Lithium effects on noradrenergic-linked adenylate cyclase activity in intact rat brain: an in vivo microdialysis study.

The effects of chronic lithium treatment on adenylate cyclase activity in intact rat brain were examined using in vivo microdialysis. Basal extracellular cyclic adenosine monophosphate (AMP) increased in a dose-dependent manner after norepinephrine was added to the perfusate. Chronic lithium treatment increased basal brain extracellular fluid cyclic AMP levels, while decreasing the magnitude of the cyclic AMP response to stimulation with 100 microM norepinephrine.     

Cyclic AMP-generating systems: regional differences in activation by adrenergic receptors in rat brain

Catecholamine, histamine, and adenosine-mediated accumulations of radioactive cyclic AMP were assessed in adenine-labeled slices from eight rat brain regions. 2-Fluoronorepinephrine, a selective beta-adrenergic agonist, elicited an an accumulation of cyclic AMP in cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, thalamus, hypothalamus, and medulla-pons. In cerebral cortex and most other brain regions, the beta-adrenergic-mediated response appeared to involve primarily beta 1-adrenergic receptors, while in cerebellum, there was a significant involvement of beta 2-adrenergic receptors. 6-Fluoronorepinephrine, a selective alpha-adrenergic agonist, elicited accumulations of cyclic AMP in all regions except cerebellum. Combinations of the two fluoro derivatives afforded in all brain regions an accumulation of cyclic AMP identical with that elicited by norepinephrine. In hypothalamus, the alpha- and beta-adrenergic responses were significantly greater than additive. In cerebral cortex, the alpha-adrenergic receptor-mediated response appeared to involve alpha 1-adrenergic receptors and to be nearly completely dependent on adenosine, while in other brain regions, the dependence of the alpha-adrenergic response on adenosine was less or absent. Combinations of 6-fluoronorepinephrine and histamine had greater than additive effects in cortex and hippocampus. The results indicate that the interactive control of cyclic AMP-generating systems by alpha-adrenergic, beta-adrenergic, adenosine, and histamine receptors differs significantly among rat brain regions.     

Altered responsiveness of adenosine 3′,5′-monophosphate-generating systems in rat cortical slices after lesions of the medial forebrain bundle

The magnitude of the accumulation of radioactive cyclic AMP elicited by norepinephrine in [¹⁴C]adenine-labeled slices of rat cortical slices was enhanced 1.4-fold in ipsilateral slices from animals with unilateral lesions of the medial forebrain bundle as compared to slices from the contralateral hemisphere. This enhanced responsiveness to norepinephrine was first manifested 2 days after the lesion, and persisted for at least 3 weeks. The level of norepinephrine in cortical tissue from the hemisphere with lesion underwent a concomitant decline to less than 20% of the level in tissue from the contralateral hemisphere. Responses of cyclic AMP-generating systems to isoproterenol and to a combination of histamine and a phosphodiesterase inhibitor were also significantly enhanced in ipsilateral cortical slices after lesions of the medial forebrain bundle, while responses to a combination of norepinephrine and the β-adrenergic antagonist, propranolol, to adenosine, veratridine, prostaglandin E₁, and a combination of serotonin and a phosphodiesterase inhibitor were not significantly altered. The response to histamine, in the presence of a phosphodiesterase inhibitor (isobutylmethylxanthine), was blocked effectively only by an H₂-antagonist, metiamide. Responses to norepinephrine after lesions of the medial forebrain bundle were enhanced in ipsilateral hippocampal slices, but not in ipsilateral slices from midbrain or cerebellum.     

Cyclic AMP-mediated potentiation of muscarinic hyperpolarization in neuroblastoma cells

Adenosine, 2-chloroadenosine and prostaglandin E₁ which are known to increase cyclic AMP in neuroblastoma cells potentiated the acetylcholine-induced muscarinic hyperpolarization of the cells without changing the resting membrane potential. The potentiation caused by 2-chloroadenosine was further augmented by Ro 20-1724, a phosphodiesterase inhibitor. A direct intracellular pressure application of cyclic AMP potentiated the muscarinic hyperpolarization without changing the resting membrane potential. Morphine which inhibits adenylate cyclase antagonized 2-chloroadenosine-induced potentiation of the muscarinic hyperpolarization. These results suggest that changes in cyclic AMP level modulate the muscarinic response of neuroblastoma cells.     

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.     

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.     

Kinetics of GTP-modulation of adenylate cyclase in gerbil cerebral cortex after bilateral ischemia

Changes in the sensitivity of adenylate cyclase and steady-state levels of cyclic AMP (adenosine 3',5'-monophosphate) occur in mammalian brain during ischemic episodes. In our previous investigation with the gerbil model of bilateral ischemia there was an indication that ischemic conditions produced an enhancement of GTP sensitivity of adenylate cyclase within the cerebral cortex. The present study employed a kinetic analysis to evaluate further the role of this GTP modulation of adenylate cyclase in the gerbil frontal cortex during periods of bilateral ischemia and recirculation. In general, after either 15-min (with or without 15-min reflow) or 60-min ischemia the Vmax to GTP (alone or with dopamine and norepinephrine) was increased. Under these conditions the ED50 for half-maximal enzyme activation was decreased, indicating a greater affinity of the transducer site for GTP during ischemia. However, if irreversible 60-min ischemia was followed by 15-min reflow the enzyme responses to GTP were now absent. An unexpected observation showed that the ED50 for GTP activation of cortical adenylate cyclase was likewise attenuated when sham-operated animals were compared to normal gerbils.     

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.     

Alteration in adenylate cyclase response to aminergic stimulation following neonatal x-irradiation

X-irradiation of the rat neonatal hippocampus produces severe alterations in the architectonic features of the mature hippocampus. The most prominent alterations is a marked depletion of the granule cells of the dentate gyrus, with a subsequent realignment of CA 4 cells. The present data also show that norepinephrine (NE), dopamine and histamine stimulation of adenylate cyclase activity is severely attenuated in the hippocampi of irradiated animals. This failure suggests that the NE fibers of irradiated subjects, although normal in content of NE, are not functional in some of their NE-effector actions.     

Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3':5'-monophosphate-generating systems, receptors, and enzymes

A particulate preparation was obtained by low speed centrifugation of guinea pig cerebral cortical homogenates prepared with a Krebs-Henseleit buffer. Light microscopic examination, using a reflected light differential interference contrast system, reveals the presence of intact neurons, axonal fragments, glial cells, and erythrocytes along with an abundance of small spherical entities (diameter about 1.1 micron) and snowman-shaped entities (diameter of larger sphere about 1.1 micron, diameter of attached smaller sphere about 0.6 micron). Many unattached smaller spherical entities are also present (diameter about 0.6 micron). Pressure filtration through 5- or 10-micron Millipore filters, followed by low speed centrifugation and resuspension, removes most of the larger entities to afford a suspension composed mainly of the small spherical and snowman-shaped entities. Electron microscopic examination reveals the presence of many synaptosomes with attached resealed postsynaptic entities. It is proposed that these correspond to the snowman-shaped entities to be termed synaptoneurosomes. Accumulations of cyclic AMP elicited by 2-chloroadenosine and histamine, and by combinations of 2-chloroadenosine, histamine, norepinephrine, and forskolin, are lower in filtered than in unfiltered preparations, whereas accumulations elicited by forskolin are unchanged. Levels of adenylate cyclase are reduced by filtration, whereas levels of phosphodiesterase are unchanged. Filtration reduces levels of markers for whole cells and endothelial cells, whereas neuronal markers such as acetylcholinesterase activity and norepinephrine uptake are increased. Levels of S-100 protein, a marker for glial cells, are not significantly decreased. There is no apparent change in the density of many receptors or ion channels. Levels of A1-adenosine and H1-histamine receptors are increased, whereas levels of so-called peripheral benzodiazepine-binding sites are decreased.     

Chronic effects of a monoamine oxidase-inhibiting antidepressant: decreases in functional alpha-adrenergic autoreceptors precede the decrease in norepinephrine-stimulated cyclic adenosine 3': 5'-monophosphate systems in rat brain

Various antidepressant drugs (monoamine oxidase inhibitors and tricyclics) enhance norepinephrine availability and lead to adaptive changes in brain noradrenergic systems, namely, decreases in the number of beta receptors and in the responsiveness of adenylate cyclase to norepinephrine monamine oxidase inhibitor, but not after 3 days, there is an increase in norepinephrine release from rat brain microsacs in response to 43 mM KCl stimulation. Microsacs prepared from 21-day clorgyline-treated animals also show a marked decrease in the inhibition of norepinephrine release caused by the alpha 2-selective agonist clonidine. These functional changes in norepinephrine release mechanisms are accompanied by a 53% reduction in brainstem alpha 2 receptor density as measured by [3H]clonidine binding. At the same time, despite findings of a decrease in beta receptor number as determined by [3H]dihydroalprenolol binding data, no significant decrease in the responses of cyclic adenosine 3': 5'-monophosphate (cyclic AMP) systems to norepinephrine stimulation is observed. Decreases in the cyclic AMP response are observed by day 35 of clorgyline treatment. The results provide direct physiological support for a change in the norepinephrine release mechanism and an effect on autoreceptors, specifically, preceding postsynaptic adaptive changes in the instance of one antidepressant, clorgyline. Difficulties in observing such changes with other antidepressants may result from the multiple nature of alpha-adrenergic receptors, especially as measured by radioactive ligand techniques; the lack of a direct relationship between physiological changes and receptors as measured by radioligand techniques; the large doses of monoamine oxidase inhibitors used in some studies; and the possible multiplicity of antidepressant molecular mechanisms.     

Histamine-activated adenylate cyclase in brain homogenates of several species

Histamine has been shown to activate cyclic AMP synthesis in brain slices and homogenates of certain species, although less is known about species differences in brain homogenates. Dutch Belted and New Zealand White rabbit brain homogenates contained a histamine-responsive adenylate cyclase similar to that of the guinea pig. In contrast, adenylate cyclase of gerbil and hamster brain exhibited little or no stimulation by histamine. Male rat hypothalamic homogenates contained adenylate cyclase, but also exhibited minimal stimulation by histamine, in disagreement with some recent reports. Detailed studies of the conditions of assay failed to resolve this discrepancy.     

Effects of naloxone and morphine on cerebral ischemia in gerbils

A therapeutic role for naloxone during stroke has been suggested, but a neurochemical site of action remains to be determined. Previous work with the gerbil cerebral cortex has shown that either bilateral secondary ischemia (60-min occlusion of the carotid arteries followed by 40-min reflow) or unilateral primary ischemia (permanent ligation of one carotid artery for 6 hr in symptomatic animals) produced deficits in both Na+, K+-ATPase (EC 3.6.1.3) activity and various parameters of activation of adenylate cyclase (EC 4.6.1.1). Pretreatment of gerbils with either naloxone or morphine failed to ameliorate or exacerbate, respectively, the neurological signs of ischemia; however, morphine did reduce mortality. Infusion of naloxone prior to ischemia afforded varying degrees of protection to forskolin, GTP analogs, and NE (norepinephrine) activation of adenylate cyclase, as well as to Na+, K+-ATPase (bilateral ischemia only). Similarly, morphine inhibited damage to basal activity of adenylate cyclase and to stimulation by NE, forskolin, and Gpp (NH)p (5'-guanylyl imidodiphosphate). Under in vitro conditions morphine increased the basal activity of adenylate cyclase but reduced responses to NE and forskolin. Furthermore, morphine injected into control gerbils elevated basal- and forskolin-elicited activities but reduced the activation of adenylate cyclase by NE.     

Isolated neuronal growth cones from developing rat forebrain possess adenylate cyclase activity which can be augmented by various receptor agonists

Isolated neuronal growth cones from neonatal rat forebrain were found to contain a high specific activity of adenylate cyclase (61 pmol cyclic AMP/min/mg protein) compared to the pelleted starting homogenate (5 pmol cyclic AMP/min/mg protein). Forskolin at 10(-4) M increased adenylate cyclase activity in both the pelleted homogenate and growth cone fraction by 70 and 217 pmol cyclic AMP/min/mg protein, respectively, over basal levels. The incremental effect of forskolin was 3-fold greater in the growth cone fraction than in the pelleted homogenate. However, relative to basal levels in each of the two fractions, forskolin increased adenylate cyclase activity in the growth cone fraction by only approx. 5-fold compared to 15-fold in the pelleted homogenate. Dopamine (10(-4) M), vasoactive intestinal polypeptide (10(-6) M) and isoproterenol (10(-5) M) also augmented adenylate cyclase activity in the two fractions. In the growth cone fraction, dopamine and vasoactive intestinal polypeptide produced a stimulation over basal levels by approx. 20 pmol cyclic AMP/min/mg protein while isoproterenol produced a stimulation of approx. 10 pmol cAMP/min/mg protein. The incremental effects of these receptor agonists in the growth cone fraction are approx. 5-fold greater than in the pelleted homogenate. The dopamine-sensitive adenylate cyclase activity in the growth cone fraction could be blocked by the compound SCH23390, a selective D1 receptor antagonist. At saturating concentrations, all combinations of dopamine, vasoactive intestinal polypeptide and isoproterenol were found to be completely additive on adenylate cyclase activity in the growth cone fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Norepinephrine depletion and responsiveness of norepinephrine-sensitive cyclic AMP generating systems in guinea pig brain.

The accumulation of radioactive cyclic AMP was examined in [¹⁴C]adeninelabeled slices of guinea pig neocortex following in vivo treatments designed to lower the levels of putative neurotransmitters such as norepinephrine, serotonin, and histamine. Lesions of the medial forebrain bundle and treatment with 6-hydroxydopamine or reserpine decreased cortical concentrations of norepinephrine in guinea pig. Based on studies in rats, enhanced responses of norepinephrine-sensitive cortical cyclic AMP-generating systems were to be expected after such treatments. Instead, no evidence for enhanced responses of cyclic AMP-generating systems to norepinephrine, serotonin, or histamine was obtained with cortical slices from the treated guinea pigs. Adenosine-sensitive cyclic AMP-generating systems in guinea pig cortical slices did become hyperresponsive after reserpine.     

Identified octopaminergic neurons modulate contractions of locust visceral muscle via adenosine 3′,5′-monophosphate (cyclic AMP)

D,L-Octopamine elevates the cyclic AMP content of the lateral oviduct of the locust, Locusta migratoria, in a dose-dependent manner with a threshold of about 10(-8) M. The effect of octopamine is potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The response is specific for octopamine and synephrine with an order of potency being octopamine = synephrine greater than metanephrine greater than tyramine greater than norepinephrine = dopamine = 5-hydroxytryptamine and the effect of octopamine is inhibited by the alpha-adrenergic receptor antagonist phentolamine. The diterpene adenylate cyclase activator forskolin also elevates cyclic AMP levels and IBMX potentiates the action of forskolin. Stimulation of the two identified octopaminergic neurons which project to the lateral oviducts results in an elevation in cyclic AMP and again this effect is blocked by phentolamine. Elevation of cyclic AMP levels in the lateral oviducts by means of IBMX, forskolin or dibutyryl cyclic AMP mimics the physiological effects of octopamine on this preparation. The results indicate that the octopaminergic control of this insect's visceral muscle is mediated via cyclic AMP.     

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 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.     

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.