Identification of an integration center for cross-talk between protein kinase C and G protein modulation of N-type calcium channels

The modulation of presynaptic calcium channel activity by second messengers provides a fine tuning mechanism for neurotransmitter release. In neurons, the activation of certain G protein-coupled receptors reduces N-type channel activity by approximately 60%. In contrast, activation of protein kinase C (PKC) results in an approximately 50% increase in N-type channel activity, and subsequent G protein inhibition is antagonized. Here, we describe the molecular determinants that control the dual effects of PKC-dependent phosphorylation. The double substitution of two adjacent PKC consensus sites in the calcium channel domain I-II linker (Thr422, Ser425) to alanines abolished both PKC-dependent up-regulation and the PKC-G protein cross-talk. The single substitution of Ser425 to glutamic acid abolished PKC up-regulation but had no effect on G protein modulation. Replacement of Thr422 with glutamic acid eliminated PKC-dependent up-regulation and mimicked the effects of PKC phosphorylation on G protein inhibition. Our data suggest that Thr422 mediates the antagonistic effect of PKC on G protein modulation, while phosphorylation of either Thr422 or Ser425 are sufficient to increase N-type channel activity. Thus, Thr422 serves as a molecular switch by which PKC is able to simultaneously trigger the up-regulation of channel activity and antagonize G protein inhibition.     

Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1

Diabetes mellitus is associated with one or more kinds of stimulus-evoked pain including hyperalgesia and allodynia. The mechanisms underlying painful diabetic neuropathy remain poorly understood. Previous studies demonstrate an important role of vanilloid receptor 1 (VR1) in inflammation and injury-induced pain. Here we investigated the function and expression of VR1 in dorsal root ganglion (DRG) neurons isolated from streptozotocin-induced diabetic rats between 4 and 8 weeks after onset of diabetes. DRG neurons from diabetic rats showed significant increases in capsaicin- and proton-activated inward currents. These evoked currents were completely blocked by the capsaicin antagonist capsazepine. Capsaicin-induced desensitization of VR1 was down-regulated, whereas VR1 re-sensitization was up-regulated in DRG neurons from diabetic rats. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate blunted VR1 desensitization, and this effect was reversible in the presence of the PKC inhibitor bisindolylmaleimide I. Compared with the controls, VR1 protein was decreased in DRG whole-cell homogenates from diabetic rats, but increased levels of VR1 protein were observed on plasma membranes. Of interest, the tetrameric form of VR1 increased significantly in DRGs from diabetic rats. Increased phosphorylation levels of VR1 were also observed in DRG neurons from diabetic rats. Colocalization studies demonstrated that VR1 expression was increased in large myelinated A-fiber DRG neurons, whereas it was decreased in small unmyelinated C-fiber neurons as a result of diabetes. These results suggest that painful diabetic neuropathy is associated with altered cell-specific expression of the VR1 receptor that is coupled to increased function through PKC-mediated phosphorylation, oligomerization, and targeted expression on the cell surface membrane.     

Characterization and possible mechanisms of alpha 2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production in HT29 cells

Preincubation with an alpha 2-adrenergic agonist sensitized subsequent forskolin- and vasoactive intestinal peptide-stimulated cyclic AMP production in HT29 cells, a human colonic adenocarcinoma cell line. Preincubation with somatostatin, another agonist negatively coupled to adenylate cyclase, sensitized forskolin-stimulated cyclic AMP production to a lesser extent. alpha 2-Adrenergic agonist preincubation also resulted in desensitization as indicated by a shift to the right in the dose-response curve of a subsequent challenge by an alpha 2-adrenergic agonist. In an effort to elucidate the mechanism for sensitization, we examined protein kinase C and the Na+/H+ antiporter. Whereas these components had marked effects on forskolin stimulation, there was no effect on sensitization. Changes in the concentration of extra-cellular Ca2+ or Mg2+ had no effect on either forskolin stimulation or sensitization. Pertussis toxin pretreatment caused a time-dependent decrease in sensitization, an attenuation of inhibition of cyclic AMP production, and a decrease in subsequent [32P]ADP-ribosylation by pertussis toxin. The time course for these three events was similar, implicating the inhibitory guanine nucleotide regulatory protein in the mechanism for alpha 2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production. In addition, pertussis toxin dramatically decreased forskolin-stimulated cyclic AMP production, although with a different time course. These results suggest that the mechanism of sensitization is via an as yet undefined sequence of biochemical events that includes the inhibitory guanine nucleotide regulatory protein, but does not include inhibition of adenylate cyclase nor activation of the Na+/H+ antiporter.     

Inhibition of cerebral protein kinase activity and cyclic AMP-dependent ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia

Studies were carried out to elucidate the mechanisms underlying the diminished phosphorylation of cerebral ribosomal protein in experimental hyperphenylalaninaemia [Roberts & Morelos (1980) Biochem. J.190, 405-419]. Administration of N(6),O(2)'-dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine, which increased phosphorylation of the S6 protein of cerebral 40S ribosomal subunits in control infant rats, did not counteract the decreased phosphorylation of this ribosomal protein resulting from intraperitoneal administration of a loading dose of l-phenylalanine. N(2),O(2)'-Dibutyryl cyclic GMP had no effect on cerebral ribosomal-protein phosphorylation in either control or hyperphenylalaninaemic animals. The phenylalanine-induced decrease in ribosomal-protein phosphorylation was associated with decreased protein kinase activity in cerebral cytosolic and microsomal preparations. However, the maximal protein kinase response to cyclic AMP added in vitro was unaltered by prior administration of phenylalanine in vivo. The heat-stable protein inhibitor of cyclic AMP-dependent protein kinases decreased the activity of these enzymes by about 90% and eliminated the phenylalanine-induced difference in protein kinase activity in the absence of added cyclic AMP. Intracisternal administration of doses of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine which increased the cyclic AMP-dependent protein kinase activity ratio in control infant rats was without effect on this index in phenylalanine-treated animals. Dibutyryl cyclic GMP had no effect on the protein kinase activity ratio in either group of animals. These results suggest that inhibition of cerebral cyclic AMP-dependent protein kinases by abnormally high concentrations of phenylalanine may contribute to the decrease in cerebral ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.     

Prostaglandin deficiency promotes sensitization of adenylyl cyclase

Inhibition of prostaglandin synthesis by the drug indomethacin suppresses the synthesis of the cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), and leads to a metabolic state comparable to type II diabetes. It was of interest whether prostaglandin-deficiency likewise causes sensitization of adenylyl cyclase, as this has been reported for the diabetic state. In liver plasma membranes of indomethacin-treated male rats, basal and forskolin-stimulated cyclic AMP synthesis remained unchanged when compared to untreated control rats. In control rats, stimulation of cyclic AMP synthesis by fluoride (2.2-fold) or glucagon (3.5-fold) was much lower than stimulation by forskolin (6.6-fold). In contrast, in indomethacin-treated rats, stimulation of cAMP synthesis by fluoride (4.6-fold) or glucagon (5.2-fold) nearly matched the stimulation by forskolin (6.4-fold). The level of alpha1-adrenergic receptors was slightly reduced, from 450 to 320 fmol/mg protein, by the indomethacin treatment. Independent of the treatment by indomethacin, stimulation of cyclic AMP synthesis by adrenaline failed, in agreement with the low density of adrenergic beta-receptors. In conclusion, PGE deficiency sensitizes adenylyl cyclase in rat liver for G protein-coupled receptors (glucagon) and also for fluoride.     

Increased sensitivity of fat cell adenylate cyclase to stimulatory agonists during fasting is not related to impaired inhibitory coupling system

In rat adipocytes, inhibition of the forskolin-stimulated cyclic AMP response by nicotinic acid and N6-phenylisopropyladenosine was unaltered by a 72 h fasting. Under assay conditions favouring inhibition, basal and forskolin-stimulated adenylate cyclase responses to inhibition by GTP and nicotinic acid were also unimpaired by fasting. Under the same conditions, however, low GTP concentrations elicited a clear activatory effect in membranes from fasted but not from fed rats. Fasting failed to alter the incorporation of [32P]ADP ribose into the alpha i-subunit of Ni and the attenuation of nicotinic acid inhibitory action that are both induced by pertussis toxin. These results, demonstrating unimpaired inhibitory control of adenylate cyclase in starved rat adipocytes, suggest that the permissive effect of fasting on the action of stimulatory receptor agonists in fat cells reflects a specific increase in the activity of the adenylate cyclase stimulatory coupling system.     

Role of Extracellular Adenosine in Ethanol-Induced Desensitization of Cyclic AMP Production

Abstract: The decrease in receptor-stimulated cyclic AMP production after chronic ethanol exposure was suggested previously to be secondary to an ethanol-induced increase in extracellular adenosine. The present study was undertaken to ascertain whether a similar mechanism was responsible for the ethanol-induced desensitization of cyclic AMP production in PC12 pheochromocytoma cells. The acute addition of ethanol in vitro significantly increased both basal cyclic AMP content and extracellular levels of adenosine. A 4-day exposure to ethanol decreased basal as well as 2-chloroadenosine- and forskolin-stimulated cyclic AMP contents. No change in cyclic AMP content was observed after a 2-day exposure of PC12 cells to ethanol. Inclusion of adenosine deaminase during the chronic ethanol treatment significantly decreased extracellular levels of adenosine, yet the percentage decrease in 2-chloroadenosine- and forskolin-stimulated cyclic AMP levels after chronic ethanol exposure was not changed by the inclusion of the adenosine deaminase. Similar results were obtained when the chronic treatment was carried out with serum-free defined media. The ethanol-induced desensitization could not be mimicked by chronic exposure of PC12 cells to adenosine analogues. A 24-h exposure of PC12 cells to 2-chloroadenosine resulted in a decrease in the subsequent ability of this adenosine analogue to stimulate cyclic AMP content, but basal and forskolin-stimulated cyclic AMP levels were increased. Similar results were obtained after a 4-day exposure of PC12 cells to 2-chloroadenosine or 5'- N -ethylcarboxamido-adenosine. The present results indicate that the ethanol-induced decrease in receptor-stimulated cyclic AMP content in PC12 cells is not due to an increase in extracellular adenosine.     

Fat cell adenylate cyclase system. Enhanced inhibition by adenosine and GTP in the hypothyroid rat

Hypothyroidism is associated with an enhanced sensitivity of rat fat cells to the inhibitory action of adenosine and adenosine agonists. The sensitivity of the forskolin-stimulated cyclic AMP response of rat fat cells to the adenosine agonist N6-phenylisopropyladenosine is amplified 3-fold by hypothyroidism. Forskolin-stimulated adenylate cyclase activity is more sensitive to inhibition by this adenosine agonist in membranes of fat cells isolated from hypothyroid as compared to euthyroid rats. Hypothyroidism does not significantly alter the number of affinity of binding sites for N6-cyclohexyl[3H]adenosine or N6-phenylisopropyladenosine in membranes of rat fat cells. GTP-induced inhibition of forskolin-stimulated adenylate cyclase was markedly enhanced in the hypothyroid state, suggesting an alteration in the inhibitory regulatory component (Ni)-mediated control of adenylate cyclase. Incubating membranes with [alpha-32P]NAD+ and preactivated pertussis toxin results in the radiolabeling of two peptides with Mr = 40,000 and 41,000 as visualized in autoradiograms of polyacrylamide gels run in sodium dodecyl sulfate. The amount of label incorporated by pertussis toxin into these two peptides (putative subunits of Ni) per mg of protein of membrane is increased 2-3-fold in the hypothyroid state. The amount of the stimulatory regulatory component, Ns, in fat cell membranes is not altered by hypothyroidism (Malbon, C. C., Graziano, M. P., and Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260). The amplified response of hypothyroid rat fat cells to the inhibitory action of adenosine appears to reflect a specific increase in the activity and abundance of Ni.     

In Vivo Evidence that Lithium Inactivates Gi Modulation of Adenylate Cyclase in Brain

In vivo microdialysis of cyclic AMP from prefrontal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 +/- 0.05 mM; n = 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin-catalyzed ADP ribosylation of alpha i (and alpha o) was increased by 20% in prefrontal cortex from lithium-treated rats, but the alpha i and alpha s contents (as determined by immunoblot) as well as the cholera toxin-catalyzed ADP ribosylation of alpha s were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin-stimulated adenylate cyclase activity.     

Inhibitory effect of a lethal toxic fragment of staphylococcal alpha-toxin on cyclic AMP-dependent protein kinase activity.

The effect of a lethal toxic fragment of staphylococcal alpha-toxin on the activity of adenosine 3',5'-monophosphate(cyclic AMP)-dependent protein kinase was examined. 1. The lethal toxic fragment produced a dose-dependent decrease in both the binding of cyclic AMP to the regulatory subunit and phosphorylation activity of cyclic AMP-dependent protein kinase obtained from rabbit skeletal muscles up to a plateau at a 50% inhibitory effect. The decrease in the activity of protein kinase observed with low doses of the lethal toxic fragment (0.1 microM) resulted from a competitive inhibition, probably by its interaction with the cyclic AMP-binding site in the regulatory subunit molecule. 2. The effects of a lethal toxic fragment and epinephrine on the cyclic AMP level and protein kinase activity were investigated in the perfused rabbit heart slices. The lethal toxic fragment attenuated the stimulation of cyclic AMP-dependent protein kinase activity ratio by epinephrine. 3. It is suggested that the specific action of a lethal toxic fragment on the cellular membrane enzymes may be attributable to the inhibition of the cyclic AMP-dependent protein kinase activity.     

Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation

Muscarinic receptors and platelet-derived growth factor (PDGF) receptors synergistically induce proliferation of airway smooth muscle (ASM), but the pathways that regulate these effects are not yet completely identified. We hypothesized that glycogen synthase kinase-3 (GSK-3), a kinase that represses several promitogenic signaling pathways in its unphosphorylated form, is cooperatively inhibited by PDGF and muscarinic receptors in immortalized human ASM cell lines. PDGF or methacholine alone induced rapid GSK-3 phosphorylation. This phosphorylation was sustained only for PDGF; however, methacholine potentiated PDGF-induced sustained GSK-3 phosphorylation. Synergistic effects of methacholine also were observed on PDGF-induced retinoblastoma protein (Rb) phosphorylation and cell proliferation. Suppression of GSK-3 inhibitory function using SB 216763 also augmented PDGF-induced Rb phosphorylation and cell cycle progression; this synergy was similar in magnitude to that seen for methacholine with PDGF. GSK-3 phosphorylation induced by methacholine required PKC, since it was abolished by GF 109203X and G? 6976; however, inhibition of PKC had no effect on cell responses to PDGF. PKC inhibition also specifically abolished the synergistic effect of methacholine on PDGF-induced GSK-3 phosphorylation and cell proliferation. Collectively, these results show that GSK-3 plays a key repressive role in ASM cell proliferation. Moreover, muscarinic receptors mediate PKC-dependent GSK-3 inhibition, and this appears to be a primary mechanism underpinning augmentation of PDGF-induced cell growth.     

Bilirubin inhibition of protein kinase: its prevention by cyclic AMP.

The effect of bilirubin on protein kinase activity has been studied. Bilirubin inhibits the binding of cyclic AMP to protein kinase. It also inhibits the phosphorylation of histone. The inhibitory action of bilirubin is competitive, and increased amounts of cyclic AMP relieved the inhibition.     

Cerebral Cortex Gsα Protein Levels and Forskolin-Stimulated Cyclic AMP Formation Are Increased in Bipolar Affective Disorder

Abstract: Experimental animal and peripheral blood cell studies point to guanine nucleotide regulatory (G) protein disturbances in bipolar affective disorder. We have previously reported elevated prefrontal cortex G_sα protein in bipolar affective disorder and have now extended these preliminary observations in a larger number of subjects, assessing the brain regional specificity of these changes in greater detail, determining the functional biochemical correlates of such changes, and evaluating their diagnostic specificity. Membrane G protein (G_sα, G_iα, G_oα, and Gβ) immunoreactivities were estimated by western blotting in postmortem brain regions obtained from 10 patients with a DSMIII-R diagnosis of bipolar affective disorder and 10 nonpsychiatric controls matched on the basis of age, postmortem delay, and brain pH. To examine whether there were functional correlates to the observed elevated G_sα levels, basal and GTPγS-and forskolin-stimulated cyclic AMP production was determined in the same brain regions. Compared with controls, G_sα (52-kDa species) immunoreactivity was significantly (p < 0.05) elevated in prefrontal (+36%), temporal (+65%), and occipital (+96%) cortex but not in hippocampus (+28%), thalamus (-23%), or cerebellum (+21%). In contrast, no significant differences were found in the other G protein subunits (G_iα, G_oα, Gβ) measured in these regions. Forskolin-stimulated cyclic AMP production was significantly increased in temporal (+31%) and occipital (+96%) cortex but not in other regions. No significant differences were apparent in basal or GTPγS-stimulated cyclic AMP production. A significant correlation (r= 0.60, p < 0.001) was observed between forskolin-stimulated cyclic AMP formation and G_sα (52 kDa) immunoreactivity when examined across these cortical regions. The observed increase in G_sα may be specific to bipolar disorders as no significant differences were detected in G_sα levels in temporal cortex from patients with either schizophrenia (n = 7) or Alzheimer's disease (n = 7). In summary, the present study confirms and extends our earlier findings and supports the notion that increased G_sα levels and possibly G_sα-adenylyl cyclase-mediated signal transduction are relevant to the pathophysiology of bipolar affective disorder.     

Inhibition of cardiac slow action potentials by 8-bromo-cyclic GMP occurs independent of changes in cyclic AMP levels

Cyclic GMP inhibits the slow inward Ca current of cardiac cells. This effect could be due to a cyclic GMP-mediated phosphorylation of the Ca channel (or some protein modifying Ca channel activity), or alternatively, to enhanced degradation of cyclic AMP owing to stimulation of a phosphodiesterase by cyclic GMP. To test the latter possibility, we examined the effect of extracellular 8-bromo-cyclic GMP on cyclic AMP levels in guinea pig papillary muscles, in parallel with electrophysiological experiments. Isoproterenol (10(-6) M) significantly increased the cyclic AMP levels and induced Ca-dependent slow action potentials. Superfusion with 8-bromo-cyclic GMP (10(-3) M) inhibited the slow action potentials induced by isoproterenol. However, muscles superfused with 8-bromo-cyclic GMP had cyclic AMP levels identical to those of muscles superfused with isoproterenol alone. Similarly, 8-bromo-cyclic GMP had no effect on the increase in cyclic AMP levels of muscles treated with forskolin (10(-6) M) or histamine (10(-6) M). We conclude that the inhibitory effect of cyclic GMP on slow Ca channels in guinea pig ventricular cells is not due to a decrease in the cyclic AMP levels. We hypothesize that a cyclic GMP-mediated phosphorylation is the most likely explanation for the Ca channel inhibition observed in this preparation.     

Melatonin Receptor-Mediated Inhibition of Cyclic AMP Accumulation in Chick Retinal Cell Cultures

Abstract: Melatonin receptors were characterized in cultured neurons and photoreceptors prepared from chick embryo retina. Cultured cells contained high-affinity 2-[¹²⁵I]iodomelatonin binding sites (K_D = 41.6 pM), similar to those in intact retina. The effects of melatonin and related indoles on cyclic AMP accumulation were examined. Melatonin (10^?7M) had no effect on basal or K⁺-stimulated cyclic AMP accumulation, but inhibited forskolin-stimulated cyclic AMP accumulation by approximately 50%. Melatonin inhibited forskolin-stimulated cyclic AMP accumulation in the presence or absence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting an effect on cyclic AMP synthesis rather than degradation. Half-maximal inhibition was observed at 5.9 × 10^?10M melatonin. The relative order of potency among melatonin analogues was 2-iodomelatonin > melatonin ≈ 6-chloromelatonin ≥ 6-hydroxymelatonin > N-acetylserotonin ≈ 5-methoxytryptophol > serotonin. The EC₅₀ value for inhibition of cyclic AMP accumulation by 2-iodomelatonin (36.7 pM) was comparable to the K_D value for binding of the radioligand, suggesting that the binding sites represent functional receptors. The inhibitory effect of melatonin was antagonized by the putative melatonin antagonists luzindole, N-acetyltryptamine, and N-(2,4-dinitrophenyl)-5-methoxytryptamine, with estimated K_B values of 0.12, 0.17, and 1 µM, respectively. At a concentration of 10 µM, N-(2,4-dinitrophenyl)-5-methoxytryptamine significantly inhibited forskolin-stimulated cyclic AMP accumulation when added alone; at 30 µM, luzindole and N-acetyltryptamine also had significant inhibitory effects. The inhibitory effect of melatonin was blocked by pretreatment with pertussis toxin. The results of this study indicate that melatonin receptors on retinal cells are coupled via inhibitory G proteins to cyclic AMP accumulation. Thus, some of the effects of melatonin on retinal physiology may be related to regulation of cyclic nucleotide metabolism.     

Tyrosine hydroxylase induction by basic fibroblast growth factor and cyclic AMP analogs in striatal neural stem cells: role of ERK1/ERK2 mitogen-activated protein kinase and protein kinase C.

Neural stem cells (NSC) with self-renewal and multilineage potential are considered good candidates for cell replacement of damaged nervous tissue. In vitro experimental conditions can differentiate these cells into specific neuronal phenotypes. In the present study, we describe the combined effect of basic fibroblast growth factor (bFGF) and dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) on the differentiation of fetal rat striatal NSC into tyrosine hydroxylase-positive cells. Tyrosine hydroxylase induction was accompanied by the activation of ERK1/ERK2 mitogen-activated protein kinase and was inhibited by the ERK1/ERK2 pathway blocker PD98059, suggesting that ERK activation may be important for this process. In addition, protein kinase C (PKC) was shown to be required for tyrosine hydroxylase protein expression. The inhibition of PKC by staurosporin, as well as its downregulation, decreased the ability of bFGF+dbcAMP to generate tyrosine hydroxylase-positive cells. Moreover, the PKC activator phorbol 12-myristate 13-acetate (PMA) together with bFGF and dbcAMP led to a significant increase in phospho-ERK1/ERK2 levels, and the percentage of beta-tubulin III-positive cells that expressed tyrosine hydroxylase increased by 3.5-fold. PMA also promoted the phosphorylation of the cyclic AMP response element binding protein that might contribute to the increase in tyrosine hydroxylase-positive cells observed in bFGF+dbcAMP+PMA-treated cultures. From these results, we conclude that the manipulation in vitro of NSC from rat fetal striatum with bFGF, cyclic AMP analogs, and PKC activators promotes the generation of tyrosine hydroxylase-positive neurons.     

Pertussis toxin blocks somatostatin's inhibition of stimulated cyclic AMP accumulation in anterior pituitary tumor cells

Somatostatin inhibits both forskolin and (-) isoproterenol-stimulated cyclic AMP accumulation in AtT-20 cells. Pretreatment of these cells with pertussis toxin prevents somatostatin's inhibitory effects on cyclic AMP production. This pretreatment also enhances the cyclic AMP response to forskolin and (-) isoproterenol without affecting basal cyclic AMP levels. The blockade of somatostatin's inhibitory effect was dependent both on the time of preincubation and concentration of pertussis toxin used. The rise in forskolin-stimulated cyclic AMP formation following pertussis toxin treatment preceded the blockade of somatostatin's inhibitory actions. The results suggest that somatostatin acts through an inhibitory guanine nucleotide regulatory protein to affect adenylate cyclase activity.     

Pertussis toxin effects on adenylate cyclase activity, cyclic AMP accumulation and lipolysis in adipocytes from hypothyroid, euthyroid and hyperthyroid rats

Adipocytes from hypothyroid rats have a decreased responsiveness to agents that activate adenylate cyclase, whereas cells from hyperthyroid rats have an increased responsiveness as compared to the controls. This is reflected in cyclic AMP accumulation as well as lipolysis. Administration of pertussis toxin to rats or its in vitro addition to adipocytes increased basal lipolysis and cyclic AMP accumulation as well as the response to norepinephrine or forskolin. The effects of thyroid status was not abolished by toxin treatment. Pertussis toxin-catalyzed ADP ribosylation of Ni was increased in adipocyte membranes from hypothyroid rats as compared to those from euthyroid rats. However, no change in sensitivity to N6-(phenylisopropyl)adenosine was observed. The data suggest that the amount of Ni might not be rate-limiting for the inhibitory action of adenosine. A consistent decrease in maximal lipolysis was observed in freshly isolated adipocytes from hypothyroid animals as compared to those from the controls. Such defective maximal lipolysis was not corrected by adenosine deaminase or in vivo administration of pertussis toxin. The relationship between cyclic AMP levels and lipolysis suggests that in fat cells from hypothyroid rats either the cyclic AMP-dependent protein kinase or the lipase activity itself may limit maximal lipolysis. There appears to be multiple effects of thyroid status on lipolysis involving factors other than those affecting adenylate cyclase activation.     

Amitriptyline-Mediated Inhibition of Neurite Outgrowth from Chick Embryonic Cerebral Explants Involves a Reduction in Adenylate Cyclase Activity

We have previously shown that amitriptyline, a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants, and that dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, or theophylline can enhance neurite outgrowth from embryonic olfactory explants. In the present study, we examined the mechanism(s) underlying amitriptyline-mediated inhibition of neurite outgrowth by studying the effects of amitriptyline on adenylate cyclase activity and cyclic AMP levels. In cultured chick embryonic cerebral explants, dibutyryl cyclic AMP or theophylline, but not dibutyryl cyclic GMP, enhanced neurite outgrowth and partially reduced the inhibitory effects of amitriptyline on neurite outgrowth. Explants treated with amitriptyline for 2 days showed decreased cyclic AMP levels that significantly correlated with the degree of neurite outgrowth. Amitriptyline inhibited both basal and forskolin-stimulated adenylate cyclase activity in vitro, but only in the presence of GTP. Taken together, these data suggest that amitriptyline inhibits the activity of adenylate cyclase via a GTP-dependent mechanism, and that the subsequent decrease in cyclic AMP level may be involved in amitriptyline-mediated inhibition of neurite outgrowth.