Biochemical analysis of mutants of a macrophage cell line resistant to the growth-inhibitory activity of interferon.

While a multiplicity of cellular and biochemical effects are mediated by interferons on cultured cells, the mechanisms involved in the direct growth-inhibitory activity of interferons remain problematic. We have previously found that variants in cAMP metabolism in a macrophage cell line, J774.2, were at least 50-fold less sensitive to the growth inhibitory activity of interferons (IFN) than the parental clone. To test the hypothesis that cAMP mediates the growth inhibition produced by IFN in these cells, interferon-resistant variants were selected and characterized with respect to cAMP synthesis and function. Approximately one-third of the IFN-resistant clones were found to be resistant to growth inhibition produced by cholera toxin, but not 8Br-cAMP. IFN was fully able to protect all of the interferon-resistant/choleratoxin-resistant (IFNr/CTr) clones against infection by vesicular stomatitis virus and markedly stimulated 2', 5'-oligodenylate synthetase activity. These IFNr/CTr variants were shown to have a defect in adenylate cyclase. The remaining IFN-resistant clones were fully susceptible to the growth-inhibitory effects of cholera toxin because their basal and stimulated adenylate cyclase activity is similar to that of the parental clone. IFN failed to protect these IFNr/choleratoxin sensitive clones against infection by vesicular stomatitis virus and failed to stimulate 2', 5-oligodenylate synthetase, suggesting that they have defective or deficient IFN receptors. In addition, IFN failed to increase intracellular cAMP levels in both IFNr/CTr and IFNr/choleratoxin sensitive clones. These results provide firm genetic and biochemical evidence that the growth inhibitory effects of IFN on this cell line are mediated by cAMP.

Adenyl cyclase of cultured mammalian cells: activation by catecholamines.

Chang's liver cells and 3T6 mouse embryo fibroblasts contain high amounts of catecholamine-sensitive adenyl cyclase, whereas HeLa cells contain relatively low amounts of activity. Both epinephrine and fluoride ion stimulate activity of each cell line. In contrast to normal liver, Chang's liver cells show greater response to epinephrine and no detectable stimulation by glucagon.

Adenosine 3',5'-monophosphate-dependent protein kinase of cultured mammalian cells.

Protein kinase was partially purified from Chang's liver cells, 3T6 mouse embryo fibroblasts, and HeLa cells. The rate of histone phosphorylation catalyzed by the kinase from each of these cell lines was stimulated two- to three-fold by 1 x 10(-6) molar adenosine 3',5'-monophosphate. The same concentration of guanosine 3',5'-monophosphate failed to stimulate these kinases.

Opioid inhibition of dopamine release from nervous tissue of Mytilus edulis and Octopus bimaculatus.

Morphine and D-Ala2-Met-enkephalin as well as other opioids suppress potassium-stimulated release of 3H-labeled dopamine from neurons tissue of two marine invertebrates, Mytilus edulis and Octopus bimaculatus. Naloxone reverses the inhibitory effects in both species. Potassium-stimulated release of 3H-labeled serotonin is not altered by opioids. It is postulated that opiate receptors and their endogenous effectors play a prominent role in regulation of transmitter release in invertebrates.

Effect of tunicamycin on the turnover of epidermal growth factor receptors in cultured calf aorta smooth muscle cells. Comparison with IMR-90 human lung fibroblasts.

Treatment of cultured calf aorta smooth muscle cells with tunicamycin, a potent inhibitor of dolichol-mediated glycosylation, resulted in progressive loss of receptors for epidermal growth factor with 50% of receptors lost after 6 h. Receptor half-life was also 6 h with cycloheximide treatment but was 12 h with either actinomycin D or camptothesin treatment. The epidermal growth factor-induced processing (internalization and/or degradation) of residual receptors remaining after tunicamycin treatment appeared to be unaltered. 50% decrease in 125I-labeled epidermal growth factor binding was observed also with IMR-90 fibroblasts upon 6 h treatment with tunicamycin, although these cells were less sensitive to inhibition by tunicamycin of glycosylation and protein synthesis.

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.

Opioid and opiate immunoregulatory processes.

The discovery of the ability of the nervous system to communicate through "public" circuits with other systems of the body is attributed to Ernst and Berta Scharrer, who described the neurosecretory process in 1928. Indeed, the immune system has been identified as another important neuroendocrine target tissue. Opioid peptides are involved in this communication (i.e., neuroimmune) and with that of autoimmunoregulation (communication between immunocytes). The significance of opioid neuropeptide involvement with the immune system is ascertained from the presence of novel delta, mu, and kappa receptors on inflammatory cells that result in modulation of cellular activity after activation, as well as the presence of specific enzymatic degradation and regulation processes. In contrast to the relatively uniform antinociceptive action of opiate and opioid signal molecules in neural tissues, the presence of naturally occurring morphine in plasma and a novel mu3, opiate-specific receptor on inflammatory cells adds to the growing knowledge that opioid and opiate signal molecules may have antagonistic actions in select tissues. In examining various disorders (e.g., human immunodeficiency virus, substance abuse, parasitism, and the diffuse inflammatory response associated with surgery) evidence has also been found for the involvement of opiate/opioid signaling in prominent mechanisms. In addition, the presence of similar mechanisms in man and organisms 500 million years divergent in evolution bespeaks the importance of this family of signal molecules. The present review provides an overview of recent advances in the field of opiate and opioid immunoregulatory processes and speculates as to their significance in diverse biological systems.

Estrogen uncouples beta-adrenergic receptor from the stimulatory guanine nucleotide-binding protein in female rat hypothalamus.

The responsiveness of adenylyl cyclase to beta-adrenergic receptor stimulation was investigated in membranes prepared from hypothalamus-preoptic area and cortex of ovariectomized female rats injected with oil vehicle or estradiol benzoate 24 or 48 h before death. Membranes from the hypothalamus-preoptic area of ovariectomized animals displayed a concentration-dependent stimulation of adenylyl cyclase when incubated with the beta-adrenergic receptor agonist, isoproterenol (10(-7)-10(-5) M). This response was suppressed in membranes from estrogen-treated animals. The effect of estrogen was observed 48 h, but not 24 h, after hormone administration. In addition, estrogen had no measurable effect on hypothalamic adenylyl cyclase activation by either GTP (10(-8)-10(-5) M) or forskolin (10(-8)-10(-6) M), on beta-adrenergic receptor density, or on antagonist binding affinity measured with the beta-adrenergic antagonist [125I]iodocyanopindolol. Analysis of isoproterenol displacement of iodocyanopindolol binding revealed that estrogen reduced agonist binding affinity in hypothalamus-preoptic area membranes. In membranes from ovariectomized controls, high affinity agonist binding to the beta-adrenergic receptor was apparent and was abolished by guanine nucleotides. However, membranes from estradiol-treated rats demonstrated only low affinity agonist binding that was unaffected by guanine nucleotides. Estradiol did not detectably alter concentrations of either cholera or pertussis toxin substrates in hypothalamus-preoptic area membranes. These data indicate that estrogen promotes a stable time-dependent desensitization of beta-adrenergic receptor activation of adenylyl cyclase in hypothalamus and preoptic area by uncoupling the receptor from the guanine nucleotide-binding protein, G8.

Estradiol plus progesterone promote glutamate-induced release of gamma-aminobutyric acid from preoptic area synaptosomes.

Treatment of ovariectomized rats with both estradiol and progesterone in vivo resulted in a marked enhancement of glutamate-induced release of newly synthesized [3H]gamma-aminobutyric acid (GABA) from synaptosomes of the preoptic area in vitro. With this treatment, as little as 0.01 nM glutamate, in vitro, enhanced release of GABA. In contrast, glutamate, in vitro, did not stimulate release of GABA from synaptosomes, obtained from rats treated with either estradiol or progesterone alone and only large concentrations of glutamate (1.0 and 10 mM) caused a modest release of GABA from synaptosomes from ovariectomized, vehicle-treated rats. Also, treatment with estradiol plus progesterone did not alter glutamate-induced release or exchange of [3H]glutamate. Glutamate-induced release of GABA was calcium-independent and attenuated by the putative chloride channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-DL-disulfonic acid. Thus, glutamate-induced, steroid-enhanced release of GABA may occur through a chloride-dependent carrier rather than by exocytosis. In addition to enhancement by glutamate, release of GABA was also enhanced by D-aspartate, an agent that is transported by the neuronal glutamate carrier. It is postulated that enhancement of glutamate-induced release of GABA, by estradiol plus progesterone in the preoptic area, represents one process by which these steroids modulate reproductive function in female rats.

Characterization of glutamate efflux from preoptic area synaptosomes.

Treatment of ovariectomized rats in vivo with ovarian steroids has been found to influence the efflux of glutamate and gamma-aminobutyric acid from preoptic area synaptosomes incubated in vitro. Since these studies indicated a possible role of the glutamate carrier in steroid-modulated release of amino acids, the present studies examined the characteristics of efflux of glutamate and of the carrier system for glutamate in synaptosomes of the preoptic area derived from ovariectomized hormone-treated rats. The efflux of [3H]glutamate from preoptic area synaptosomes, was induced by glutamate and by the glutamate carrier agonist, D-aspartate; the putative glutamate carrier antagonist dihydrokainate failed to block this efflux. Dihydrokainate inhibited the uptake of glutamate but it was less effective than D-aspartate. The excitatory amino acid receptor agonists, N-methyl-D-aspartate and kainate were without effect while quisqualate modestly stimulated the efflux of [3H]glutamate. Efflux of [3H]glutamate, induced by glutamate itself or by D-aspartate was not blocked by the excitatory amino acid receptor antagonists, D-2-amino-5-phosphonovaleric acid, 6,7-dinitroquinoxaline-2,3-dione or kynurenate. Glutamate-induced efflux of [3H]glutamate did not require external Ca2+. Glutamate altered neither the basal nor the potassium-induced increases in the intrasynaptosomal concentration of Ca2+ as measured by the fura-2 method. Glutamate-induced efflux of [3H]glutamate was blocked by the putative chloride channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. It is concluded that the glutamate-induced efflux of [3H]glutamate in synaptosomes of the preoptic area is a carrier-mediated process that does not require activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Striatal met-enkephalin concentration increases following nigrostriatal denervation.

Following specific lesion of the nigrostriatal dopaminergic pathways in rat brain, striatal met-enkephalin on the lesioned side increased to 245% of that on the non-lesioned side. This increase was evident only after a lag period of 7 days and the increase was maintained for at least 2 months after lesion. By contrast, there was no change in striatal somatostatin or vasoactive intestinal polypeptide concentration, indicating that the effect was not a generalised one. Levels of all three of these neuropeptides were unchanged in frontal cortex. These findings support the concept of a dopaminergic-enkephalinergic functional interrelationship in the striatum. In addition, the findings provide evidence that, following destruction of nigrostriatal dopaminergic neurons, not only is there a gradually developing postsynaptic dopamine receptor supersensitivity but also a compensatory alteration in the enkephalinergic system.

Binding sites for [3H]SCH 23390 in retina: properties and possible relationship to dopamine D1-receptors mediating stimulation of adenylate cyclase.

Bovine, rat and chick embryo retinal membranes contain high affinity, saturable and stereospecific binding sites for the selective dopamine D1-receptor antagonist, [3H]SCH 23390 (R-(+)-8-chloro-2,3,4,5-tetrahydro-3- methyl-5-phenyl-lH-3-benzazepin-7-ol). Saturation studies and Scatchard analyses showed a single class of [3H]SCH 23390 binding sites with Kd (apparent dissociation constant) values of 0.5-1.4 nM for the different species studied. A high ratio of specific to non-specific binding was found over a wide range of radioligand concentrations. The Bmax (binding site number) for [3H]SCH 23390 in calf retina was 307 +/- 38 fmol/mg protein, significantly greater than Bmax values previously obtained for binding of [3H]spiroperidol and [3H]ADTN (2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene) to dopamine D2-receptors in calf retina. Relative affinities (Ki values) of dopamine antagonists for calf retinal [3H]SCH 23390 binding sites were similar to those reported for [3H]SCH 23390 binding in rat striatum and also were in general agreement with potencies for antagonism of retinal dopamine-stimulated adenylate cyclase. However, they differed markedly from the relative affinities for retinal D2-receptor sites. Additional data indicated that SCH 23390 did not bind significantly to retinal D2- or serotonergic receptors and had 30- to 80-fold less affinity for alpha 2-noradrenergic than for the [3H]SCH 23390 sites. Competition studies indicated a high degree of selectivity for dopamine agonist over other agonists for [3H]SCH 23390 binding sites with Ki values in the range expected for interaction with dopamine receptors mediating stimulation of adenylate cyclase. Affinity for dopamine was decreased in the presence of the GTP analogue, Gpp(NH)p. In the presence of sodium ions the affinities of dopamine agonists for [3H]SCH 23390 binding sites were markedly and selectively decreased; the sensitivity to dopamine for stimulation of adenylate cyclase activity was also decreased in the presence of sodium ions. Modulation by sodium ions was previously observed for D2- but not for a D1-receptor interaction. It is proposed that [3H]SCH 23390 binds to a unique class of receptors, most likely D1-receptors coupled to adenylate cyclase in retina. [3H]SCH 23390 provides a potent new tool for study of these receptors. In retina D1-receptors positively coupled to cyclase as well as D2- and other receptors that may be negatively coupled to cyclase, appear to be regulated by sodium ions as well as by guanine nucleotides.

Regulation of tryptophan hydroxylase activity by a cyclic AMP-dependent mechanism in rat striatum.

Evidence is presented indicating that a cAMP-dependent mechanism activates tryptophan hydroxylase (TrpH), the rate-limiting enzyme for serotonin (5-HT) biosynthesis. Forskolin, a selective activator of adenylate cyclase, stimulated 5-HT formation in synaptoneurosome preparations of rat striatum, substantia nigra, hypothalamus, and amygdala. Further studies of striatum revealed that the forskolin-induced activation of serotonin synthesis is readily reversible. Also, it may be self-limited by a mechanism of desensitization, since after an initial exposure to forskolin followed by removal, a re-exposure of synaptoneurosomes to forskolin was no longer stimulatory. In contrast to these results for 5-HT synthesis, forskolin-induced stimulation of dopamine synthesis persisted following removal of forskolin; hence the response was not rapidly reversible or desensitized. In soluble extracts of striatum, 8-thiomethyl-cyclic AMP enhanced TrpH activity, supporting a direct role of cyclic AMP and cyclic AMP-dependent protein kinase in regulating TrpH. In agreement with previous reports, 8-thiomethyl cyclic AMP also stimulated tyrosine hydroxylase activity in soluble striatal extracts. We conclude that cyclic AMP is an important regulator of TrpH, in addition to its known effects on tyrosine hydroxylase.

Interaction of LSD and other hallucinogens with dopamine-sensitive adenylate cyclase in primate brain: regional differences.

The influence of D-lysergic acid diethylamide (LSD) and mescaline on adenylate cyclase activity was studied in homogenates of Cebus and rhesus monkey anterior limbic cortex (ALC), frontal cortex (FC), caudate nucleus and retina. Previous studies have shown these tissues to contain dopamine-stimulated adenylate cyclase (AC). In addition, we are now reporting the presence of a dopamine-sensitive adenylate cyclase in the auditory cortex. AC of ALC and auditory cortex was stimulated by LSD and mescaline, whereas activity of FC, caudate nucleus and retina was not stimulated by the same agents. In contrast to regional specificity for stimulation, LSD was capable of antagonizing dopamine-stimulated activity in all brain regions examined. LSD and mescaline produced similar maximal stimulation (about 70%) of AC of ALC homogenates, but the EC50 for LSD (0.43 micrometer) was about one-tenth that for mescaline (4.5 micrometer). Similar relative potencies were also observed for the auditory cortex enzyme. Although much weaker than LSD, methamphetamine also produced a dose-dependent stimulation of ALC AC. Both agonist and antagonist effects of the hallucinogens appear to involve interaction with dopamine receptors; LSD- or methamphetamine-stimulated activity in ALC was blocked by haloperidol and fluphenazine, which are dopamine antagonists, but not by phentolamine, an alpha-receptor blocker. Antagonism of dopamine by LSD in both ALC and FC was found to be competitive and mescaline was an effective but weaker antagonist than was LSD. In addition, neither histamine--nor Gpp(NH)p--stimulated activity of FC was inhibited by LSD. It is proposed that the occurrence of dopamine agonistic action of hallucinogens in only certain regions of primate brain may provide a basis for at least some of the behavioral effects of LSD, mescaline and methamphetamine in primates.

Pertussis toxin blocks depressant effects of opioid, monoaminergic and muscarinic agonists on dorsal-horn network responses in spinal cord-ganglion cultures.

After chronic exposure of mouse spinal cord-ganglion explants to morphine, the acute depressant effects of opioids on sensory-evoked dorsal-horn network responses are markedly attenuated, and characteristic cord discharges can then occur even in the presence of greater than 100-fold higher opioid concentrations. The present study demonstrates that a remarkably similar degree of tolerance to opioids develops in these cord-ganglion explants after exposure to pertussis toxin (PTX). The usual acute depressant effects of serotonin, norepinephrine and oxotremorine on dorsal-horn discharges are also similarly attenuated in PTX-treated cultures. PTX is known to interfere with the guanine nucleotide protein Gi that is required for opioid, alpha 2-adrenergic and muscarinic receptor-mediated inhibition of adenylate cyclase in various cells. We have previously found that in cord-dorsal root ganglion explants agents which elevate intracellular cAMP also attenuate opioid depressant effects. Furthermore, these explants contain an opioid-inhibited adenylate cyclase system, and chronic exposure to morphine as well as PTX increases adenylate cyclase activity. These findings together with the present results suggest that the neuromodulatory effects of opioid, monoaminergic and muscarinic agonists on primary afferent networks in the spinal cord may be mediated by binding to neuronal receptor subtypes that are negatively coupled via Gi to a common pool of adenylate cyclase.

Modulation of adenylate cyclase activity of mouse spinal cord-ganglion explants by opioids, serotonin and pertussis toxin.

Organotypic cultures of fetal mouse spinal cord-ganglion explants (2-4 weeks in vitro) contain forskolin-stimulated adenylate cyclase (AC) activity that is inhibited by levorphanol and other opioid agonists in a dose-dependent manner. Inhibition by levorphanol no longer occurs if sodium is omitted from the incubation and the levorphanol inhibition is blocked by the opioid antagonist, naloxone. These findings together with the ineffectiveness of dextrorphan indicate that the opioid inhibition of forskolin-stimulated AC is receptor mediated. Both the delta- and kappa-receptor subtypes appear to be involved since the selective delta-opioid agonist, [D-Pen2, D-Pen5]enkephalin, and the selective kappa-opioid agonist, t-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]-benzene acetamide (U-50,488H) are both effective at nanomolar concentrations. In contrast, the selective mu-opioid agonist, Tyr-D-Ala-Gly-N-MePhe-Gly-ol, has no significant effect even at micromolar concentrations. Both cord and ganglion components of the explants contain opioid-sensitive AC. Forskolin-stimulated AC of the explants is also inhibited by serotonin and carbachol. The serotonin effect appears to be mediated by 5-HT1A receptors, based on relative agonist and antagonist selectivity. Chronic exposure of cultures to morphine results in enhanced basal and forskolin-stimulated AC as well as attenuation of opioid-inhibition of AC assayed in the presence of forskolin; treatment of explants with pertussis toxin causes similar changes in the AC system. The inhibitory effect of serotonin is also attenuated by the pertussis toxin treatment. Basal AC activity of the explants (assayed without forskolin present) is stimulated to a small but significant extent by opioids and by serotonin. The opioid stimulatory effect is markedly enhanced following either morphine or pertussis toxin treatment of the explants. The attenuation of opioid- and serotonin-inhibition of AC produced by chronic exposure to pertussis toxin and the attenuation of opioid inhibition produced by exposure to morphine are consonant with the attenuation of opioid and monoaminergic depression of sensory evoked dorsal horn network responses after similar chronic treatments. It is proposed that the inhibitory effects of opioids and serotonin on these neurons are mediated by receptors that are negatively coupled via a pertussis toxin sensitive Gi protein to AC. Furthermore, alterations of AC with chronic morphine treatment may be involved in the development of physiologic tolerance to opioids.

Occurrence of the opiate alkaloid-selective mu3 receptor in mammalian microglia, astrocytes and Kupffer cells.

Evidence is presented for occurrence of opiate alkaloid-selective, opioid-peptide-insensitive receptor binding sites, labeled with [3H]morphine, in primary cultures of cat microglia and cat astrocytes, as well as on highly purified preparations of rat Kupffer cells. These receptors have been designated mu3 on the basis of their close similarity to receptors first found to be present on human peripheral blood monocytes. Exposure of the microglia to morphine and etorphine caused marked quantifiable changes in cellular morphology, including assumption of a more rounded shape and retraction of cytoplasmic processes; in contrast, several opioid peptides were without effect on morphology. The effects of morphine on microglial morphology were blocked by the opiate antagonist naloxone. These effects of drugs on morphology were as predicted for action via the mu3 receptor. Opiate alkaloid binding sites previously detected on the rat C6 glioma cell line were also characterized here as of the mu3 receptor subtype. It is proposed that mu3 receptors have broad distribution in different macrophage cell types of bone marrow lineage, including microglia and Kupffer cells. Furthermore, these receptors are not restricted to cells of bone marrow lineage, since they are also present on astrocytes.

Interaction of morphine, etorphine and enkephalins with dopamine-stimulated adenylate cyclase of monkey amygdala.

Adenylate cyclase activity (AC) of homogenates of monkey amygdaloid nucleus was approximately doubled in the presence of dopamine (10 micrometer). Morphine, etorphine, and several enkephalin analogs (met-enkephalin, D-ala2-met-enkephalin, and D-met2, pro5-enkephalinamide) were capable of inhibiting the stimulation of AC produced by dopamine (90-100% with etorphine or D-ala2-met-enkephalin). Unlike morphine and etorphine, the peptides exhibited bell-shaped dose-response curves for this inhibition with maximal effects at approximately 1 X 10(-7) M, but negligible effects at 1 X 10(-5) M. Under the conditions studied, only etorphine inhibited basal AC. Naloxone antagonized the inhibitory effects of each of the opioids tested, and dextrorphan, an inactive L-(+)-opiate, failed to inhibit the dopamine response. Together these data indicate that the effects were mediated via the classically described stereospecific opiate receptor. The relative order of potency (etorphine greater than enkephalins greater than morphine) was similar to that previously reported for the binding affinities of these drugs in rat brain homogenates. The influence of narcotic agents on dopamine stimulated AC was eliminated by either freezing the amygdaloid tissue or preincubating the homogenate at 4 degrees C; the dopamine responses, however, could still be elicited. The narcotic receptor interaction with the adenylate cyclase thus appears to be distinct from and more labile than that of the dopamine receptor. Gpp(NH)p-stimulated AC was not inhibited by morphine. It is postulated that the inhibition involves interaction of opiate receptors with catalytic units of dopamine-stimulated AC, but not with other cyclase species which may provide the major component of Gpp(NH)p-stimulated activity in amygdala.

Presence and characterization of nociceptin (orphanin FQ) receptor binding in adult rat and human fetal hypothalamus.

High affinity and saturable nociceptin (orphanin FQ) receptors were detected and characterized in adult rat and human fetal hypothalamic membranes, utilizing [125I]Tyr12-nociceptin as ligand. Nociceptin bound with picomolar affinity, dynorphin A with nanomolar affinity, naloxone and dynorphan A(1-8) with micromolar while des-Tyr1-dynorphin (dynorphin A(2-17)), several other opioids, morphine and benzomorphans failed to compete for binding at 1-10 microM. Gpp(NH)p together with sodium ion markedly decreased binding, consistent with involvement of a G protein-linked receptor.

Forskolin stimulates the conversion of tyrosine to dopamine in catecholaminergic neural tissue.

Forskolin stimulates the conversion of tyrosine to dopamine in slices and synaptosomes from rat striatum, in synaptosomes from rat hypothalamus, and in slices from bovine retina. In striatal synaptosomes, there is an approximately 80% stimulation of dopamine formation from tyrosine, but no effect with DOPA as substrate, suggesting that the effect is on tyrosine hydroxylase. Stimulation is saturable with a half-maximal effect at under 1 microM forskolin. Forskolin stimulation is additive with, and hence independent of , activation due to KCl, but not that due to dibutyryl-cyclic AMP. Stimulation of dopamine synthesis by forskolin may provide an approach for elucidating the regulation of the adenylate cyclase system associated with catecholaminergic nerve endings.