The effect of chronic ethanol consumption on muscarinic receptors in rat brain

Ethanol (15% v/v) was administered in the drinking water to male Wistar rats over period of 3 months. Binding properties of muscarinic receptors were studied in synaptosomes from selected brain areas using [³H]quinuclidinyl benzilate and its displacement by the selective antagonist, pirenzepine and the agonist, carbachol. Dissociation constants (K_d) of all three ligands in the cerebral cortex, hippocampus and striatum of ethanol-treated groups did not differ from those in controls. Density of [³H]quinuclidinyl benzilate binding sites in the cortex of ethanol-treated animals was approx. 50% higher than in controls (2.06 ± 0.2 and 1.32 ± 0.2 pmol/mg of protein respectively, mean ± SD, n = 6, P < 0.001). This was largely attributable to an increase in M₁ binding sites as shown by pirenzepine displacement studies. In the hippocampus and striatum binding capacity of muscarinic receptors was not affected by ethanol treatment. Synthesis of acetylcholine in cerebral cortex prisms from ethanol-treated animals was not inhibited under resting conditions, but stimulation of synthesis by high K⁺ concentration was significantly altenuated by comparison with controls. These results suggest that chronic ethanol consumption induces changes in cholinergic neurotransmission in selected brain areas.     

Effects of Chronic Ethanol Treatment on the β-Adrenergic Receptor-Coupled Adenylate Cyclase System of Mouse Cerebral Cortex

Chronic ingestion of ethanol, which produced tolerance and physical dependence, resulted in altered function of the cerebral cortical beta-adrenergic receptor-coupled adenylate cyclase system in mice. Although there was no change in basal adenylate cyclase activity, or in the activity of the digitonin-solubilized catalytic unit, stimulation of adenylate cyclase activity by the nonhydrolyzable guanine nucleotide analog guanylylimidodiphosphate [Gpp(NH)p] was reduced in brains of ethanol-fed animals. Ethanol added in vitro increased adenylate cyclase activity, and this enhancement, in the presence of Gpp(NH)p, was also reduced in cortical membranes of ethanol-fed mice. Furthermore, the maximal response to isoproterenol was decreased, and the EC50 for isoproterenol stimulation of adenylate cyclase activity was increased in ethanol-fed animals. The results are consistent with a qualitative or quantitative defect in the function of the stimulatory guanine nucleotide-binding protein (Ns), as well as in the beta-adrenergic receptor, after chronic ethanol exposure. In part, these changes appear to be similar to those that occur during heterologous desensitization of various receptor systems, and may be associated with dependence on or tolerance to ethanol.     

Differential regulation of beta-adrenergic receptor-coupled adenylate cyclase by thyroid hormones in rat liver and heart: possible role of corticosteroids

Thyroid hormone regulation of beta-adrenergic receptor-coupled adenylate cyclase activity was studied in rat liver and heart particulate fractions. Thyroidectomy (Tx) increased isoproterenol-stimulated cAMP accumulation in the liver and decreased it in the heart. Administration of L-thyroxine (L-T4) or L-3,3',5-triiodothyronine (L-T3) reversed these changes in both liver and heart. The changes observed in liver beta-receptor-coupled adenylate cyclase activity after Tx were similar to those reported after adrenalectomy (ADX). Thus the hypothesis was considered that these changes with altered thyroid status are produced indirectly through alteration in adrenal corticosteroids. Hydrocortisone in Tx rats decreased liver isoproterenol-stimulated adenylate cyclase activity but had no significant effect on the heart. Serum corticosterone levels were decreased significantly (by 34%) in Tx rats, as compared to euthyroid rats. Administration of L-T4 to Tx rats doubled the serum corticosterone levels. In Tx-ADX rats, L-T4 had no significant effect on liver beta-receptor-coupled adenylate cyclase. However, L-T4 significantly increased heart beta-receptor-coupled adenylate cyclase in these animals. Dexamethasone, but not deoxycorticosterone, decreased liver isoproterenol-stimulated cAMP accumulation in Tx animals to the same extent as was observed with L-T4 and hydrocortisone. Thus overall the results indicate that in the liver, as opposed to the heart, thyroid hormones regulate beta-adrenergic receptor-coupled adenylate cyclase indirectly through corticosteroids. Glucocorticoid rather than mineralocorticoid activity seems to be responsible for this regulation.     

A possible role of adenylate cyclase in the longterm dietary regulation of insulin secretion from rat islets of Langerhans

1. Adenylate cyclase activity and patterns of insulin release in response to various concentrations of glucose were determined in islets of Langerhans isolated from starving, fed, or glucose-loaded rats. 2. Basal and glucagon-stimulated activities of adenylate cyclase were lower in islets from starved than from fed rats. The minimum glucose concentration required for stimulation of insulin secretion was higher, whereas the maximum secretory response to glucose was lower, in islets from starved than from fed rats. 3. Adenylate cyclase activity in islets of Langerhans obtained from fed rats loaded with glucose by intermittent intravenous or intraperitoneal injections over 5h was significantly higher than that seen in islets from normal fed rats. Islets obtained from glucose-loaded rats required a lower glucose concentration for stimulation of insulin secretion and attained a higher maximal response to glucose stimulation than those derived from fed rats. 4. Incubation in vitro of islets isolated from normal fed rats, for periods of 1 to 24h in the presence of high concentrations of glucose resulted in an activation of adenylate cyclase that occurred progressively from 2 to 7h and which was maintained during 24h of incubation. The increase of adenylate cyclase activity in isolated islets incubated for 4h in the presence of glucose was not prevented by addition of cycloheximide or actinomycin D. Galactose or 2-deoxyglucose was ineffective in increasing adenylate cyclase activity, and pyruvate (20mm) was less effective than glucose. 5. It is suggested that glucose or a glucose metabolite may exert long-term effects on islet cell adenylate cyclase.     

Decreased response with age of the cardiac catecholamine sensitive adenylate cyclase system

The cardiac β-adrenergic coupled adenylate cyclase system was examined in young and old male Wistar rats. The concentration of binding sites for (?) ³H-DHA in membranes prepared from cardiac ventricles was 21.1 ± 2.78 (SD) fmoles/mg protein in 3–4 month old rats (young rats) and 31.2 ± 2.20 fmoles/mg protein in 24 month old rats (old rats). The dissociation constant, K_D was 4.3 ± 1.8 nM and 6.7 ± 1.7 nM for young and old rats, respectively. Various compounds were used to study the characteristics of activation of adenylate cyclase in homogenates from cardiac ventricles. Basal adenylate cyclase was reduced 30% in old animals compared to young (6.1 pmoles/min/mg protein in 24 month vs. 8.6 pmoles/min/mg protein in 3–4 month). (?)Isoproterenol (10^?5M) alone stimulated adenylate cyclase greater than two-fold in young rats (10.6 pmoles/min/mg protein above basal) and this stimulation was 34% lower in old animals. GppNHp (100 μM), fluoride (10 mM), and forskolin (100 μM) activation of adenylate cyclase above basal was reduced 38, 37, and 34%, respectively, in the old animals. No significant changes between the two groups were noted in the apparent affinity of GppNHp either alone or in the presence of (?)isoproterenol nor in the affinities of catecholamine agonists for activation of cyclase. These results suggest a reduction in the amount of functional regulatory protein or possibly cyclase in 24 month old rat ventricular tissue compared to 3–4 month old tissue. However, this data does not rule out the possibility of altered molecular interactions of a full complement of regulatory protein (s) with β-adrenergic receptor and/or catalytic adenylate cyclase.     

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3',5'-monophosphate system to hormonal stimulation during chronic ingestion of DL-ethionine.

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3',5'-monophosphate system were examined in premalignant liver from rats chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissues quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AM content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 +/- 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 +/- 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 +/- 0.04; ethionine 0.55 +/- 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 +/- 7%; ethionine, 15 +/- 1.5%) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethionine ingestion was bilogically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (control, 185 +/- 24 pg/ml; ethionine, 1532 +/- 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14 -fold increase over basal, to 8.63 +/- 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 +/- 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of protaglandin E1 or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue. In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.     

Effect of pentobarbital dependence on adenylate cyclase activity and calmodulin levels in rat cerebral cortex

The effect of calcium (Ca2+) on the adenylate cyclase activity and calmodulin level of cerebral cortex was determined in pentobarbital dependent rats and age matched controls. Female Sprague-Dawley rats were made dependent and maintained on pentobarbital by eating a mixture of pentobarbital and rat chow (350 mg pentobarbital/30 g chow). Ca2+ activated then inhibited the adenylate cyclase activity associated with a 20,000 X g particulate fraction from pentobarbital dependent and age matched control rats. The values for one-half maximal stimulation and inhibition by Ca2+ did not differ significantly in either cortical preparation. However, the ability of Ca2+ to activate adenylate cyclase from pentobarbital dependent animals was significantly decreased (p less than 0.05) when compared to control animals. Pentobarbital (10(-4) - 10(-3) added to particulate fractions from naive control rats did not alter the ability of Ca2+ to activate adenylate cyclase. The calmodulin levels in the particulate fraction from pentobarbital dependent animals (30.2 +/- 6.7 ng calmodulin/mg protein) did not differ significantly when compared to control (33.0 +/- 4.7 ng/mg). By contrast, the calmodulin levels (37.9 +/- 5.9 ng/mg) in the 20,000 X g supernatant from cortex of pentobarbital dependent animals was significantly greater than the level in the supernatant from control animals (28.6 +/- 2.6 ng/mg). The ability of forskolin, dopamine, GTP or forskolin plus GTP (all at a concentration of 100 microM) to activate adenylate cyclase was significantly decreased in particulate preparations from pentobarbital dependent animals. In summary, our data show that alterations in calmodulin levels and a decreased responsivity of adenylate cyclase occur in animals physically dependent on pentobarbital.     

Changes in rat testicular adenylate cyclase activities and gonadotrophin binding during unilateral experimental cryptorchidism

Unilaterally cryptorchid rats were examined at 3, 8, 15, 22 and 28 days after operation. There was a selective decrease in the adenylate cyclase (ATP pyrophosphate--lyase (cyclizing), EC 4.6.1.1) responses to gonadotrophin stimulation in the abdominal testis. This was associated with a parallel decrease in specific FSH and LH binding. There was no reduction in the response of testicular adenylate cyclases to prostaglandin (PG) E-1 or fluoride stimulation, indicating that both the GTP binding protein (N-component) and the catalytic subunit of the adenylate cyclase complexes were intact. The reduction in FSH-responsive adenylate cyclase activity in the abdominal testis was not due to a change in the Km for adenylate cyclase activation, but was due to a reduction in maximal velocities. Unilateral cryptorchidism was also associated with a rapid decline in soluble Mn2+-dependent adenylate cyclase activity in germ cells (spermatids). By 3 days after operation there was an 82% decrease in germ cell adenylate cyclase activity. The loss of soluble Mn2+-dependent adenylate cyclase activity was associated with a parallel decrease in Sertoli cell secretion of androgen binding protein, indicating that Sertoli cell factors may be important for the maintenance of germ cell adenylate cyclase activity. The desensitization of the gonadotrophin--responsive adenylate cyclases and the loss of gonadotrophin receptors in Leydig and Sertoli cells were not due to changes in plasma gonadotrophin values because LH concentrations were within normal limits and plasma FSH was only marginally elevated in the cryptorchid rats. No significant alterations of any of these parameters were seen in the scrotal testis of unilaterally cryptorchid rats when compared to values for intact controls.     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the ?EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F^?, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in ?EFA livers. When glucagon was used in vitro at 1–1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in ?EFA membranes than in +EFA. Total Na⁺, K⁺ (Mg²⁺)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent K_m and V_max. 5′-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

Glycogenolytic responsiveness to glucagon, epinephrine, vasopressin and angiotensin II in the liver of developing hypothyroid rats. A comparative study of in vitro hormonal binding and in vivo biological response

Both dose-response curves and time-courses of plasma glucose levels after single maximal doses showed that in vivo glycogenolytic responsiveness to glucagon and epinephrine was significantly higher in developing hypothyroid rats, whereas it remained unchanged after vasopressin and angiotensin II injections. In contrast with the decreased basal activity of phosphorylase(a), the glucagon-stimulated activity increased in hypothyroid rats, whereas it was only slightly modified under vasopressin stimulation. Daily thyroxine treatment abolished these abnormalities. Thus, there is a close correlation between glucose output and enzyme activation. The maximal binding capacity of [3H]vasopressin and [125I]glucagon was significantly decreased in hypothyroid rats, without changes in the apparent dissociation constant of hormone from its specific receptor. Daily thyroxine treatment also abolished this deficit, which moreover appeared to be independent of possible changes in plasma hormone levels. With respect to glucagon action, neither basal nor Gpp(NH)p-stimulated adenylate cyclase activities were affected in hypothyroid rats. Glucagon-sensitive adenylate cyclase activity and the apparent activation constant appeared to be unaffected. The apparent discrepancy between the results obtained from in vivo and in vitro experiments is discussed on the basis of different membrane transducing phenomena and related intracellular mechanisms underlying the biological response to hormonal stimulation.     

Direct Effects of Chronic Ethanol Exposure on β-Adrenergic and Adenosine-Sensitive Adenylate Cyclase Activities and Cyclic AMP Content in Primary Cerebellar Cultures

The direct effects of chronic ethanol exposure on adenylate cyclase activity and cyclic AMP content were investigated in primary cerebellar cultures. By morphological criteria these cultures mainly contain granule cells with some astrocytes, and each cell type appears to contain both beta-adrenergic and adenosine-sensitive adenylate cyclase systems. Chronic treatment of the primary cerebellar cultures with 120 mM ethanol for 6 days caused a reduction in the stimulation of cyclic AMP content by isoproterenol and by the adenosine analogue 2-chloroadenosine. Kinetic analysis indicated that the chronic ethanol treatment decreased maximal activation of adenylate cyclase, as well as increased the EC50 values for norepinephrine and 2-chloroadenosine. Activation of norepinephrine-stimulated adenylate cyclase activity by in vitro ethanol was significantly enhanced after the chronic ethanol exposure. However, the chronic treatment did not alter activation of the 2-chloroadenosine-stimulated enzyme by in vitro ethanol. A similar difference in the response to in vitro ethanol after the chronic treatment was observed when cyclic AMP content of the intact cells was measured. The present data indicate that chronic ethanol exposure causes a selective increase in the sensitivity of adenylate cyclase to ethanol in some brain cells and a more generalized desensitization of receptor-stimulated cyclic AMP production.     

Exposure to intermittent high altitude induces different changes in adenylyl cyclase activity in hearts of young and adult Wistar rats

This study investigates changes of adenylyl cyclase activity in the heart of young and adult Wistar rats exposed to experimental conditions simulating high altitude hypoxia as a model for interpretation of some adaptive changes of adenylyl cyclase observed in human. The exposure of rats to intermittent high altitude (IHA) hypoxia (5000 m) showed significant adaptive changes. The right ventricular weight and the ratio of right/left ventricular weights of adult rats exposed to IHA were significantly increased when compared to appropriate controls; adaptive changes of cardiac adenylyl cyclase being dependent on the age of the animals. The isoprenaline-stimulated activity was higher in the left than in the right ventricle, and in both ventricles it was higher in young rats than in adult rats. When compared to controls, isoprenaline stimulation was decreased in the right ventricles of adapted young rats and, by contrast, it was increased in the left ventricles of adapted adult rats. This decrease and increase of adenylyl cyclase activity evoked by isoprenaline was paralleled by forskolin-induced adenylyl cyclase activity in these experimental groups. It seems therefore that the changes in the pattern of total adenylyl cyclase activity observed under IHA hypoxia may at least be partially explained by the changes of beta-adrenergic receptor susceptibility following IHA hypoxia.     

Exposure to Intermittent High Altitude Induces Different Changes in Adenylyl Cyclase Activity in Hearts of Young and Adult Wistar Rats

Abstract

This study investigates changes of adenylyl cyclase activity in the heart of young and adult Wistar rats exposed to experimental conditions simulating high altitude hypoxia as a model for interpretation of some adaptive changes of adenylyl cyclase observed in human. The exposure of rats to intermittent high altitude (IHA) hypoxia (5000 m) showed significant adaptive changes. The right ventricular weight and the ratio of right/left ventricular weights of adult rats exposed to IHA were significantly increased when compared to appropriate controls; adaptive changes of cardiac adenylyl cyclase being dependent on the age of the animals. The isoprenaline‐stimulated activity was higher in the left than in the right ventricle, and in both ventricles it was higher in young rats than in adult rats. When compared to controls, isoprenaline stimulation was decreased in the right ventricles of adapted young rats and, by contrast, it was increased in the left ventricles of adapted adult rats. This decrease and increase of adenylyl cyclase activity evoked by isoprenaline was paralleled by forskolin‐induced adenylyl cyclase activity in these experimental groups. It seems therefore that the changes in the pattern of total adenylyl cyclase activity observed under IHA hypoxia may at least be partially explained by the changes of beta‐adrenergic receptor susceptibility following IHA hypoxia.     

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3′,3′-monophosphate system to hormonal stimulation during chronic ingestion of dl-ethionine

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3′,5′-monophosphate system were examined in premalignant liver from rat chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissue quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AMP content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 ± 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 ± 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 ± 0.04; ethionine 0.55 ± 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 ± 7%; ethionine, 15 ± 1.5 %) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethione ingestion was biologically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (contro, 185 ± 24 pg/ml; ethionine, 1532 ± 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14-fold increase over basal, to 8.63 ± 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 ± 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of prostaglandin E₁ or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue.In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.     

Sensitivity of rat brain adenylate cyclase to activation by calcium and ethanol after chronic exposure to ethanol

Rats were fed ethanol (Lieber-DeCarli diet) for three weeks. Stimulation of cerebellar adenylate cyclase by calcium was measured in control (pair-fed), chronic-alcohol and alcohol-withdrawn animals. No differences in the sensitivity or maximal stimulation of this enzyme were observed among these groups. Ethanol $\text{in}$,$\text{vitro}$ (1%) stimulated brain adenylate cyclase approximately 50% in the presence or absence of calcium. Chronic alcohol exposure $\text{in}$,$\text{vivo}$ did not alter the sensitivity of adenylate cyclase to stimulation by alcohol $\text{in}$,$\text{vitro}$.     

DEVELOPMENTAL AND REGIONAL VARIATIONS IN NEUROTRANSMITTER-SENSITIVE ADENYLATE CYCLASE SYSTEMS IN CELL-FREE PREPARATIONS FROM RAT BRAIN

Investigations have been carried out on regional and developmental variations in the properties of adenylate cyclase systems in participate preparations from rat brain. EGTA was routinely included in the assay medium to minimize differences in the state of activation of these systems resulting from variations in their exposure to endogenous Ca²⁺. At birth, adenylate cyclase activity was much higher in the hindbrain-medullary preparations than in comparable fractions from cerebellum, cerebral cortex or subcortex (including midbrain, corpus striatum, hypothalamus and hippocampus). Adenylate cyclase activity increased during early development in preparations from all areas of the brain. Maximal levels were reached at 14 days of age or later. These levels were not greatly altered in the young adult animal, except in the hindbrain-medullary area, where a decrease in activity was observed. Adenylate cyclase systems in cerebral cortical and subcortical preparations were activated by norepinephrine and dopamine throughout development. Serotonin also stimulated adenylate cyclase activity in these preparations from young animals but was much less effective in comparable fractions from adult rats. The response to dopamine was diminished with age in cerebral cortical preparations, but not in subcortical fractions. The responses to norepinephrine increased in both brain regions during early development. Adenylate cyclase systems in particulate preparations from the cerebellum and hindbrain-medullary areas exhibited relatively poor responses to the biogenic amines. Detailed studies of the properties of the cerebral cortical adenylate cyclase systems revealed enhancement of activity by Ca²⁺ and F^? at all stages of development with the maximal activation at 2–3 weeks of age. The results suggest that developmental differences in hormonal sensitivity of adenylate cyclase systems from diverse areas of the brain are related to changes in the proportions of the receptor-enzyme complexes responsive to the different biogenic amines.     

The effect of age and cholesterol on the rat lung beta-adrenergic system

To assess the influence of membrane lipid composition on beta-adrenergic receptor number and adenylate cyclase activity in aging, we investigated the effect of cholesteryl hemisuccinate on these parameters in lung membranes of 3-, 12-, and 24-month-old CDF (F-344) rats. When cholesteryl hemisuccinate (0.5 mg/ml) was incubated with lung membranes, beta-adrenergic receptor density was increased by 70%. This effect was the same for each age group studied and indicated that the density of both basal and CHS-sensitive receptors is unaltered in rat lung with age. Forskolin, NaF, p[NH]ppG, and isoproteronol-stimulated adenylate cyclase activity is 30% lower in lung membranes from aged rats. Since enzyme activity is affected by the lipid environment and membrane composition often changes with age, we assessed adenylate cyclase activity following cholesteryl hemisuccinate incorporation. There was up to a 75% decrease in adenylate cyclase activity following cholesteryl hemisuccinate incorporation in lung membranes in each of the three age groups. In untreated membranes, there was no significant difference in cholesterol or lipid phosphate content with age. These data suggest that cholesterol content does not account for alterations in senescent rat lung adenylate cyclase activity.     

Quantitation of epinephrine- and glucagon-sensitive adenylate cyclases of rat liver implications of alterations of enzymatic activities during preparation of particulate fractions and membranes

Stimulated and basal adenylate cyclase activities from livers of young and old rats were lower in particulates than in homogenates. Particulates were compared to homogenates by reconstituting the suspensions to the volume of the homogenates from which they were derived; enzyme activities in paired homogenates and particulates therefore reflected the same amounts of membrane-bound enzyme. The magnitude of the losses of hormone-sensitive activities in particulates was dependent on the age and sex of the animals and the concentrations of hormone. Particulates from 3-month-old animals showed glucagon-( (1 · 10^?5 M) and epinephrine-sensitive (1 · 10^?4 M) activities which were 67 and 78% of homogenate activities, respectively; particulates from 24-month-old animals had activities relative to homogenates of 55% for glucagon and as low as 32% for epinephrine. The glucagon dose vs. response curve in particulates and membranes showed maximal activity at 1 · 10^?7 M glucagon while in homogenates activity increased linearly with increasing glucagon concentrations up to 1 · 10^?5 M. Losses of basal and anion-stimulated activities were similar at both ages. Fluoride and azide stimulations relative to basal activities were greater in particulates than in homogenates, while relative epinephrine activity was lower in particulates, suggesting qualitative alteration of adenylate cyclase during preparation of particulates. These studies show that adenylate cyclase activity in rat liver is presently best quantitated in homogenates and suggest caution in comparisons of enzyme activities based on particulates or membranes prepared from animals of differing physiologic states.     

Adenylate cyclase activity in rat submandibular gland during postnatal development

Adenylate cyclase activity was measured in homogenates of submandibular glands of 1 to 42 day old rats. During this period of time the gland reached its final stage of differentiation. Adenylate cyclase activity was higher in the glands of one day old rats than in those of 7 and 14 day old animals. Between 14 and 28 days of age the enzyme activity more than doubled and approached the level that characterized the glands of adult animals. Fluoride (10mM) stimulated the enzyme activity in all age groups but the stimulation was less in the case of one day old rats as compared to older animals. Isoproterenol (10^?4 M) stimulated adenylate cyclase by 50–60% in the gland of adult rats but had no effect on the enzyme activity in 7 to 28 day old animals. Administration of isoproterenol for 5 days to 9 day old rats increased the weight of the submandibular gland by 70 per cent. Total adenylate cyclase activity increased parallel with the weight of the gland but the specific activity of the enzyme remained unchanged. It is concluded that during the postnatal development of the submandibular gland the rapid increase in adenylate cyclase activity occurs after weaning and it coincides with an accelerated rate of functional differentiation of the acinar cells.     

Regulation of adenylate cyclase in cultured cardiocytes from neonatal rats by adenosine and other agonists

The presence of adenosine receptors coupled to adenylate cyclase in cultured cardiocytes from atria and ventricles from neonatal rats is demonstrated in these studies. N-Ethylcarboxamideadenosine (NECA), l-N⁶-phenylisopropyladenosine (PIA), and 2-chloroadenosine (2-cl-Ado) stimulated adenylate cyclase in a concentration-dependent manner in both cultured atrial and ventricular cells. The order of potency of stimulation was NECA > PIA > 2-cl-Ado. The stimulation of adenylate cyclase by NECA was enhanced by guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine in both these cells. Other agonists such as epinephrine, norepinephrine, dopamine, F^?, and forskolin were also able to stimulate adenylate cyclase, although the extent of stimulation by these agents was higher in ventricular than in atrial cells. The stimulation of adenylate cyclase by epinephrine and norepinephrine was inhibited by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol, and haloperidol inhibited dopamine-stimulated adenylate cyclase activity to the same extent. Forskolin, at its maximal concentration, potentiated the stimulatory effect of epinephrine, norepinephrine, and dopamine on adenylate cyclase in both atrial and ventricular cardiocytes, but the interaction of NECA with epinephrine, norepinephrine, or dopamine was different in atrial and ventricular cells. The stimulation by an optimal concentration of NECA was additive with maximal stimulation by the catecholamines in atrial cells but not in ventricular cells. The data suggest the existence of adenosine “Ra” and catecholamine receptors in cultured atrial and ventricular cardiocytes. It can be postulated that adenosine in addition to its role as a potent vasodilator might regulate cardiac performance through its interaction with “Ra” receptors associated with adenylate cyclase. The difference in the mode of interaction of adenosine with catecholamines in atrial and ventricular cells suggests that the mechanism by which these agents activate adenylate cyclase may be different in these cells.