N-phenylisopropyladenosine stimulates in normal and inhibits in adrenalectomized rats the low KM cyclic AMP phosphodiesterase in the brain

In crude rat brain membranes, apparent adenylate cyclase activity tested in the absence of phosphodiesterase inhibitors was dose-dependently inhibited by the adenosine ‘R-site’ agonist N⁶-phenylisopropyladenosine (N⁶-PIA). In membranes from adrenalectomized rats, however, N⁶-PIA induced, under the same conditions, an activation. However, in the presence of the phosphodiesterase inhibitor Ro-20-1724, brain adenylate cyclase responsiveness to N⁶-PIA resulted in a dose-dependent inhibition in both sham-operated and adrenalectomized rats. Thus, the low K_M cyclic AMP phosphodiesterase activity present in these brain membranes was investigated. Although this activity (tested in the presence of GTP) was unaltered by adrenalectomy, the dose-response curve of this enzyme to low concentrations of N⁶-PIA showed an activation in sham-operated and an inhibition in adrenalectomized rats, two effects which were suppressed by sodium (80 mM). These results showing that N⁶-PIA modulates both adenylate cyclase and cyclic AMP phosphodiesterase in the brain, provide an additional argument for a potential role of adenosine in the regulation of cyclic AMP metabolism in normal as well as in pathological brain.     

Adenosine receptors in the mammalian central nervous system

1. 1. Adenosine by interaction with discrete extracellular recognition sites can modulate cyclic AMP formation and cell firing in the mammalian CNS.

2. 2. The effects of adenosine on cyclic AMP formation are mediated through two extracellular recognition sites: a high affinity (Kd = 10⁻⁹ M) site designated A-1, activation of which results in an inhibition of adenylate cyclase activity and a lower affinity site (Kd = 10⁻⁶ M) designated A-2, activation of which stimulates adenylate cyclase activity. Stable radiolabeled analogs of adenosine have been used to label A-1 receptors in mammalian brain.

3. 3. Adenosine and its stable analogs are potent inhibitors of neurotransmitter release.

4. 4. In addition to being phosphodiesterase inhibitors, the alkylxanthines are also adenosine antagonists, stimulating neurotransmitter release and increasing cell firing by antagonism of the effects of endogenous adenosine. These effects have been attributed to the presence of an inhibitory purinergic tone.

5. 5. Adenosine and related purines have been implicated in the mode of action of several centrally active drugs including anxiolytics, antidepressants and analgesics.

6. 6. Future progress in understanding the potential physiological role of adenosine in the mammalian CNS will depend on the availability of more potent and specific adenosine antagonists, ligands specific for the A-2 receptor, and a better understanding of the factors that regulate adenosine availability.

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.     

Effect of denervation on cyclic nucleotide metabolism in different types of skeletal muscle of the rat

Slow-twitch soleus and fast-twitch extensor digitorum longus muscles of the rat were denervated unilaterally by sciatic nerve section at mid-thigh level. Activities of adenylate cyclase, guanylate cyclase, low Km and high Km cyclic AMP phosphodiesterase, and cyclic GMP phosphodiesterase were compared on the same, freshly prepared homogenates of denervated and shamoperated contralateral muscles one, two, three, or five days after surgery. As an early consequence of denervation, cyclic AMP metabolism was differentially affected in these different types of skeletal muscle. The adenylate cyclase activity of soleus muscle increased significantly by the second day following denervation and continued to rise through the fifth day, while this enzyme did not increase in denervated extensor digitorum longus even by the fifth day. The high Km cyclic AMP phosphodiesterase was already increased by day one in the denervated soleus, but not until the fifth day in the denervated extensor digitorum longus. Parallel increases beginning the first day were observed for the low Km cyclic AMP phosphodiesterase in both muscles. Since the activity of cytosolic cyclic AMP-dependent protein kinase of soleus muscle was also increased two days following denervation, the changes in cyclic AMP synthetic and degradative enzymes apparently result in a rise in intracellular cyclic AMP concentration. Alterations of the cyclic GMP enzymes following denervation were similar in the soleus and extensor digitorum longus, but were delayed relative to the increases in activity in the cyclic AMP enzymes.     

Human retinal pigment epithelial cells in culture possess A2-adenosine receptors

Adenosine agonists cause a marked stimulation in cyclic AMP accumulation in whole human retinal pigment epithelial (RPE) cells in the presence of adenosine deaminase and papaverine, a phosphodiesterase inhibitor. N-Ethylcarboxamidoadenosine (NECA) stimulates cyclic AMP accumulation 16.1-fold above basal with an EC50 of 2.5 x 10(-7) M. It is also an effective (1.9-fold) stimulator of adenylate cyclase activity in RPE membrane preparations and a modest (1.22-fold) stimulator in the presence of forskolin in RPE cell membranes prepared from freshly isolated porcine RPE. N6-Cyclopentyladenosine (CPA) and N6-phenylisopropyladenosine (PIA) also increase cyclic AMP levels with EC50s of 4.9 x 10(6) M (8.9-fold above basal) and 3.5 x 10(-6) M (8.0-fold above basal) respectively. This potency order (NECA greater than PIA greater than CPA) is typical of A2-adenosine receptors. The relatively A1-selective agonists 10(-7) M indicating that RPE cells do not have A1-receptors which inhibit adenylate cyclase. Three adenosine receptor antagonists, BW-A1433U, 8-cyclopentyltheophylline and 8-sulfophenyltheophylline, blocked the NECA-induced stimulation of cyclic AMP accumulation with IC50s of 0.36 microM, 1.5 microM, and 75 microM respectively. Since alteration of cAMP levels has been demonstrated to affect several RPE functions, including cell migration, resorption of subretinal fluid, and phagocytosis, adenosine may play a significant regulatory role in RPE.     

Adenylate cyclase and the search for new compounds with the clinical profile of lithium

It is possible to evaluate the beta-adrenergic receptor-adenylate cyclase complex in the human periphery by measuring the plasma cyclic AMP rise after adrenergic agonists. A clinical trial of the beta 2 adrenergic agonist salbutamol in depression provided an opportunity to test whether adrenergic receptor subsensitivity does occur during clinical antidepressant treatment. After 1 and 3 weeks of oral salbutamol treatment, depression scores declined significantly in 11 depressed patients, while the plasma cyclic AMP response to i.v. salbutamol declined over 60%. The results support the concept that receptor sensitivity changes occur during human antidepressant therapy. Data are presented that Li, too, markedly reduces activity of beta-adrenergic adenylate cyclase in humans. The effect was evaluated by studying the effect of Li at therapeutic serum concentrations on the plasma cyclic AMP response to subcutaneous epinephrine. The Li effect is specific, since the plasma cyclic AMP response to glucagon is not inhibited. In rat cortical slices Li inhibition of noradrenaline-induced cyclic AMP accumulation is clearly demonstrable only at concentrations close to 2 mM Li. However, fresh human brain slices from edges of surgically-removed tumors show Li inhibition at 1 mM Li concentrations. These results imply that in brain as well as periphery, human noradrenergic adenylate cyclase is inhibited by therapeutic concentrations of Li. Demeclocyclin, a tetracycline-derived antibiotic, was found to inhibit noradrenaline-sensitive adenylate cyclase in rat cortical slices and to inhibit amphetamine-induced hyperactivity in rats in an open field. Clinical trials should search for new compounds with the clinical profile of Li.     

Muscarinic-dopaminergic synergism on retinal cyclic AMP formation

The cholinergic agonist, carbachol, produces a small increase in cyclic AMP concentration in the isolated rat retina, and markedly potentiates dopamine-stimulated cyclic AMP formation. This effect of carbachol is mediated through a muscarinic receptor, is calcium-independent, and is not due to inhibition of phosphodiesterase activity. Activation of miscarinic receptors may potentiate dopaminergic responses in the retina by enhancing coupling of the dopamine receptor to adenylate cyclase.     

Alterations of catecholamine-sensitive adenylate cyclase in gerbil cerebral cortex after bilateral ischemia

Alterations in central concentrations of cyclic nucleotides occur in mammalian brain during ischemic episodes. Our aim was to evaluate the catecholamine sensitivity of adenylate cyclase using homogenates of the gerbil cerebral cortex during periods of bilateral ischemia and recirculation. After 15-min ischemia without recirculation, the sensitivity of adenylate cyclase was usually enhanced to norepinephrine (NE) and dopamine (DA) alone or in the presence of guanosine triphosphate (GTP) but not 5′-guanylyl imidodiphosphate [Gpp(NH)p]. After 60-min ischemia (no recirculation), the sensitivity of the enzyme to NE and DA showed either no change from sham controls or a slight decrease. Responses, however, in the presence of GTP remained elevated. When the animals were subjected to 15-min ischemia followed by 15-min recirculation, enzyme activation by NE and DA with or without GTP and Gpp(NH)p was usually greater than controls. After 60-min ischemia plus 15-min recirculation, however, enzyme responsiveness to catecholamines and NaF was attenuated. No changes in either high and low K_m cyclic AMP phosphodiesterase or in guanylate cyclase were observed during ischemic episodes. The results indicate that the catecholamine-elicited activity of adenylate cyclase became elevated as a consequence of a short duration of ischemia. Longer periods of ischemia thought to be irreversible showed deficits in adenylate-cyclase sensitivity to catecholamines unless GTP was present. In the presence of GTP, enzyme sensitivity was restored as long as recirculation was prevented. Thus adverse consequences of recirculation on neuronal membranes or receptors could be evaluated by their state of GTP sensitivity.     

Evidence for FMRF-amide as a neurotransmitter in the gill of Aplysia californica

In Aplysia californica, multiple regulatory mechanisms are involved in the actions of neurotransmitters on the gill. Neurotransmitter receptors and adenylate cyclase were examined in a particulate fraction of gill homogenates. The neuropeptide FMRF-amide stimulated enzyme activity 7- to 8-fold (EC50, 1 microM) via receptors that were pharmacologically distinct from those for dopamine and serotonin. FMRF-amide augmented cyclic AMP levels in slices of gill tissue with a time course similar to that for adenylate cyclase activation. Increases in cyclic AMP levels produced by the neuropeptide were potentiated by the phosphodiesterase inhibitor theophylline. Physiological responses to neuropeptides and cyclic AMP analogues were examined in a perfused, isolated gill preparation. Phasic contractions evoked by FMRF-amide (EC50, 0.1 microM) were mimicked by membrane-permeable analogues of cyclic AMP. Comparison of FMRF-amide effects on adenylate cyclase and gill behavior suggests an association between cyclic AMP and phasic contractions. In addition, FMRF-amide-like immunoreactivity, detected by antisera raised against the neuropeptide, was found in nerve fibers innervating the gill. These findings indicate that in Aplysia, FMRF-amide or a closely related peptide neurotransmitter may be involved in the physiological regulation of gill behavior.     

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)     

Developmental changes in carbonic anhydrase and adenylate cyclase in quaking mice

The present investigation focuses on the developmental changes in the activity and levels of carbonic anhydrase, adenylate cyclase and 3′,5′-cyclic adenosine monophosphate (cAMP) in the Quaking mouse mutant in different regions of the brain.Carbonic anhydrase activity was found to be lower than normal in the forebrain of the C57/B Quaking mouse. The deficit in forebrain carbonic anhydrase was restricted to subcortical structures and was reflected equally in the membrane and soluble fractions, indicating that neither pool was affected selectively. However, no difference in carbonic anhydrase activity was observed in purified myelin from Quaking and control mice. Investigation of the changes in carbonic anhydrase activity as a function of age showed a cessation in enzyme accumulation in Quaking mice at around 20 days postnatally, suggesting an abnormality in cellular development. A tritiated acetazolamide binding assay was used to quantitate the amount of enzyme present. The amount of carbonic anhydrase parallelled enzyme activity, suggesting that the defect in the Quaking animals was at the level of the control of enzyme synthesis.Similar studies on cyclic AMP metabolism showed a higher than normal adenylate cyclase activity in the upper brain stem region of Quaking mice of ages between 19 and 40 days. Adenylate cyclase activity was stimulated by norepinephrine in both control and Quaking animals. The increased adenylate cyclase activity in the Quaking mice was in contrast to a lower cyclic AMP level and could not be accounted for by an alteration in phosphodiesterase activity.     

Alterations in Cyclic AMP Concentration and Adenylate Cyclase Activity in Specific Brain Areas of Rats with Inherited Hypothalamic Diabetes Insipidus (Brattleboro Rats)

The concentration of cyclic AMP (cAMP) and the activity of sodium-fluoride-stimulated adenylate cyclase was measured in 29 microdissected brain areas of homozygous Brattleboro rats and their Long-Evans control rats. In ten of the investigated brain areas a decreased cAMP level was measured in Brattleboro rats. It was particularly decreased in the supraoptic nucleus, cingulate and parietal cortex, hippocampus, habenula and organum vasculosum laminae terminalis. Significantly lower cAMP levels were also found in the periventricular nucleus, bed nucleus of the stria terminalis, area postrema and locus coeruleus. An increased cAMP concentration was detected only in the subcommissural organ of Brattleboro rats. In most brain areas, where cAMP was decreased, sodium fluoride-stimulated adenylate cyclase activity was significantly increased (supraoptic nucleus, parietal cortex, periventricular nucleus, bed nucleus of the stria terminalis, locus coeruleus) or unchanged (hippocampus, habenula, organum vasculosum laminae terminalis). The coincidence of alterations in cAMP concentration and adenylate cyclase activity in brain areas of Brattleboro rats with relatively dense vasopressinergic innervation and/or vasopressin receptor population in control rats, suggests an influence of brain vasopressin on the cAMP-adenylate cyclase second messenger system.     

A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function.

Previous studies have shown that chronic morphine increases levels of the G-protein subunits Gia and Goa, adenylate cyclase, cyclic AMP-dependent protein kinase, and certain phosphoproteins in the rat locus coeruleus, but not in several other brain regions studied, and that chronic morphine decreases levels of Gia and increases levels of adenylate cyclase in dorsal root ganglion/spinal cord (DRG-SC) co-cultures. These findings led us to survey the effects of chronic morphine on the G-protein/cyclic AMP system in a large number of brain regions to determine how widespread such regulation might be. We found that while most regions showed no regulation in response to chronic morphine, nucleus accumbens (NAc) and amygdala did show increases in adenylate cyclase and cyclic AMP-dependent protein kinase activity, and thalamus showed an increase in cyclic AMP-dependent protein kinase activity only. An increase in cyclic AMP-dependent protein kinase activity was also observed in DRG-SC co-cultures. Morphine regulation of G-proteins was variable, with decreased levels of Gia seen in the NAc, increased levels of Gia and Goa in amygdala, and no change in thalamus or the other brain regions studied. Interestingly, chronic treatment of rats with cocaine, but not with several non-abused drugs, produced similar changes compared to morphine in G-proteins, adenylate cyclase, and cyclic AMP-dependent protein kinase in the NAc, but not in the other brain regions studied. These results indicate that regulation of the G-protein/cyclic AMP system represents a mechanism by which a number of opiate-sensitive neurons adapt to chronic morphine and thereby develop aspects of opiate tolerance and/or dependence. The findings that chronic morphine and cocaine produce similar adaptations in the NAc, a brain region important for the reinforcing actions of many types of abused substances, suggest further that common mechanisms may underlie psychological aspects of drug addiction mediated by this brain region.     

Pertussis toxin lesioning of the nucleus caudate-putamen attenuates adenylate cyclase inhibition and alters neuronal electrophysiological activity

Receptor-mediated inhibition of brain adenylate cyclase activity has been well characterized at the biochemical level. However, less understood is how these, typically modest, inhibitory effects on cyclase activity correlate with the electrophysiological activity of brain preparations. In addressing this question, we injected pertussis toxin (PT) into the nucleus caudate-putamen of intact rats, and observed a subsequent attenuated inhibition of adenylate cyclase activity in caudate membranes, which correlated with altered electrophysiological activity in this nucleus. PT completely abolished and electrophysiological activity in this nucleus. PT completely abolished and significantly reduced, respectively, dopamine D2 and opioid receptor-mediated inhibition of adenylate cyclase. In addition, pretreatment of rat caudate nuclei with PT attenuated the amount of in vitro ADP-ribosylation of 41,000 and 39,000 Da PT substrates measured in caudate membranes. Extracellular recording of the spontaneous activity of caudate neurons revealed that PT pretreatment significantly increased firing rates above those of cells recorded from sham-operated and unoperated controls. Furthermore, a significantly greater number of cells pretreated with PT displayed interspike intervals less than 50 ms, reflecting 'burst-like' activity. In short, the inactivation of G-proteins serving as PT substrates in rat caudate-putamen renders caudate cells more likely to fire spontaneously, and to fire in bursting, rather than uniform, patterns of activity. These observations suggest that PT substrates identical or similar to those which regulate adenylate cyclase, play a significant role in governing the electrophysiological behavior of intact caudate neurons.     

Lateral difference in responsiveness of norepinephrine-sensitive cyclic AMP-generating systems of rat cerebral cortex with iron-induced epileptic activity

Responsiveness of norepinephrine-sensitive cyclic AMP-generating systems was examined in slices of different cortical areas of rats showing electrographic spike and wave complexes after unilateral injection of ferrous chloride solution into the sensorimotor cortex. Accumulation of cyclic AMP elicited by norepinephrine was greater on the injection side of the cortex than on the other. Similar lateral differences were detected in cyclic AMP levels antagonized by phentolamine or propranolol, in which 8-phenyltheophylline almost completely inhibited the elicitation of cyclic AMP accumulation by a norepinephrine-propranolol combination but not by a norepinephrine-phentolamine combination. These results suggest that alterations in cyclic AMP generation through the beta-adrenoceptor-coupled adenylate cyclase system and through alpha-adrenergic activation of the adenosine receptor-coupled adenylate cyclase system are closely related to the electrographic activity of iron-induced epilepsy.     

Effects of Ca2+ and calmodulin on cyclic nucleotide metabolism in neurosecretosomes isolated from ox neurohypophyses

In neurosecretosomes, isolated from ox neurohypophyses, both guanylate and adenylate cyclase activity was shown to be predominantly membrane-bound. Membrane-bound adenylate cyclase was inhibited by increasing the ionized calcium concentration from 10(-7) M to 10(-5) M, but was stimulated by calmodulin in the presence of 10(-7) M and 10(-5) M ionized calcium. In contrast, neither calcium ions nor calmodulin affected the activity of membrane-bound guanylate cyclase. Soluble cyclic AMP and cyclic GMP phosphodiesterase activities increased with increasing ionized calcium concentration (10(-7) M to 10(-3) M). At 10(-7) M ionized calcium concentration, both soluble phosphodiesterase activities were stimulated by calmodulin. Both the membrane-bound phosphodiesterase activities were inhibited by a high ionized calcium concentration (10(-3) M) and not affected by calmodulin.     

Regional cyclic AMP systems during secondary ischemia in gerbils: Influence of anesthetic agents

The effect of three modes of anesthesia was evaluated with regard to regional damage to central cyclic nucleotide systems in the gerbil brain as a consequence of bilateral ischemia (clamping the common carotids) followed by various periods of recirculation. The injection of thiopental as much as 90 min before stroke prevented damage to chemical activation [catecholamines, guanosine triphosphate (GTP), or forskolin] of adenylate cyclase. However, the basal enzyme activity was lower in all brain regions whether thiopental was administered to stroke or sham-operated animals. Injection of ketamine drastically shortened the survival times of gerbils undergoing stroke followed by recirculation. About 90% of the animals could tolerate a maximum of only 15 min stroke with 15 min recirculation. At this time frame the patterns of activation of adenylate cyclase in only the olfactory tubercle and hippocampus were altered. When procaine was used as a local anesthetic agent during surgery, damage to catecholamine-, GTP-, or forskolin-activated adenylate cyclase was evident to varying degrees in the frontal cortex, hippocampus or olfactory tubercle, but not in the nucleus accumbens and olfactory bulb of gerbils subjected to 60-min stroke followed by 15 or 150 min of recirculation. The degree of enzyme damage was neither correlated with the fed vs. fasted state of the animal nor with the whole blood concentration of glucose. A depression in the amplitude of visually evoked potentials correlated to neurological signs and to enzyme damage. During anesthesia, ketamine increased steady-state concentrations of cyclic AMP in the frontal cortex and hippocampus from gerbil brains that had been rapidly inactivated by microwave irradiation. Thiopental increased steady-state cyclic AMP in only the olfactory tubercle. Cyclic GMP concentrations were unchanged by any anesthetic agent. In animals completely recovering from anesthesia and occluded for a brief period followed by 10 min of reflow, steady-state concentrations of only cyclic AMP were augmented.     

Somatostatin pretreatment desensitizes somatostatin receptors linked to adenylate cyclase and facilitates the stimulation of cyclic adenosine 3':5'-monophosphate accumulation in anterior pituitary tumor cells

Somatostatin-14 (SRIF) inhibits both hormone- and forskolin-stimulated cyclic adenosine 3':5'-monophosphate (cyclic AMP) formation in tumor cells of the mouse anterior pituitary (AtT-20/D16-16). However, long-term pretreatment of cells with SRIF modifies the responsiveness of this system in two ways: The response of adenylate cyclase to stimulatory agents is enhanced, whereas the ability of SRIF to inhibit stimulated cyclic AMP formation is reduced. The supersensitive adenylate cyclase response and the SRIF desensitization were dependent on the concentration and duration of SRIF pretreatment. Enhancement of forskolin-stimulated cyclic AMP formation occurred within 4 hr, whereas that of corticotropin-releasing-factor-, (-)-isoproterenol-, and vasoactive intestinal peptide-induced cyclic AMP accumulation required 16 hr of pretreatment. The elevated responses to each of these stimulants were due to increases in their maximal ability to stimulate cyclic AMP formation. Cycloheximide treatment blocked the enhanced cyclic AMP response induced by SRIF pretreatment, suggesting a requirement for protein synthesis. In membrane preparations, SRIF pretreatment facilitated activation of adenylate cyclase by forskolin, sodium fluoride, and guanosine 5'-(beta,tau-imido)-triphosphate without affecting basal activity. These results suggest that desensitization of an inhibitory input to adenylate cyclase is accompanied by a supersensitivity of adenylate cyclase to stimulatory agents through a process requiring protein synthesis.     

The role of cAMP in regulation of electrical activity of the neuroendocrine caudodorsal cells of Lymnaea stagnalis

The neuroendocrine caudodorsal cells (CDCs) of the pond snail Lymnaea stagnalis release a number of peptides, including the ovulation hormone, caudodorsal cell hormone (CDCH), during a period of high electrical activity (the CDC-discharge). Earlier studies have shown that during the CDC-discharge adenylate cyclase activity is increased, and that the cyclic adenosine monophosphate (cAMP) analogue 8-chlorophenylthio (8-CPT)-cAMP induces exocytosis and release of peptides from the CDCs. Here, we have investigated the role of cAMP, adenylate cyclase and phosphodiesterase in determining the state of excitability of the CDCs. The cAMP analogue 8-CPT-cAMP induced long-lasting discharges in CDCs. Simultaneous inhibition of the phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX) and activation of the adenylate cyclase by forskolin gave similar results. These agents also induced discharges of CDCs in dissociated cell culture, indicating that the responses to an increase of cAMP were an endogenous property of the cells. The CDC-afterdischarge can be induced by a period of repetitive electrical stimulation. Inhibition of the phosphodiesterase-activity by IBMX did not change the resting membrane potential, but increased the proportion of preparations that responded to this stimulation with an afterdischarge by more than 200%. It is suggested that cAMP-regulating enzymes are involved in stimulus-response coupling of the afterdischarge in CDCs. The induction of an after discharge probably requires both a low phosphodiesterase-activity and the activation of the adenylate cyclase. The low excitability of the CDCs following an afterdischarge might originate from a refractoriness in the activation of the adenylate cyclase.     

Receptor-linked cyclic amp systems in rat neostriatum: Differential localization revealed by kainic acid injection

Various receptor-linked cyclic AMP systems were measured in rat neostriatum 2--14 days after selective destruction of neuronal cell bodies and dendrites by micro-injection of 3 microgram of kainic acid. Basal adenylate cyclase activity was reduced by up to 56% in the injected side and the sensitivity to dopamine was abolished. Up to 84% of cyclic nucleotide phosphodiesterase activity, hydrolyzing either cyclic AMP or cyclic GMP, was destroyed by kainic acid injection. Specific binding of [3H]etorphine and [3H]spiroperidol was reduced by up to 62% in the injected side, while non-specific binding was unchanged. All of these changes were time-dependent, and were greatest 7--14 days after kainic acid treatment. On the other hand, intrastriatal kainic acid injection caused no change in the steady-state concentration of cyclic AMP in striatal slices, or in the in vivo cyclic AMP content in the striatum of rats killed by microwave irradiation. Receptor-mediated increases in cyclic AMP accumulation in striatal slices were either unchanged or markedly potentiated by kainic acid treatment. The maximum response to adenosine was unchanged, while the response to isoprenaline was increased up to 3.7-fold, the response to dopamine increased up to 6.7-fold, and the response to PGE1 increased up to 30-fold. The effect of dopamine in kainic acid-treated striatal slices was no longer blocked by fluphenazine, but was blocked by propranolol, suggesting an interaction of dopamine with a beta-adrenoceptor in kainic acid-treated slices. The results suggest differential cellular localizations of the various receptor-linked cyclic AMP systems in rat neostriatum. Some dopamine and opiate receptors, as well as most of the phosphodiesterase activity, are associated with local neuronal elements, while beta-adrenoceptor, adenosine and PGE1 alterations in cyclic AMP are not. The potentiation of the beta-adrenoceptor and PGE1 responses suggests that they may occur in glial cells. In addition, the pool of adenylate cyclase destroyed by kainic acid appears to make little contribution to normal levels of cyclic AMP in the tissue.