervous system, whether it applies to all inhibitory neurotransmitters8,9 and the implications of this regulation for stimulatory neurotransmission.KeywordsAdenylate CyclasePertussis ToxinInositol PhosphateAdenylate Cyclase ActivityAdenosine AnalogueThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Regulation of neuronal adenylate cyclase.

It appears that several components function in a spirit of integrated cooperation toward the intracellular regulation of neurotransmitter responsiveness. We have demonstrated that cytoskeletal proteins might interact with GNs and that GNs and GNi might interact with one another. At this juncture, it appears that both of these phenomena might occur only in cells of neural origin. Calmodulin and antidepressants may also affect adenylate cyclase in nervous tissue alone. The effects of AAGTP are different in nervous tissue from other tissues, and experiments with that nucleotide have led to the discovery of a new, 32 kDa GTP-binding protein which appears only in neural crest cells. Appreciation of the intricacies of signal transduction through the adenylate cyclase system are developing along with our understanding of that system. When combined with the complexity of neurotransmitter responsiveness, comprehension of the combined systems remains in its infancy, destined to grow as well as to surprise and delight all who are interested.

Exchange of guanine nucleotide between GTP-binding proteins that regulate neuronal adenylate cyclase.

GTP-binding proteins have been demonstrated to stimulate and inhibit rat brain adenylate cyclase without the prior addition of hormone. Exposure of rat cerebral cortex membranes to hydrolysis-resistant GTP analogs results in inhibition (or stimulation) of adenylate cyclase, which persists subsequent to buffer washing. The hydrolysis-resistant GTP photoaffinity probe P3-(4-azidoanilido)-P1-5' GTP (AAGTP) can promote a similar persistent inhibition of adenylate cyclase, and, after removal of unbound AAGTP and subsequent UV photolysis, AAGTP is covalently linked to the 40-kDa inhibitory GTP binding protein, GNi (inhibitory guanine nucleotide binding regulatory subunit of adenylate cyclase). Under conditions where the persistent inhibition of adenylate cyclase is overcome by subsequent incubation with 5'-guanylyl imidodiphosphate or NaF, AAGTP bound to the 40-kDa GNi protein is diminished while that bound to the 42-kDa stimulatory GTP-binding protein (GNs) is increased. Additionally, we have identified a 32-kDa protein that binds AAGTP with an affinity similar to that of GNs. This protein does not appear to be a byproduct of proteolysis as demonstrated by Staphylococcus aureus V8 protease digestion experiments, and it is not a substrate for ADP-ribosylation by bacterial toxins. The sum of the AAGTP bound by the GNi and GNs proteins is constant, and the transfer of nonphotoactivated AAGTP to GNs from GNi is stable to buffer washing. Furthermore, this alteration in the AAGTP-labeling pattern corresponds to the shift in adenylate cyclase from inhibition to stimulation. These data raise the possibility that hydrolysis-resistant GTP analogs might be exchanged directly between the GNi and GNs and that there exists some interaction between those proteins in the regulation of adenylate cyclase activity.

Regulation of neuronal adenylate cyclase by microtubule proteins.

Regulation of neuronal adenylate cyclase by microtubule proteins.

Neuronal membrane enzymes in rat lines selected for differential motor impairment by ethanol

Neuronal membrane enzyme activities were determined in naive and ethanol-treated (30 min after 2 g/kg) male and female rats of lines developed for more (ANT) and less (AT) ethanol-induced motor impairment. Ethanol did not affect acetylcholinesterase, (Na+K)-ATPase or 5′-nucleotidase activities, but adenylate cyclase activities were lowered in both cerebellum and cerebrum. Cerebral acetyl-cholinesterase activities were higher in ANT than AT rats. No consistent line difference was observed regarding (Na+K)-ATPase activities. Slightly higher cerebellar 5′-nucleotidase activities were found in the ANT line. Cerebellar adenylate cyclase levels were substantially higher in the AT line. No line differences were displayed in the activation of adenylate cyclase activity by dopamine or norepinephrine. Is is concluded that ethanol in vivo may inhibit neuronal adenylate cyclase activity and that cerebellar phosphorylation may be a regulator of motor impairment. Cholinergic mechanisms may also be connected to the ethanol-induced motor impairment.

Cellular site of opiate dependence.

Experiments on whole animals, guinea pig isolated ileum, individual neurones in situ and cultured neuroblastoma--glioma hybrid cells indicate that opiate dependence and associated tolerance develop within neurones bearing specific opiate receptors (opiate-sensitive neurones). The essential change appears to be an hypertrophy of the cyclic AMP system, in response to inhibition by opiate of a neuronal adenylate cyclase.

Prostaglandins and Opioids

In guinea-pig small intestine, rat brain in vitro and neuroblastomaXglioma hybrid cells, opioids specifically inhibit the action of E prostaglandins. In the whole rat, E prostaglandins, administered centrally, antagonize the antinociceptive action of morphine. E prostaglandins also antangonize the induction of opioid tolerant/dependence. In turn, tolerance/dependent preparations respond with added intensity to E prostaglandins. The antagonism between opioids and E prostaglandins does not occur at the opioid receptor; but, certainly in some preparations and probably in others, this antagonism occurs at the coupling or catalytic unit of a neuronal adenylate cyclase that opioids inhibit and E prostaglandins stimulate. The proposition that antagonism of E prostaglandin at appropriate neurons in the brain is part of the natural mechanism of opioid analgesia remains possible, but unproven, and is worth continued investigation.