Activation of Cyclic AMP-Generating Systems in Brain Membranes and Slices by the Diterpene Forskolin: Augmentation of Receptor-Mediated Responses

The diterpene forskolin markedly activates adenylate cyclase in membranes from various rat brain regions and elicits marked accumulations of radioactive cyclic AMP in adenine-labeled slices from cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, hypothalamus, thalamus, and medulla-pons. In cerebral cortical slices, forskolin has half-maximal effects at 20-30 microM on cyclic AMP levels, both alone and in the presence of the phosphodiesterase inhibitor ZK 62771. The presence of a very low dose of forskolin (1 microM) can augment the response of brain cyclic AMP-generating systems to norepinephrine, isoproterenol, histamine, serotonin, dopamine, adenosine, prostaglandin E2, and vasoactive intestinal peptide. Forskolin does not augment responses to combinations of histamine-norepinephrine adenosine-norepinephrine, or histamine-adenosine. For norepinephrine and isoproterenol in rat cerebral cortical slices and for histamine in guinea pig cerebral cortical slices, the presence of 1 microM-forskolin augments the apparent efficacy of the amine, whereas for adenosine, prostaglandin E2, and vasoactive intestinal peptide, the major effect of 1 microM-forskolin is to increase the apparent potency of the stimulatory agent. In rat striatal slices, forskolin reveals a significant response of cyclic AMP systems to dopamine and augments the dopamine-elicited activation of adenylate cyclase in rat striatal membranes. The activation of cyclic AMP systems by forskolin is rapid and reversible, and appears to involve both direct activation of adenylate cyclase and facilitation and/or enhancement of receptor-mediated activation of the enzyme.     

Effects of Cyclic AMP Analogues and Phosphodiesterase Inhibitors on K+-Induced [3H]Noradrenaline Release from Rat Brain Slices and on Its Presynaptic α-Adrenergic Modulation

The possible role of cyclic AMP in the presynaptic alpha-adrenoceptor-mediated modulation of [3H]noradrenaline (NA) release induced by 13 mM K+ from superfused rat cerebral cortex slices was investigated. Both dibutyryl-cyclic AMP (db-cAMP) and 8-bromo-cyclic AMP (8-Br-cAMP) dose-dependently (10(-4) - 10(-2) M) enhanced K+-induced (3H]NA release, maximally to about 160% of control. In contrast, db-cAMP had no effect on calcium-induced [3H]NA release in the presence of the calcium ionophore A 23187. Surprisingly, the phosphodiesterase (PDE) inhibitors 3-isobutyl-1-methylxanthine (IBMX). 7-benzyl-IBMX, 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) appeared to inhibit K+-induced [3H]NA release in a dose-dependent (10(-5) - 10(-3) M) manner. At a concentration of 10(-4) M, AK 62771 caused an inhibition of [3H]NA release by 30%, and this inhibitory effect was not affected by 10(-6) M phentolamine nor by 10(-3) M db-cAMP or 10(-4) M theophylline. Theophylline by itself enhanced [3H]NA release to about 135% of control. The inhibitor effect of the alpha-adrenoceptor agonist oxymetazoline (1 micro M) and the enhancing effect of the antagonist phentolamine (1 micro M) on [3H]NA release were significantly decreased in the presence of 10(-3) M db-cAMP or 8-Br-cAMP, whereas 10(-4) M ZK 62771 had no effect. In the presence of 10(-2) M NaF, a potent activator of adenylate cyclase, the inhibitory effect of oxymetazoline (1 micro M) on [3H]NA release was significantly decreased. The data obtained with the cyclic AMP analogues support the hypothesis that activation of presynaptic alpha-receptors modulating NA release results in an inhibition of a presynaptic adenylate cyclase. Possible causes for the anomalous effects of th PDE inhibitors are discussed.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

The effect of repeated electroconvulsive shock treatment and chronic lithium feeding on the release of norepinephrine from rat cortical vesicular preparations

The effect of repeated electroconvulsive shock (ECS) treatment and chronic LiCl feeding on calcium-dependent, K+-evoked release of [3H] norepinephrine from rat cortical vesicular preparation was studied. There was no significant effect of either acute or repeated ECS treatment on [3H]norepinephrine release in cortical vesicles obtained from animals treated for either 1 or 10 days. Release of norepinephrine was examined over a range of CaCl2 concentrations. Clonidine effectively inhibited release of [3H]norepinephrine in cortical vesicles obtained from control and ECS-treated animals. K+-evoked release of [3H]norepinephrine at low (0.2 mM) and high (1.0 mM) CaCl2 concentrations was significantly increased in cortical vesicles obtained from LiCl-treated animals. Clonidine effectively inhibited release of [3H]norepinephrine in cortical vesicles obtained from both control and LiCl-fed animals. These results suggest a possible common mechanism of action of antidepressant drug therapy on presynaptic release of norepinephrine from nerve terminals.     

Role of Adenylate Cyclase in Presynaptic α2-Adrenoceptor- and μ-Opioid Receptor-Mediated Inhibition of [3H]Noradrenaline Release from Rat Brain Cortex Slices

Rat brain cortex slices, prelabelled with [3H]noradrenaline, were superfused and exposed to electrical biphasic block pulses (1 Hz; 12 mA, 4 ms) or to the Ca2+ ionophore A 23187 (10 microM) in the presence of 1.2 mM Ca2+. Forskolin (10 microM), 8-bromo-cyclic AMP (300 microM), and dibutyryl-cyclic AMP (300 microM) facilitated both the electrically evoked and A 23187-induced [3H]noradrenaline release, whereas the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX, 300 microM) and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771, 30 microM) enhanced the electrically evoked release only. The inhibitory effects of clonidine (1 nM-1 microM) and the facilitatory effect of phentolamine (0.01-10 microM) on the electrically evoked [3H]noradrenaline release were strongly reduced in the presence of 8-bromo-cyclic AMP. Clonidine (1 microM) reduced and phentolamine (3 microM) enhanced A 23187-induced [3H]noradrenaline release, provided that the slices were simultaneously exposed to forskolin. The inhibitory effects of morphine (1 microM) and [D-Ala2-D-Leu5]enkephalin (DADLE, 0.3 microM), like that of the Ca2+ antagonist Cd2+ (15 microM), on the electrically evoked release of [3H]noradrenaline were not affected by 8-bromo-cyclic AMP. Moreover, morphine and DADLE did not inhibit A 23187-induced release in the absence or presence of forskolin. These data strongly suggest that in contrast to presynaptic mu-opioid receptors, alpha 2-adrenoceptors on noradrenergic nerve terminals are negatively coupled to adenylate cyclase and may thus reduce neurotransmitter release by inhibiting the feed-forward action of cyclic AMP on the secretion process.     

Regulation of Depolarization-Dependent Release of Neurotransmitters by Adenosine: Cyclic AMP-Dependent Enhancement of Release from PC12 Cells

We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2-chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM-dependent phospholipid N-methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2-chloroadenosine did not alter the release of [3H]norepinephrine [(3H]NE) in the absence of depolarization. However, depolarization-dependent release of [3H]NE was markedly elevated by short (1-20 min) pretreatments with adenosine or 2-chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2-chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus-secretion coupling process.     

1-Isoamyl-3-isobutylxanthine: a remarkably potent agent for the potentiation of norepinephrine, histamine, and adenosine-elicited accumulations of cyclic AMP in brain slices.

1-Isoamyl-3-isobutylxanthine (EC₅₀ t 5 μM) potentiates by 2 to 6-fold the accumulations of cyclic AMP elicited in guinea pig cerebral cortical slices by norepinephrine, histamine, and adenosine. In addition, the xanthine derivative causes a 2 to 3-fold elevation of basal levels of cyclic AMP. 1-Isoamyl-3-isobutylxanthine has no effect on accumulations of cyclic AMP elicited by histamine or adenosine in the presence of a potent phosphodiesterase inhibitor, ZK 62771. The xanthine derivative retards the disappearance of cyclic AMP after a prior stimulation by adenosine. The results indicate that 1-isoamyl-3-isobutylxanthine is an extremely potent and effective inhibitor of phosphodiesterases involved in the regulation of cyclic AMP levels in guinea pig cerebral cortical slices. The 1-benzyl, 1-isoamyl, and 1-isobutyl derivatives of 3-isobutylxanthine potentiate the accumulation of cyclic AMP elicited by adenosine, while the 1-methyl derivative and 1-isoamyl-3-methylxanthine are inhibitory undoubtedly because of blockade of adenosine-receptors by these compounds. Xanthines with bulky 1- and 3- substituents appear to be relatively weak adenosine-antagonists and relatively specific and potent agents for inhibition of phosphodiesterases involved in cyclic AMP metabolism in brain tissue.     

Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.     

Modulation of γ-Aminobutyric Acid Release in Cerebral Cortex by Fluoride, Phorbol Ester, and Phosphodiesterase Inhibitors: Differential Sensitivity of Acetylcholine Release to Fluoride and K+ Channel Blockers

In this study we have used fluoride as a tool to investigate the involvement of G protein-coupled effector systems in the regulation of the depolarization-induced release of gamma-aminobutyric acid (GABA) from rat cerebral cortex. To distinguish among the activating effects of NaF on G proteins linked to different effectors, such as adenylate cyclase, polyphosphoinositide phospholipase C, and K+ channels, agents specific to these effectors have been used in parallel. NaF induced a marked dose-dependent facilitation of the K(+)-evoked release of [14C]GABA, with an EC50 of 1.26 mM, increasing release by 103% at 5 mM NaF. No effect on basal release was seen up to 3 mM NaF, and no modulation of [3H]acetylcholine (ACh) release was seen up to 5 mM NaF. Phorbol 12,13-diacetate (PDA) produced a similar dose-dependent facilitation of the K(+)-evoked release of [14C]GABA, potentiating the release of [14C]GABA by 50% at 10 microM PDA. The phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine (IBMX) and theophylline, inhibited the K(+)-evoked release of [14C]GABA, and IBMX reversed the NaF facilitation of GABA release in a dose-dependent manner (pA2 2.57). The K+ channel blocker (IA current) tetrahydroaminoacridine (THA), which markedly inhibits the K(+)-evoked release of [14C]GABA, also reversed the NaF facilitatory effect, but the release of [3H]ACh was less sensitive to the inhibitory effect of THA. On the other hand, the K+ channel blocker, tetraethylammonium, which has no effect on the release of [14C]GABA, caused a significant facilitation of K(+)-evoked release of [3H]ACh. From these studies, it is concluded that GABA release in cerebral cortex is subject to regulation by G protein-linked effector systems that are distinct from those affecting the release of [3H]ACh in cerebral cortex.     

Affinities of Histamine H1-Antagonists in Guinea Pig Brain: Similarity of Values Determined from [3H]Mepyramine Binding and from Inhibition of a Functional Response

Affinity constants for five antagonists at histamine H1-receptors in guinea pig brain have been determined from inhibition of the potentiation by histamine of the adenosine-induced accumulation of cyclic AMP in cerebral cortical slices. This action of histamine appeared to be mediated solely through H1-receptors. The affinity constants obtained were similar to those determined on peripheral H1-receptors and from inhibition of high-affinity [3H]mepyramine binding. This provides strong evidence that at least some of the [3H]mepyramine binding sites in guinea pig brain can be identified with functional H1-receptors.     

A MODULATING ROLE OF PITUITARY-ADRENAL AXIS IN CEREBRAL METABOLISM OF ADENOSINE 3'',5''-MONOPHOSPHATE

Abstract— The effect of adrenalectomy or hypophysectomy on the metabolism of adenosine 3',5'-monophosphate (cyclic AMP) in the cerebral cortex of male Wistar rats was investigated.
The bilateral removal of adrenal glands reduced significantly the activity of cerebral adenylate cyclase [EC 4.6.1.1]. whereas that of cyclic 3'.5'-nucleotide phosphodiesterase [EC 3.1.4.17] remained unchanged. The formation of cyclic AMP measured in cerebral cortical slices from adrenalectomized or hypophysectomized rats was also diminished. Decreases in the activity of adenylate cyclase and formation of cyclic AMP following adrenalectomy were antagonized by in vivo administration of dexamethasone or aldosterone, while those observed in hypophysectomized rats were restored by ACTH or dexamethasone. It is suggested that the pituitary adrenal axis has a modulating role in the metabolism of cerebral cyclic AMP, possibly by changing adenylate cyclase activity.     

Adenosine activating A(2A)-receptors coupled to adenylate cyclase/cyclic AMP pathway downregulates nicotinic autoreceptor function at the rat myenteric nerve terminals

In addition to the somatodendritic region, myenteric motoneuron terminals are endowed with nicotinic autoreceptors. We aimed at investigating the effect of nicotinic receptor (nAChR) activation on [3H]-acetylcholine ([3H]-ACh) release from longitudinal muscle-myenteric plexus of the rat ileum and to evaluate whether this could be modulated by adenosine, an endogenous neuromodulator typically operating changes in intracellular cyclic AMP. The nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 1-30 microM, 3 min) increased [3H]-ACh release in a concentration-dependent manner. DMPP (30 microM)-induced [3H]-ACh outflow was attenuated by hexamethonium (0.1-1 mM), tubocurarine (1-5 microM), or by removing external Ca2+ (plus EGTA, 1 mM). In contrast to veratridine (0.2-10 microM)-induced [3H]-ACh release, the DMPP (30 microM)-induced outflow was resistant to tetrodotoxin (1 microM) and cadmium (0.5 mM). Pretreatment with adenosine deaminase (0.5 U/mL) or with the adenosine A(2A)-receptor antagonist, ZM 241385 (50 nM), enhanced nAChR-induced transmitter release. Activation of A(2A) receptors with CGS 21680C (3 nM) reduced the DMPP-induced release of [3H]-ACh. CGS 21680C (3 nM) inhibition was prevented by MDL 12,330A (10 microM, an adenylate cyclase inhibitor) and by H-89 (10 microM, an inhibitor of protein kinase A), but was potentiated by rolipram (300 microM, a phosphodiesterase inhibitor). DMPP-induced transmitter release was decreased by 8-bromo-cyclic AMP (1 mM, a protein kinase A activator), rolipram (300 microM), and forskolin (3 microM, an activator of adenylate cyclase). Both MDL 12,330A (10 microM) and H-89 (10 microM) facilitated DMPP-induced release of [3H]-ACh. The results indicate that nAChR-induced [3H]-ACh release is triggered by the influx of Ca2+, independent of voltage-sensitive calcium channels, presumably directly through nAChRs located on myenteric axon terminals. It was also shown that endogenous adenosine, activating A(2A) receptors coupled to the adenylate cyclase/cyclic AMP transducing system, is tonically downregulating this nAChR-mediated control of [3H]-ACh release.     

Effects of forskolin and analogues on nicotinic receptor-mediated sodium flux,voltage-dependent calcium flux,and voltage-dependent rubidium efflux in pheochromocytoma PC12 cells

1. Forskolin, a naturally occurring diterpene that activates adenylate cyclase, HL706, a water-soluble derivative of forskolin (6 beta-[(piperidino)acetoxy]-7-desacetylforskolin) that is less potent than forskolin in activating adenylate cyclase, and 1,9-dideoxyforskolin, an analogue that does not activate adenylate cyclase, were examined for effects on the nicotinic receptor-mediated 22Na+ flux, a high potassium-induced 45Ca2+ flux through L-type calcium channels, and a high potassium-induced 86Rb+ efflux through a calcium-dependent potassium channels in PC12 cells. 2. Forskolin and analogues at 30 microM completely blocked carbamylcholine-elicited flux of 22Na+ through the nicotinic receptor-gated channel. 1,9-Dideoxyforskolin had an IC50 value of 1.6 microM with forskolin and HL706 being two- to three fold less potent. 3. Forskolin and its analogues appear to be noncompetitive blockers of the neuronal nicotinic receptor-channel complex in PC12 cells, but unlike many noncompetitive blockers, did not markedly enhance desensitization. Instead, forskolin, but not HL706 or 1,9-dideoxyforskolin, slightly antagonized the desensitization evoked by high concentrations of carbamylcholine. N-Ethylcarboxamidoadenosine, an adenosine analogue that elevates cyclic AMP and 8-bromo-cyclic AMP had no effect on desensitization. 4. Forskolin, HL706, and 1,9-dideoxyforskolin in the presence of carbamylcholine inhibited the binding of a noncompetitive blocker, [3H]perhydrohistrionicotoxin, to the muscle-type nicotinic receptor-channel complex in Torpedo electroplax membranes with IC50 values of 20 microM. Forskolin had no effect on [3H]perhydrohistrionicotoxin binding in the absence of carbamylcholine, while HL706 and 1,9-dideoxyforskolin still inhibited binding in the absence of carbamylcholine. 5. Forskolin, but not HL706 or 1,9-dideoxyforskolin had a slight inhibitory effect on the binding of [125I]alpha-bungarotoxin to acetylcholine recognition sites in Torpedo membranes. 1,9-Dideoxyforskolin at 30 microM, but not forskolin or HL706, markedly inhibited depolarization-evoked 45Ca+ flux and 86Rb+ efflux in PC12 cells, suggesting that 1,9-dideoxyforskolin has nonspecific inhibitory effects on a variety of ion channels.     

Regulation of Noradrenaline Release from Rat Occipital Cortex Tissue Chops by α2-Adrenergic Agonists

Noradrenaline (NA) and the alpha 2-adrenergic agonists clonidine, BHT-920, and UK 14304-18 inhibit potassium-evoked release of [3H]NA from rat occipital cortex tissue chops with similar potencies. NA (10(-5) M) was most effective as up to 85% inhibition could be observed compared with 75%, 55%, and 35% for UK 14304-18, clonidine, and BHT-920, respectively, all at 10(-5) M. Potassium-evoked release was enhanced by both forskolin (10(-5) M) and 1 mM dibutyryl cyclic AMP. Pretreatment of tissue chops with 1 mM dibutyryl cyclic AMP in the presence of 3-isobutyl-1-methylxanthine partially reversed the alpha 2-adrenergic agonist inhibition of NA release. No reversal of inhibition was observed following pretreatment with 10(-5) M forskolin. The effects of clonidine, BHT-920, UK-14308-18, and NA on cyclic AMP formation stimulated by (a) forskolin, (b) isoprenaline, (c) adenosine, (d) potassium, and (e) NA were examined. Only cAMP formation stimulated by NA was inhibited by these alpha 2-adrenergic agonists. These results suggest that only a small fraction of adenylate cyclase in rat occipital cortex is coupled to alpha 2-adrenergic receptors. These results are discussed in relation to recent findings that several alpha 2-adrenergic receptor subtypes occur, not all of which are coupled to the inhibition of adenylate cyclase, and that alpha 2-adrenergic receptors inhibit NA release in rat occipital cortex by a mechanism that does not involve decreasing cyclic AMP levels.     

Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain

—The accumulation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) was studied in cell-free homogenates of guinea pig brain. Homogenates, prepared in Krebs-Ringer buffer, responded markedly to the addition of neurohormones with an increased rate of cyclic AMP synthesis; preparations from cerebellum, cerebral cortex, and hippocampus responded to a degree approximating that achieved with slices of these areas of guinea pig brain. Adenylatc cyclase activity was seen only when cyclic AMP was measured by a [³H]adenine prelabelling technique or when total cyclic AMP was measured by radioimmunoassay; [³²P]ATP did not serve as a substrate for this preparation of the enzyme. The adenylate cyclase was paniculate and required a Krebs Ringer buffer; use of tris, or tris with Mg²⁺ and Ca²⁺, resulted in a preparation totally devoid of hormonal stimulation. Digestion by purified beef heart cyclic nucleotide phosphodiesterase, Dowex chromatography, solubility in Ba(OH)₂-ZnSO₄ mixtures, and two thin layer chromatographic systems demonstrated that the product of the hormonally stimulated adenylate cyclase preparation was cyclic AMP. The selectivity of hormonal stimulation and the adrenergic character of the hormonal receptors from different brain areas were maintained in the cell-free preparation. However, simultaneous stimulation with two different neurohormones resulted in additive responses, rather than in the potentiation observed in preparations of slices of brain.     

Regulation of nicotine-evoked dopamine release from PC12 cells.

The nature of second messengers involved in the nicotine-evoked release of dopamine from PC12 cells was examined. Calmidazolium, a calmodulin inhibitor, abolished the nicotine-evoked release. A23187, a Ca2+ ionophore, enhanced dopamine release, and this was inhibited by calmidazolium. Further, 2', 5'-dideoxyadenosine abolished both the nicotine- and A23187-evoked release. Forskolin, dibutyryl-cyclic AMP, and rolipram (a cyclic AMP phosphodiesterase inhibitor) all enhanced dopamine release. 1, 9-Dideoxyforskolin, a forskolin analog which does not activate adenylate cyclase, did not alter dopamine release. These results suggest an obligatory role for Ca2+ and calmodulin-sensitive adenylate cyclase in the nicotine-evoked release process.     

Action of the cardiac alpha 1-adrenergic receptor. Activation of cyclic AMP degradation

Using purified rat ventricular myocytes and membranes prepared from them, we have previously found that alpha 1-adrenergic stimulation causes decreased cyclic AMP accumulation and decreased activation of cyclic AMP-dependent protein kinase. We have now analyzed the mechanism by which alpha 1 stimulation is linked to cyclic AMP metabolism. In an adenylate cyclase assay in which carbachol inhibits the stimulatory effect of norepinephrine, the addition of prazosin (alpha 1-antagonist) has no effect on the response to norepinephrine. In membranes prepared from myocytes treated with pertussis toxin, norepinephrine competes for alpha 1-receptors (assessed by [3H]prazosin binding) with two components, binding to the high affinity component being sensitive to exogenous GTP, exactly as in membranes prepared from control myocytes. In intact cells labeled with [3H]adenine in which carbachol antagonizes the norepinephrine response, prazosin enhances accumulation of [3H]cyclic AMP due to norepinephrine. Treatment of cells with pertussis toxin eliminates inhibition by carbachol but does not alter prazosin's capacity to enhance the norepinephrine response. Addition of phosphodiesterase inhibitors eliminates this effect of alpha 1 blockade. In [3H]adenine-labeled cells loaded with [3H]cyclic AMP by prior treatment with isoproterenol, alpha 1-adrenergic stimulation enhances disappearance of [3H]cyclic AMP. Measurements of cellular cyclic AMP give results similar to those obtained with the adenine labeling technic. We conclude that occupation of the myocyte alpha 1-receptor results in stimulation of cyclic AMP phosphodiesterase activity.     

Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets.

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.     

Streptozotocin-induced diabetes produces alterations in adenylate cyclase in rat cerebrum, cerebral microvessels and retina

Eight weeks following streptozotocin-induced diabetes mellitus in rats, the sensitivity of adenylate cyclase to dopamine (DA) and norepinephrine (NE) was reduced in homogenates of retina. Furthermore, the activation of adenylate cyclase in cerebral microvessels (capillaries) by NE, 5'-guanylyl imidodiphosphate (alone or with NE) and forskolin was reduced in diabetic rats versus appropriate controls. In diabetic rats enzyme sensitivity to only NE was attenuated in homogenates of cerebral cortex and cortical piaarachnoid. No differences between controls and diabetics were noted with respect to guanylate cyclase or cyclic AMP phosphodiesterases. The damage observed in retina and microvessels may play an important pathogenic role in diabetes-induced blindness and stroke.     

Enhancement of depolarization-dependent neurosecretion from PC12 cells by forskolin-induced elevation of cyclic AMP

The effects of elevated intracellular cyclic AMP on the release of neurotransmitters was studied using the clonal pheochromocytoma cell line, PC12, and forskolin, a direct activator of adenylate cyclase. Intracellular cyclic AMP concentrations ranging from 8 to 400 times basal levels were achieved with 0.1 to 100 uM forskolin. Unstimulated release of neurotransmitters was unchanged by any concentration of forskolin. However, K+-stimulated release of both norepinephrine (NE) and acetylcholine was enhanced by 0.1 to 10 uM forskolin. Release of NE elicited by depolarization with carbachol and veratridine also was enhanced by 1 uM forskolin. Enhancement of release was reversed by higher concentrations of forskolin, especially in the presence of a phosphodiesterase inhibitor (RO 20-1724) which caused very large increases in cyclic AMP content. The enhancement of transmitter release from the PC12 cells occurred without concomitant changes in agonist-stimulated ion flux through the acetylcholine receptor ion channel, or in depolarization-dependent uptake of 45Ca++. Thus, increasing the cyclic AMP content of PC12 cells fails to initiate neurosecretion but appears to facilitate some element in the secretion process subsequent to Ca++ influx.