Effects of biogenic amines on the concentration of adnosine 3',5'-monophosphate in bovine superior cervical ganglion.

The accumulation of cyclic AMP elicited in bovine superior cervical ganglia by several biogenic amines in the presence of theophylline was investigated in vitro. The response to dopamine (10^?4M), after 6 min of incubation, was found to exceed that induced by equimolar concentrations of norepinephrine. Experiments with an inhibitor of dopamine-beta-oxidase showed that prior conversion of dopamine to norepinephrine was not necessary for the activation of ganglionic adenylate cyclase. The effect of norepinephrine could be prevented by pre-incubation with a combination of alpha- and beta-adrenergic antagonists; the cyclic AMP increase caused by epinephrine was predominantly beta-sensitive. Tyramine, 5-hydroxytryptamine and histamine caused a moderate increase in the concentration of cyclic AMP. The effect of histamine, which could be reduced by pre-incubation with an antihistamine, burimamide, was found to be additive to that of dopamine and norepinephrine.     

Binding of alpha-and beta-adrenergic ligands to cerebral cortical membranes: effect of 6-hydroxydopamine treatment and relationship to the responsiveness of cyclic amp-generating systems in two rat strains.

The binding of an α-adrenergic antagonist, WB 4101 and of a β-adrenergic antagonist, dihydroalprenolol, to cerebral cortical membranes fromo two rat strains, Sprague-Dawley and F-344, was examined after intraventricular administration of 6-hydroxydopamine. Cyclic AMP-generating systems in cerebral cortical slices from Sprague-Dawley rats respond to norepinephrine with an accumulation of cyclic AMP which is increased twofold after 6-hydroxydopamine treatment, while the cyclic AMP systems in slices from F-344 rat respond to norepinephrine with a relatively large accumulation of cyclic AMP which is not further increased after 6-hyxdroxydopamine treatment. The binding of WB 4101 to cerebral cortical membranes was similar in both rat strains and was unchanged after 6-hydroxydopamine treatment. The binding of dihydroalprenolol to membranes was similar in both rat strains and increased about 25% in both strains after 6-hydroxydopamine. Thus, the number of binding sites for these ligands does not correlate well with the magnitude of responses of cyclic AMP-generating systems to norepinephrine. Activity of cyclic AMP-phosphdiesterases in cerebral cortical homogenates was similar in both rat strains and was unchanged after 6-hydroxydopamine. Fluoride-stimulated adenylate cyclase activity in cerebral cortical homogenates appeared to be slightly higher in F-344 rats compared to Sprague-Dawley rats before but not after 6-hydroxydopamine treatment suggesting that changes in responsiveness of the cyclic AMP system might not be dependent upon changes in receptor density but in a post-receptor alteration such as adenylate cyclase activity.     

Sertraline, a selective inhibitor of serotonin uptake, induces subsensitivity of beta-adrenoceptor system of rat brain

Subacute administration (b.i.d. for 4 days) of sertraline, a potent and selective inhibitor of serotonin uptake, was found to reduce cyclic AMP generation by the norepinephrine receptor-coupled adenylate cyclase in rat limbic forebrain slices and decrease the number of beta-adrenoceptors in rat cerebral cortex without affecting the affinity of [3H]dihydroalprenolol binding. Co-administration of sertraline and the serotonin agonist, quipazine, at doses at which neither agent had an effect, resulted in desensitization of norepinephrine receptor-coupled adenylate cyclase and down-regulation of beta-adrenoceptors. These findings suggest that increased serotonergic activity may be involved in the induction of subsensitivity of the beta-adrenoceptor system of rat brain by sertraline.     

Effect of selective monoamine oxidase inhibition by clorgyline and deprenyl on the norepinephrine receptor-coupled adenylate cyclase system in rat cortex

The consequences of selective monoamine oxidase (MAO) inhibition on the norepinephrine(NE)-sensitive adenylate cyclase system were determined in slices of rat cerebral cortex. The chronic administration of clorgyline, which selectively inhibited the activity of MAO-A, caused a significant decrease in the responsiveness of the noradrenergic cyclic AMP-generating system. The noradrenergic subsensitivity was accompanied by a significant decrease in the density of beta-adrenoceptors, as measured by 3H-dihydroalprenolol (DHA) binding, without altering the Kd value. However, selective inhibition of MAO-B by deprenyl did not alter the sensitivity of the cyclic AMP-generating system to NE or the specific DHA binding. The basal levels of cyclic AMP in the cortex were unaltered by the drugs. Since inhibition of MAO-A, but not MAO-B, increases the availability of NE, the results support the hypothesis that a persistent NE-receptor interaction is one of the prerequisites for the in vivo densitization of the NE-sensitive adenylate cyclase and the concomitant down-regulation of the number of beta-adrenoceptors in brain.     

Effect of narcotic analgesics on plasma cyclic AMP levels in male mice.

Systemic administration of morphine, as well as pethidine and pentazocine, increased plasma cyclic AMP levels in male mice. Intracerebroventricular administration of morphine and β-endorphin also increased plasma cyclic AMP levels. The effect of morphine and β-endorphin was antagonized by naloxone indicating the involvement of opiate receptors in the central nervous system for the effect of morphine on cyclic AMP levels. Tolerance was shown to develop to the action of morphine in increasing the plasma cyclic AMP. The morphine-induced increase in plasma cyclic AMP was abolished by treatment with propranolol or pentolinium or by adrenalectomy but not by treatment with atropine, phentolamine, 6-hydroxydopamine or α-methyltyrosine. These findings suggest that morphine increases plasma cyclic AMP levels mainly by releasing catecholamines, from the adrenal medulla, and these activate adenylate cyclase via the stimulation of β-adrenoceptors.     

Differential effects of chronic administration of desipramine on the cyclic AMP response in cortical slices and membranes in the rat

Chronic administration of desipramine resulted in a significant decrease in the degree of stimulation of production of cyclic AMP by noradrenaline, isoproterenol and forskolin in slices of the cerebral cortex of the rat. In preparations of membranes from similarly treated animals, GppNHp- and forskolin-stimulated adenylate cyclase activity was increased, while isoproterenol-, manganese- and sodium fluoride-stimulated activity was unchanged. It is concluded that chronic administration of desipramine exerts effects on the adenylate cyclase system additional to its action on beta-adrenergic receptors, possibly by modifying the catalytic or guanyl nucleotide binding units of the enzyme.     

Neuronal localization of the enhanced adenylate cyclase responsiveness to catecholamines in the rat cerebral cortex following reserpine injections.

Rats were injected daily with reserpine (2.5 mg/kg) for four days. The animals were sacrificed 4 hr subsequent to the last injection. Studies were performed as a means to determine alterations in catecholamine sensitivity of adenylate cyclase in the cerebral cortex following reserpine. In incubated tissue slices from reserpinized animals a greater accumulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in response to norepinephrine and isoproterenol was seen. In control animals both α-(phentolamine) and β-blocking agents (propranolol) inhibited norepinephrine-induced cyclic nucleotide accumulation, while in the reserpine-injected rats only propranolol was effective. The accumulation of cyclic AMP elicited by isoproterenol was inhibited by propranolol alone whether or not reserpine was administered. In cortical homogenates adenylate cyclase responsiveness to norepinephrine, isoproterenol and dopamine was enhanced in animals pretreated with reserpine. An identical observation was found in isolated neuronal fractions, although increased sensitivity of the enzyme to dopamine was now absent. Neither a- nor β-blocking agents were effective in broken cellular preparations. These data are interpreted to suggest that following depletion of catecholamines by reserpine, the β-receptor component of adenylate cyclase in the cortical neurones becomes hypersensitive to stimulation by catecholamines.     

Cyclic AMP in the rat jugular vein: relationship to vascular relaxation.

Most blood vessels contract to norepinephrine whereas the rat jugular vein relaxes. In the present study, we sought to determine if differences in the adenylate cyclase system accounted for the differing beta adrenergic responses of blood vessels. Cyclic AMP levels in the rat jugular vein incubated in vitro were lower than levels in the femoral vein or mesenteric artery. In the jugular vein, papaverine, an inhibitor of phosphodiesterase, produced a small, but not significant increase in cyclic AMP levels although in the femoral vein and mesenteric artery, cyclic AMP levels were doubled and tripled, respectively. Norepinephrine increased cyclic AMP only in jugular veins contracted with serotonin or in the presence of papaverine, whereas a marked increase in cyclic AMP occurred in the other vessels in the absence of serotonin or papaverine. In the presence of papaverine, the increase in cyclic AMP by norepinephrine in the jugular vein was less than in the other tissues. Neither the β₁-receptor antagonist, practolol, nor the β₂-receptor antagonist, N-isopropylmethoxamine, blocked the increase in jugular vein cyclic AMP produced by norepinephrine in the presence of papaverine. However, practolol and N-isopropylmethoxamine added together, significantly attenuated the norepinephrine-induced increase in cyclic AMP. These data indicate that relaxation of the rat jugular vein to norepinephrine is not associated with higher resting cyclic AMP levels, or a more responsive β-adrenergic-sensitive adenylate cyclase system.     

Some effects of oestrogens on [3H]-mepyramine binding in rat brain

Oestrogen depress [3H]-mepyramine binding in the amygdala, hypothalamus and cerebral cortex of rats. These effects of oestrogens are prevented by concurrent administration of inhibitors of cyclic 3'5' AMP phosphodiesterase, suggesting that the hormones act by reducing the activity of adenylate cyclase.     

Action in vitro and in vivo of chlorpromazine and haloperidol on cyclic nucleotide systems in mouse cerebral cortex and cerebellum.

Three different procedures were utilized to study the action of chlorpromazine (CPZ) and haloperidol on adenylate cyclase-cyclic nucleotide systems in the mouse cerebral cortex and cerebellum. Using incubated tissue slices which in both tissues demonstrate an activation of adenylate cyclase by norepinephrine (NE), CPZ was found to be more effective than haloperidol in reducing this response. With the second method it was shown that adenylate cyclase in homogenates was stimulated in both tissues by NE while dopamine (DA) was active in only the cerebral cortex. With this broken cellular preparation, CPZ exerted greater antagonism than haloperidol on both the NE- and DA-sensitive enzymes. Moreover, the DA-responsive enzymes were blocked by both agents to a greater extent than was observed with the NE-induced stimulation. In the third experiment haloperidol and three doses of CPZ were injected into mice in vivo, at specified intervals; thereafter the tissues were rapidly inactivated by focused microwave irradiation (0.5 sec), and cyclic AMP and cyclic GMP levels were subsequently determined. In general, the acute injections ($\text{1}\text{2}$–8 hr) of CPZ and haloperidol diminished the steady-state levels of the two nucleotides. Subchronic administration (24–48 hr) usually resulted in elevated cyclic AMP amounts. The data indicate that under both in vitro and in vivo conditions, neuroleptics inhibit neurohumorally-induced activation of adenylate cyclase. Subchronic injections, however, suggest other factors become prominent i.e. inhibition of phosphodiesterase or enhanced receptor sensitivity.     

Pituitary adenylate cyclase-activating polypeptide (PACAP)-evoked increase in cyclic AMP production in chick cerebral cortex: lack of a role of the protein kinase C pathway

Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a potent activator of cyclic AMP formation in the chick brain. The peptide also stimulates inositol phosphates accumulation and protein kinase C (PKC) activity in the chick cerebral cortex. In this work, we analyzed whether PACAP38-induced increase in cyclic AMP production in the chick cerebrum can be modified by a PKC pathway. 4Beta-phorbol 12,13-dibutyrate (4beta-PDB), a PKC activator, did not significantly affect the PACAP38-evoked increase in [3H]cyclic AMP production in [3H]adenine-prelabeled slices of the chick cerebral cortex. Of the tested PKC inhibitors, i.e. chelerythrine, H-7, NPC-15437 and staurosporine, only chelerythrine markedly decreased, in a concentration-dependent manner, the PACAP38-activated cyclic AMP accumulation in the chick cerebrum. These results suggest that (1) the process of cyclic AMP production stimulated by PACAP in the cerebral cortex of chick is not PKC-dependent, and that (2) chelerythrine, a widely used PKC inhibitor, influences the intracellular signaling pathway(s) associated with PACAP receptors in the chick brain in a way not involving PKC.     

Forskolin as an activator of cyclic AMP accumulation and lipolysis in rat adipocytes

Forskolin increased cyclic AMP accumulation in isolated adipocytes and markedly potentiated the elevation of cyclic AMP due to isoproterenol. In adipocyte membranes, forskolin stimulated adenylate cyclase activity at concentrations of 0.1 microM or greater. Forskolin did not affect the EC50 for activation of adenylate cyclase but did increase the maximal effect of isoproterenol. Neither the soluble nor particulate low-Km cyclic AMP phosphodiesterase activity was affected by forskolin. Low concentrations of forskolin (0.1-1.0 microM), which significantly elevated cyclic AMP levels, did not increase lipolysis, whereas similar increases in cyclic AMP levels due to isoproterenol elevated lipolysis. Forskolin did not inhibit the activation of triacylglycerol lipase by cyclic AMP-dependent protein kinase or the subsequent hydrolysis of triacylglycerol. Higher concentrations of forskolin (10-100 microM) did increase lipolysis. Both the increased cyclic AMP production and lipolysis due to forskolin were inhibited by the antilipolytic agents insulin and N6-(phenylisopropyl)adenosine. Hypothyroidism reduced the ability of forskolin to stimulate cyclic AMP production and lipolysis. These results indicate that forskolin increases cyclic AMP production in adipocytes through an activation of adenylate cyclase. Lipolysis is activated by forskolin but at higher concentrations of total cyclic AMP than for catecholamines.     

Changes in apomorphine-induced stereotypy as a result of subacute neuroleptic treatment correlates with increased D-2 receptors, but not with increases in D-1 receptors

Administration of haloperidol (5 mg/kg i.p.), cis-flupenthixol (2.5 mg/kg i.p.) or sulpiride (2 X 100 mg/kg i.p.) daily for 21 days followed by a 3-day drug withdrawal period caused equivalent cerebral dopamine receptor supersensitivity as judged by enhanced apomorphine-induced stereotypy. These treatments also produced equivalent rises in the number of adenylate cyclase-independent dopamine receptors (D-2) in both striatal and mesolimbic tissue as assessed by specific [3H]spiperone and [3H]N,n-propylnorapomorphine (NPA) binding. No change in the dissociation constant (KD) was apparent in response to neuroleptic treatment. However, only repeated administration of cis-flupenthixol caused an increase in the number of adenylate cyclase-linked dopamine receptors (D-1) in striatum as assessed by enhanced [3H]piflutixol binding and increased dopamine-stimulated cyclic AMP formation. The dissociation constant for [3H]piflutixol binding was unchanged by cis-flupenthixol administration. No change in D-1 receptor numbers or dopamine stimulation of adenylate cyclase occurred in mesolimbic tissue. Repeated treatment with sulpiride or haloperidol was without effect on either [3H]piflutixol binding to D-1 receptors or cyclic AMP formation. In conclusion, increased apomorphine-induced stereotypy following subacute neuroleptic treatment correlates with changes in D-2 receptor numbers, but not with changes in D-1 receptors.     

Effects of narcotic analgesic drugs on the cyclic adenosine 3',5'-monophosphate-adenylate cyclase system in rat brain.

1. The concentrations of cyclic adenosine 3',5'-monophosphate (cyclic AMP), measured in discrete brain areas removed from rats killed by microwave irradiation, rose transiently in most areas after the administration of morphine. The most pronounced changes, however, were found 2 h after doses of either 10 or 60 mg/kg morphine when cyclic AMP levels declined significantly in the hypothalamus, medulla and cerebellum. In morphine-tolerant rat brains there were no decreases in cyclic AMP levels. 2. Basal adenylate cyclase activity in crude nerve-ending fractions from discrete areas of rat brain was unaffected by the addition of active analgesic agonists, antagonists or inactive isomers to the assay medium in vitro, except for a nonspecific inhibition at drug concentrations of 1 mM. 3. The acute administration of morphine or levorphanol, but not dextrorphan produced transient increases in basal cyclase activity of crude nerve-ending preparations from midbrain and striatum. In morphine-tolerant rats, these changes in basal adenylate cyclase activity were no longer seen.     

Desensitization of H2-like histamine receptors stimulating cyclic AMP formation in the chick cerebral cortex

Histamine potently stimulates cyclic AMP formation in slices of the chick cerebral cortex. Pretreatment of the tissue slices with 10 microM histamine for 2-30 min led to a time-dependent attenuation (when compared with values observed in the control tissue) of the cyclic AMP response produced by subsequent re-stimulation with 1, 10 or 100 microM histamine. The observed histamine-induced desensitization appears to be specific and homologous as the increase in cyclic AMP formation evoked by both forskolin or pituitary adenylate cyclase-activating polypeptide (PACAP) in slices pretreated with 10 microM histamine for 15 min was unchanged. It is concluded that in the chick cerebral cortex, H2-like receptors linked to cyclic AMP-generating system undergo rapid homologous desensitization.     

Inhibition of 3',5'-nucleotide phosphodiesterase and the stimulation of cerebral cyclic AMP formation by biogenic amines in vitro and in vivo.

A number of compounds that have been shown to be potent inhibitors of phosphodiesterase in brain homogenates were examined for their ability to potentiate biogenic amine-stimulated cyclic AMP formation in mouse cerebral cortex slices and in chick cerebral hemispheres in vivo. Of the drugs examined at a concentration of 200 μM only 4-(3-butoxy-4-methoxy)-2-imidazolidinone (Ro20-1724) and 2-amino-6-methyl-5-oxo-4-n-propyl-4,5,-dihydro-s-triazolo(1,5-a)pyrimidine (ICI63197) significantly potentiated the cyclic AMP response to biogenic amines. Theophylline. papaverine and medazepam were ineffective both in vivo and in vitro. Histamine and dopamine were completely inactive in stimulating cyclic AMP formation in mouse cerebral cortex slices but in the presence of 200 μM Ro20-1724 or ICI63197, significant (2–3 fold) accumulations of the nucleotide were produced by both of these amines. Prostaglandin E₁ and 5-hydroxytryptamine did not enhance cyclic AMP formation either in the presence or absence of Ro20-1724. However, this phosphodiesterase inhibitor greatly potentiated the effect of adenosine. It is suggested that the discrepancy between the effects of drugs on phosphodiesterase in broken cell and whole cell preparations may be related to differential inhibition of different forms of phosphodiesterase in brain or to the relative lipophilic nature of the compounds examined.     

The use of cyclic AMP efflux studies in attempts to determine the effects of morphine on cyclic AMP formation in striatal slices

The effect of morphine and naloxone on basal and forskolin-stimulated efflux of cyclic AMP from rat striatal slices was examined. Neither morphine nor naloxone had any consistent effect on the basal efflux of cyclic AMP. Forskolin produced a time and dose-dependent enhancement of cyclic AMP efflux. Neither morphine nor naloxone affected this forskolin-enhanced release. These results suggest that measurements of cyclic AMP released from brain slices do not accurately reflect effects on adenylate cyclase inhibition by opiates.     

mu-Opioid receptor-regulated adenylate cyclase activity in primary cultures of rat striatal neurons upon chronic morphine exposure.

Rat striatal neurons cultured in serum-free, hormone-supplemented medium, were exposed to 10 microM morphine for several hours or days before intracellular cyclic AMP production was measured. Dopamine D1 receptor- and beta-adrenoceptor-stimulated cyclic AMP production were profoundly increased upon morphine exposure (up to 150% of control). In contrast, cyclic AMP production induced by direct activation of the catalytic unit of adenylate cyclase with forskolin remained unaffected. Interestingly, the relative inhibitory effect of the mu-opioid receptor agonist [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) on dopamine D1 receptor-stimulated cyclic AMP production was unchanged after exposure to morphine. On the other hand, unlike mu-opioid receptors chronically exposed to morphine, beta-adrenoceptors mediating activation of adenylate cyclase were rapidly desensitized upon prolonged exposure of the neurons to isoprenaline. It is suggested that tolerance to morphine may be caused by the fact that morphine is acting against up-regulated signal transduction mechanisms rather than by mu receptor desensitization. Moreover, this enhanced effector system function may be involved in opioid dependence. The adaptive changes following morphine treatment appear to be independent of possible alterations at the level of dopaminergic or noradrenergic nerve terminals which are not present in primary cultures of rat striatum.     

Mu and delta opiate receptors in neuronal and astroglial primary cultures from various regions of the brain--coupling with adenylate cyclase, localisation on the same neurones and association with dopamine (D1) receptor adenylate cyclase.

Primary cultures, enriched in neurones or astroglial cells, from three phylogenetically different regions of the brain of the rat, the cerebral cortex, the striatum and the brain stem, were used to investigate the presence of opiate receptors, coupled to adenylate cyclase. Morphine was used as a mu-receptor agonist and [D-Ala2, D-Leu5]-enkephalin (DADLE) was used as a delta-receptor agonist. In the neuronal cultures, both ligands inhibited the prostaglandin (PG)E1-stimulated intracellular accumulation of cyclic AMP dose-dependently, with the most prominent effects seen in the cultures of striatum and with DADLE being more potent than morphine. The opiate receptor antagonist, naloxone reversed the effects. Morphine and DADLE, added together, inhibited the PGE1-stimulated accumulation of cyclic AMP, less than the sum of the effects of each drug. Therefore, it might be that these opioid receptors are localized together on the same neurone. Striatal neurones contained dopamine receptors coupled to cyclic AMP, as second messenger. It was shown that the D1 (dopamine) receptor-stimulated activity of adenylate cyclase was inhibited by the mu and delta opioid receptor ligands. Thus, interactions at the level of adenylate cyclase seem to exist between D1, mu and delta opiate receptors. In the astroglial enriched cultures, DADLE inhibited the PGE1-induced accumulation of cyclic AMP, however, with a less prominent effect in the brain stem cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic adenosine 3',5'-monophosphate in rat cerebral cortical slices: effects of methoxamine and clonidine.

In incubated slices of cerebral cortex from Sprague-Dawley rats, methoxamine and clonidine have no effect on basal levels of cyclic AMP. Methoxamine effectively inhibits the noradrenaline-stimulated formation of cyclic AMP. The inhibitory constant for methoxamine was 12.6 mumol/l. In the presence of 100 mumol/l adenosine, methoxamine does not inhibit the activity of noradrenaline, but is capable to activate alpha-adrenergic receptors leading to enhanced formation of cyclic AMP. The mechanism by which adenosine alters adrenergic receptors to become methoxamine-sensitive is not known. Clonidine inhibits the effect of noradrenaline alone or in combination with adenosine on the cyclic-AMP-generating system. It does not, as reported earlier, enhance the activity of submaximal concentrations of the beta-adrenergic agonist isoproterenol. These data do not support the concept of adrenergic receptors which require both, alpha- and beta-stimulation for maximal activation of adenylate cyclase.