Somatostatin inhibition of anterior pituitary adenylate cyclase activity: different sensitivity between male and female rats

Somatostatin (SRIF) is a potent inhibitor of growth hormone (GH) secretion. Although cyclic AMP (cAMP) has been suggested as intracellular mediator of SRIF action, a complete characterization of its effect and the different sensitivity between male and female animals, has not yet been carried out. In this study SRIF inhibited basal and GH-releasing factor (GRF) stimulated anterior pituitary adenylate cyclase activity with a greater effectiveness in male than in female glands. Similarly SRIF reduction of forskolin-stimulated anterior pituitary adenylate cyclase activity, was more pronounced in male than in female animals. By using pertussis toxin (PTX), which uncouples inhibitory receptors from adenylate cyclase catalytic subunit, SRIF inhibition of both basal and forskolin-stimulated adenylate cyclase activity was nearly abolished. These results show that anterior pituitary SRIF receptors are coupled in an inhibitory fashion with adenylate cyclase enzyme, and that male rat adenohypophyses are more responsive to SRIF inhibition.     

Agonist-induced desensitization of adenylyl cyclase activity mediated by 5-hydroxytryptamine7 receptors in rat frontocortical astrocytes

Our previous study has demonstrated that astrocytes derived from the rat frontal cortex contain 5-hydroxytryptamine (5-HT)₇ receptors positively coupled to adenylyl cyclase. In this study, we observed a desensitization of 5-HT₇ receptors induced by a treatment with agonists (0.1, 1, and 10 μM, 0.5 to 3.5 h). Maximum responses, but not the EC₅₀ values, in the concentration–response curve of 5-HT-induced cyclic AMP formation were decreased after pretreatment with 5-HT. Pretreatment with 5-carboxamidotryptamine (5-CT) elicited a potent desensitization of 5-HT-induced cyclic AMP formation. Neither 2-methyl-5-HT nor α-methyl-5-HT caused the desensitization. When the astrocytes were treated with isoproterenol, N-ethylcarboxamidoadenosine, and dibutyryl cyclic AMP (all of which increase intracellular cyclic AMP levels), 5-HT-induced cyclic AMP responses were not affected. Conversely, adenylyl cyclase activity mediated by either isoproterenol or N-ethylcarboxamidoadenosine was attenuated by pretreatment with each of these agonists, but not 5-HT. In addition, our study showed that the administration of 5-HT, 5-CT, and 8-hydroxy-2-(di-n-propylamino)tetralin to the astrocytes stimulated cyclic AMP formation both in the presence and absence of forskolin, whereas in neuron-rich cultures of the frontal cortex, these agonists did not change basal cyclic AMP levels and decreased forskolin-stimulated cyclic AMP formation. Neurons may predominantly contain 5-HT_1A receptors that are negatively coupled to adenylyl cyclase. These results suggest that 5-HT₇ receptors are highly expressed in astrocytes but not in neuronal cells, and that pretreatment with their agonists results in a homologous desensitization of the receptors.     

Corticotropin-releasing factor desensitization of adrenocorticotropic hormone release is augmented by arginine vasopressin

The desensitization of corticotropin-releasing factor (CRF)-stimulated ACTH release from and intracellular cyclic AMP accumulation in anterior pituitary cells was investigated in primary cultures of rat pituitary cells and in a mouse tumor cell line (AtT 20/D16-16). CRF potently stimulated ACTH secretion and cyclic AMP accumulation in both preparations. When primary cultures were preincubated with 100 nM CRF, 50% desensitization of ACTH release occurred within 4 hr while similar desensitization of cyclic AMP accumulation occurred by 1 hr. This desensitization was manifested as a reduced maximal CRF response. Concentrations of CRF as low as 1 to 10 pM induced desensitization. Pretreatment did not affect ACTH content nor did it alter the ability of epinephrine or forskolin to stimulate ACTH release. Pretreatment of AtT 20 cells, which are a homogeneous population of corticotrophs, with 100 nM CRF reduced the subsequent ability of CRF, but not of isoproterenol or of forskolin, to stimulate ACTH release and cyclic AMP formation. Preincubation of AtT 20 cells with isoproterenol did not affect the cyclic AMP or ACTH release responses induced by CRF, but did desensitize beta-adrenergic receptors. Arginine vasopressin (AVP) was a weak ACTH-releasing factor, but AVP enhanced CRF-directed ACTH release from primary cultures. When these cells were preincubated with both CRF and AVP, CRF desensitization occurred with lower concentrations of CRF than when CRF desensitization was induced by preincubating the cells with CRF alone. The ED50 for CRF-induced desensitization was 700 +/- 150 pM when the cells were exposed to CRF alone, and 20 +/- 15 pM when 1 microM AVP was added during the preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Forskolin and Melatonin on Cyclic AMP Generation in Pars Tuberalis Cells of Ovine Pituitary

The pineal indoleamine, melatonin, acts on specific secretory cells of the pars tuberalis of the sheep pituitary. Using pars tuberalis cells in primary culture melatonin inhibited forskolin-stimulated cyclic AMP production in both a time - and dose-dependent manner, but the nature of the melatonin response was critically dependent upon the stimulatory concentration of forskolin used. Forskolin alone stimulated dose-dependent cyclic AMP accumulation, which reached an equilibrium state after 15 min. This was maintained for up to 3 h, indicating a lack of desensitization to forskolin. Melatonin (1 μM) inhibited this response by greater than 80% at all doses. However, 100 μM forskolin reduced both the affinity and the number of the melatonin receptors, relative to untreated controls. Consistent with this, melatonin was 100 times less potent at inhibiting forskolin-stimulated cyclic AMP production, when titrated against 100 μM forskolin as compared to 1 μM forskolin. The response to 1 μM forskolin could be potentiated by 10 μM phorbol 12,13 myristate acetate, but not by calcium ionophore (A23187). This provides evidence for the interaction of the phosphatidylinositol pathway with the cyclic AMP system in these cells. Nevertheless melatonin can inhibit both the potentiated and non-potentiated response with equal effect.     

17-beta Oestradiol Pretreatment of Mouse Striatal Neurons in Culture Enhances the Responses of Adenylate Cyclase Sensitive to Biogenic Amines

Embryonic striatal neurons from the mouse grown in primary culture (6 day old culture) were used in order to investigate the effects of 17-beta oestradiol (17-beta E2) on biogenic amine-sensitive adenylate cyclases. Pretreatment (28 h) of intact cells with 17-beta E2 (10-9 M) enhanced cyclic AMP production induced by either dopamine, isoproterenol, serotonin, or 2-chloro-adenosine (maximal effective concentrations). These effects of 17-beta E2 on biogenic amine-sensitive adenylate cyclases occurred after several hours (8 h at least) and were seen in most cases with a concentration as low as 10-11 M (EC50: 10-10 M). They were additive with those induced by phenol red (5.6 microg/l) and chemically specific since 17alpha-oestradiol, 2(OH)17-beta E2, progesterone, and dexamethasone were without effect. In addition, they were not seen in cells which had been pretreated (30 h) with cycloheximide or alpha-amanitin, suggesting an involvement of de novo protein synthesis. Since 17-beta E2 did not influence cyclic AMP production induced by either forskolin or manganese ions, the stimulatory effects of 17-beta E2 pretreatment on biogenic amine-sensitive adenylate cyclases were not linked to an increase in the amount of enzyme catalytic units. 17-beta E2 pretreatment enhanced twofold the number of beta-adrenergic receptors (as estimated by the specific binding of (125I)iodocyanopindolol) but did not, in contrast, affect either the number or the affinity of dopaminergic receptors (as estimated by (125I)SCH 23982 binding). Therefore, the enhancing effects of 17-beta E2 pretreatment on biogenic amine-sensitive adenylate cyclases could be related either to an increased number of coupled receptors or to modifications of the adenylate cyclase transducing system (occurring probably at the G-protein level) or to a combination of the two.     

Chronic infusion of agents that increase cyclic AMP concentration enhances the regeneration of mammalian peripheral nerves in vivo

Our previous investigation indicates that forskolin, a robust activator of adenylate cyclase, promotes sensory nerve regeneration in amphibians. The present study was designed to determine if forskolin had a similar effect in mammals. We also wished to test the hypothesis that cyclic AMP modulates nerve regeneration by comparing the effects of chronically infused forskolin with the effects of infused dibutyryl cyclic AMP, 8-bromo cyclic AMP, and the phosphodiesterase inhibitor, theophylline. Our results indicated that all agents promoted some aspect of regeneration. The two which presumably generated the largest increase in cyclic AMP concentration, forskolin and 8-bromo cyclic AMP, had the most profound effect on axonal elongation. All agents decreased the time to sprout initiation, but theophylline produced the largest decrease and its effect was mimicked by caffeine, a methylxanthine with limited ability to inhibit phosphodiesterase. This suggests that sprout formation may be triggered by an increase in intraaxonal free Ca2+, possibly modulated by cyclic AMP. The role of cyclic AMP in axonal elongation remains to be determined, but may be associated with stimulation of protein synthesis in the nerve cell body.     

Peptidergic modulation of G-protein coupled cyclic-AMP accumulation in the rat caudate nucleus.

Somatostatin, substance P, and vasoactive intestinal polypeptide were incubated in an adenylate cyclase assay with a particulate fraction of caudate-putamen tissue of the rat in order to examine the effect of the neuropeptides on G-protein coupled adenylate cyclase in vitro. Somatostatin induced an enhancement of cyclic AMP formation in presence of guanine nucleotides and cholera toxin but inhibited pertussis toxin and forskolin enzyme stimulation. Pertussis toxin and cholera toxin also depressed forskolin-induced stimulation as described previously. Somatostatin was able to antagonize these inhibitory effects of both toxins. On the contrary, substance P reduced GTP and cholera toxin stimulated striatal adenylate cyclase, without affecting forskolin activation. In our preparation, VIP did not influence basal adenylate cyclase activity or the stimulation by guanine nucleotides, cholera toxin, and pertussis toxin. VIP potently inhibited the enhancement of cyclic AMP formation by forskolin and completely antagonized the inhibitory effect of cholera toxin on forskolin activation. These results suggest that neuromodulatory effects of somatostatin, substance P, and VIP are mediated by the inhibitory as well as stimulatory guanine nucleotide proteins G-i and G-s coupled to an adenylate cyclase system.     

Ontogeny of beta-adrenergic receptor-mediated cyclic AMP generating system in primary cultured neurons.

The developmental changes in the beta-adrenergic receptor/cyclic AMP generating system were examined using mouse cerebral cortical neurons in primary culture. During neuronal growth in vitro, the number of binding sites for [3H]dihydroalprenolol (DHA) showed a tendency to increase (Bmax), while the affinity (Kd) for [3H]DHA did not show any noticeable changes. Basal and isoproterenol-stimulated adenylate cyclase activities as well as the activation of adenylate cyclase by 5'-guanylylimidodiphosphate (GppNHp), NaF and forskolin showed progressive and parallel increases during neuronal growth on a polylysine-coated surface. The treatment of primary cultured neurons with islet-activating protein (IAP), one of the pertussis toxins, attenuated the inhibitory effect of carbachol, a muscarinic agonist, on isoproterenol-induced activation of adenylate cyclase activity. These results indicate that primary cultured neurons possess a cyclic AMP generating system coupled with beta-adrenergic and muscarinic receptors, which is regulated via stimulatory and inhibitory GTP-binding proteins, respectively. The results described above also suggest that the beta-adrenergic receptor, stimulatory and inhibitory types of GTP-binding proteins and adenylate cyclase may develop in a parallel fashion during neuronal growth on a polylysine-coated surface.     

β-adrenoceptor control of cardiac adenylyl cyclase during development: agonist pretreatment in the neonate uniquely causes heterologous sensitization, not desensitization

In the adult, increased stimulation of postsynaptic receptor sites produces compensatory desensitization that reduces tissue responsiveness. During development, however, responses in most systems increase with age and with the maturation of neuronal inputs. In the current study, we examined whether agonist-induced desensitization of cardiac β-adrenergic receptor signaling mediated through adenylyl cyclase could be elicited in 6-, 15- and 25-day-old rats, and in adults. In each case, animals were pretreated with isoproterenol daily for four days preceding the experiment, and on the fifth day, cardiac membrane preparations were examined. Fifteen and 25-day-old animals and adults all exhibited desensitization, as demonstrated by a diminished cyclase response to isoproterenol in vitro. However, in 6-day-old animals, the enzymatic response to isoproterenol was enhanced by chronic pretreatment. Measurements of the G-protein-sensitive component of cyclase (decrement in activity obtained with deletion of GTP from the reaction mixture, stimulatory response to fluoride) indicated heterologous desensitization in the older animals, evidenced by diminished dependence on GTP and reduced response to fluoride; the 6-day-old animals showed enhanced GTP dependence and augmentation of the fluoride response. Uniquely in 6-day-old animals, the total catalytic activity of adenylyl cyclase, measured with forskolin-Mn²⁺, was markedly elevated by chronic isoproterenol pretreatment, whereas it was unaffected in older animals. These data suggest that regulation of receptor signaling is completely different early in neonatal life. Instead of producing desensitization of responses, agonist exposure promotes receptor signaling by enhancing expression and/or catalytic efficiency of adenylyl cyclase. In older animals, the predominant effect is heterologous desensitization mediated at the level of G-proteins. These developmental differences are likely to be important in the maintenance of tissue responsiveness during the period in which innervation develops, as well as in the ability of neurotrophic input to ‘program’ the responsiveness of target tissues.     

Beta-adrenergic stimulation of protein (arginine) methyltransferase activity in cultured cerebral cells from embryonic mice

Several adrenergic effectors and neurotransmitters were tested as potential regulators of myelin basic protein (MBP) and histone methyltransferase activities. Both enzymes were specifically activated by beta-adrenergic agonists in a stereospecific manner. Cyclic AMP (but not AMP) stimulated the enzymes to the same extent as did the beta-adrenergic agonist, (-) isoproterenol. The studies suggest that beta-adrenergic agonists stimulate adenylate cyclase thereby causing an increased production of cyclic AMP which stimulates the methyltransferases. Cycloheximide addition to the reaction mixture did not affect the stimulation due to cyclic AMP, indicating that new protein synthesis is not involved in the cyclic AMP stimulation of the methyltransferases. Thyroid hormone (T3) has been shown to stimulate MBP methyltransferase [Amur et al, 1984] and could exert its stimulatory effect through beta-adrenergic-dependent systems. But the beta-adrenergic antagonist, propranolol, did not block the stimulation by T3, suggesting that the effect of T3 is not mediated through beta-adrenergic-dependent systems. Thus, the methylation of MBP seems to be regulated both by T3 and by neurotransmitters and/or hormones mediating their effects through cyclic AMP production, whereas the methylation of histones seems to be regulated only by the latter.     

Sexual experience alters D1 receptor-mediated cyclic AMP production in the nucleus accumbens of female Syrian hamsters

Drugs of abuse produce long-term changes in dopamine neurotransmission and receptor-effected intracellular signaling. Similar changes in neuronal activity are mediated by motivated behaviors. To explore cellular mechanisms underlying these neuroadaptations following sexual experience, cyclic AMP accumulation following stimulation of D1 dopamine receptors, G-proteins, and adenylate cyclase was compared in the nucleus accumbens and caudate nucleus of sexually naive and experienced female hamsters following sexual behavior. Direct stimulation of adenylate cyclase with forskolin or indirectly by activation of G-proteins with Gpp(NH)p produced dose-dependent increases in the formation of cyclic AMP in the nucleus accumbens and caudate nucleus, with no effects of sexual experience on these measures. Specific D1 receptor stimulation increased Gpp(NH)p-induced adenylate cyclase activity in the nucleus accumbens and caudate nucleus of all animals. Interestingly, this stimulation was further enhanced only in membranes from the nucleus accumbens, but not from the caudate nucleus, of sexually experienced hamsters compared to the response of naive females. These results demonstrate that sexual behavior experience can sensitize mesolimbic dopamine pathways and that this sensitization occurs through an increase in D1 receptor-mediated signaling.     

The cyclic AMP second messenger system in man: the effects of heredity, hormones, drugs, aluminum, age and disease on signal amplification

The intracellular effects of a number of hormonal signals are mediated by the cyclic AMP second messenger system in man and the ubiquitous distribution of hormone-stimulated adenylate cyclase suggests the importance of this enzyme complex in normal aging and pathophysiological states. Various vectors including heredity, endogenous catecholamines, steroid hormones, and drugs affect the activity of hormone-stimulated adenylate cyclase in man. The effect of heredity was studied using lymphocytes obtained from monozygotic twin pairs and age and sex-matched sib pairs. Only for forskolin-stimulated activity is a significant proportion of individual variance attributable to heredity, suggesting the relative stability of the catalytic subunit. Beta-adrenergic and prostaglandin E-1 activity are "state" characteristics and their activities are controlled by environmental parameters. A significant reduction in isoproterenol-stimulated cyclic AMP accumulation between the menses and luteal phase of the menstrual cycle is observed in lymphocytes obtained from 11 female subjects. The lowest level of beta-adrenergic receptor activity is associated with the highest levels of progesterone and estradiol hormone levels in blood. Lithium at therapeutic concentrations markedly inhibits adenylate cyclase activity in platelet membranes. Moreover, marked individual differences are observed in sensitivity to lithium as determined by Dixon plot derived Ki values for 9 normal, healthy subjects. Human adenylate cyclase obtained from platelets and lymphocytes is activated by micromolar amounts of aluminum in the presence of NaF. Irreversible activation of adenylate cyclase by aluminum is suggested as a possible mechanism of this metal's neurotoxicity. The biochemical basis for the age-associated decline in beta-adrenergic responsiveness in man is discussed. Several investigations suggest a deficit at two levels in the adenylate cyclase complex: an impaired coupling of the receptor/N protein subunits and an additional lesion distal to the receptor at the level of N/C coupling. Perfusion studies with salbutamol suggest that the decline in beta-adrenergic sensitivity is general and not restricted to lymphocytes. Possible abnormalities in cyclic AMP signal amplification and recognition in various disease states is discussed. Increased prostaglandin E-1-stimulated cyclic AMP accumulation is observed in lymphocytes obtained from patients with Alzheimer's disease compared to age-matched controls and correlated with severity of the disease state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serotonin-1C sites in the choroid plexus are not linked in a stimulatory or inhibitory way to adenylate cyclase

The association of the serotonin recognition sites in the pig choroid plexus (5-HT-1C sites) with an adenylate cyclase was examined. The interaction of serotonin and mianserin with [3H]mesulergine binding was not affected by the stable GTP analogue GppNHp. The binding of [3H]serotonin to choroid plexus membranes was also unaffected by GppNHp while a dose-dependent decrease was observed in pig cortical and hippocampal membranes. The porcine choroid plexus contained a forskolin- and histamine-sensitive adenylate cyclase. Serotonin, however, was ineffective in this preparation. While forskolin-stimulated adenylate cyclase in the rat hippocampus was inhibited by serotonin forskolin-stimulated adenylate cyclase in the choroid plexus was insensitive to serotonin. These results indicate that the serotonin recognition sites in the choroid plexus are not linked in a stimulatory or inhibitory way to an adenylate cyclase, in contrast with other 5-HT-1 receptor subtypes.     

Evidence for a presynaptic adenylate cyclase system facilitating [3H]norepinephrine release from rat brain neocortex slices and synaptosomes

The effects of drugs known to enhance intracellular cyclic AMP levels on depolarization-induced [3H]norepinephrine release from superfused rat neocortical slices and synaptosomes were investigated. The adenylate cyclase activator forskolin, the membrane-permeating cyclic AMP analogues 8-bromo-cyclic AMP and dibutyryl cyclic AMP, as well as the phosphodiesterase inhibitors isobutylmethylxanthine and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrolidone (ZK 62771) enhanced the electrically evoked release of [3H]norepinephrine from superfused rat brain neocortex slices. 8-Bromo-cyclic GMP was without effect on the electrically evoked release. When [3H]norepinephrine release was enhanced by prolonging the electrical pulse duration from 2 msec to 10 msec, the relative inhibitory effect of the Ca2+ channel blocker Cd2+ and the relative facilitatory effect of the K+ channel blocker 4-aminopyridine remained unaffected. In striking contrast, the relative facilitatory effects of forskolin and 8-bromo-cyclic AMP were strongly reduced, whereas the effect of ZK 62771 was almost doubled. When veratrine-induced release of [3H]norepinephrine from cortex synaptosomes was examined, the facilitatory effects of forskolin, 8-bromo-cyclic AMP, and ZK 62771 were even more pronounced than in brain slices. The data strongly support the hypothesis that a presynaptic adenylate cyclase system plays a facilitatory role in the stimulus-secretion coupling process in central noradrenergic nerve terminals.     

Desensitization of the dopaminergic system in bovine retina following incubation with high potassium.

The effect of potassium depolarization on dopamine D1 receptor activity in bovine retina was investigated. Preincubation of bovine retinas in buffer containing high KCl (56 mM) as compared to a low KCl control buffer resulted in a significant decrease in dopamine-stimulated adenylate cyclase activity with no change in basal or GTP-stimulated adenylate cyclase activity. The apparent Vmax for dopamine was decreased from 102 +/- 15 pmol/min/mg protein in retinas preincubated in high KCl to 71 +/- 11 pmol/min/mg protein in control retinas (n = 5). The apparent Ka for dopamine stimulation of the enzyme did not change. The potassium-induced desensitization could be blocked by preincubation with the dopamine antagonist cis-flupenthixol suggesting that the desensitization was caused by the release of dopamine. The rapid desensitization was not accompanied by a change in D1 receptor density as assessed by binding of [3H]SCH23390 nor in agonist binding as assessed by competition of the selective D1 agonist, SKF38393, for [3H]SCH23390 binding. The potassium-induced desensitization was mimicked by preincubation of retinas in control medium containing isobutylmethylxanthine or dibutyryl cyclic AMP. Incubation of retinas in 56 mM KCl also led to a decrease in activation of adenylate cyclase by vasoactive intestinal polypeptide. These results strongly suggest that potassium depolarization leads to a very rapid heterologous desensitization of adenylate cyclase in bovine retinas.     

The effect of capsaicin on the adenylate cyclase activity of rat brain.

The effect of capsaicin on the adenylate cyclase activity in different regions of the rat brain (preoptic area of the hypothalamus, cerebral cortex and cerebellum) was investigated. Capsaicin added in vitro (10(-7)-10(-5) M) increased the adenylate cyclase activity of different brain regions. Following systemic capsaicin desensitization adenylate cyclase activity was significantly increased in the preoptic area. The enhanced adenylate cyclase activity in the preoptic area was inhibited by the vitro addition of capsaicin or 5-HT, whereas desensitization did not affect the in vitro activating effect of capsaicin in other brain regions (cerebral cortex, cerebellum). It is assumed that the pharmacological effect of capsaicin in the preoptic area is mediated through the activation of adenylate cyclase. Since capsaicin induces irreversible impairment of the function of warmsensitive hypothalamic neurons it is assumed that adenylate cyclase is involved in maintaining normal thermoregulatory functions.     

Somatostatin inhibition of VIP- and isoproterenol-stimulated cyclic AMP accumulation in dissociated cells from rat cerebral cortex

Freshly dissociated cerebral cortex cells from adult rats have been used in the present study to determine if dual regulation of cyclic AMP levels by inhibitory and stimulatory agents can be expressed in the mature brain. Somatostatin, an inhibitory agent, barely affected the basal cyclic AMP metabolism while vasoactive intestinal peptide (VIP) and isoproterenol, two stimulatory agents enhanced cyclic AMP production. However, this increase was depressed by somatostatin, which decreased the efficiency, but not the potency, of the effects of the two stimulatory agents on cyclic AMP accumulation.     

Refractory hypothalamic adenylate cyclase in anorectic tumor-bearing rats: implications for NPY-induced feeding

Although isoproterenol stimulated adenylate cyclase activity in hypothalamic membranes taken from freely-feeding, food-restricted or nonanorectic tumor-bearing rats, the response was greatly reduced in anorectic tumor-bearing rats. The addition of NPY to the membrane preparation inhibited adenylate cyclase activity in hypothalamus taken from freely-feeding and food-restricted rats, but NPY-inhibitory activity was significantly reduced in both groups of tumor-bearing rats. These results suggest that cyclic AMP formation is refractory in anorectic tumor-bearing rats, and that NPY-induced inhibition of hypothalamic adenylate cyclase is reduced in tumor-bearing rats prior to the onset of significant anorexia. Therefore, NPY-induced feeding may be reduced in tumor-bearing organisms due to a dysfunction in the cyclic AMP second messenger system.     

Regulation of choline acetyltransferase in primary cell cultures of spinal cord by neurotransmitter L-norepinephrine

Neurotransmitter L-norepinephrine increased up to 8-fold the activity of choline acetyltransferase (CAT), the enzyme responsible for the synthesis of acetylcholine, in mouse spinal cord cells in culture grown for several days. The increase of CAT activity by L-norepinephrine was mediated by a beta-adrenergic receptor in the same manner as the response of intracellular cyclic AMP. Derivatives of cyclic AMP caused an increase of CAT activity to the level similar to that of L-norepinephrine. A cyclic AMP phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX), enhanced the elevation of CAT activity by L-norepinephrine. These results indicate that L-norepinephrine stimulated the synthesis of CAT molecules via the action of cyclic AMP. The pretreatment of cells with 5-fluoro-2'-deoxyuridine (FdU) markedly diminished the numbers of satellite cells and, in parallel, the responses of CAT activity to L-norepinephrine. The increase of cyclic AMP by L-norepinephrine was also reduced by pretreatment of the cells with FdU. In contrast, co-cultures of spinal cord with heart muscle markedly (30-fold) stimulated CAT activity both with and without pretreatment of FdU. The addition of L-norepinephrine and co-cultures with heart muscle showed an additive effect. These observations indicate that the stimulatory effect of L-norepinephrine on CAT activity is mostly, if not only, mediated via the interaction with satellite cells, and that the increase of CAT activity by L-norepinephrine is based on a mechanism different from that of co-cultures with heart muscle cells.     

Vasoactive intestinal peptide is a secretagogue in bovine chromaffin cells pretreated with pertussis toxin.

Vasoactive intestinal peptide (VIP) evokes little or no secretion of catecholamines from cultured bovine chromaffin cells. However, pretreatment of chromaffin cells with pertussis toxin (PTX, 100 ng/ml for > or = 4 h) revealed that VIP is a secretagogue. In PTX-treated cells catecholamine secretion evoked by VIP occurs with minimal elevation of cyclic AMP and is only slightly enhanced by cyclic nucleotide phosphodiesterase inhibitors. Forskolin, a direct activator of adenylate cyclase, causes delayed secretion of catecholamines from chromaffin cells treated with PTX, but only with pronounced elevation of cyclic AMP levels. Stimulation of catecholamine secretion by histamine, known to activate phosphatidylinositol-specific phospholipase C in chromaffin cells, is also enhanced by preincubation of the cells with PTX. These results suggest that in the bovine chromaffin cell a PTX-sensitive G-protein mediates tonic inhibition of secretion, possibly by preventing activation of phospholipase C.