31164261
Guanine nucleotide binding regulatory proteins and adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. Immunodetection of Gs and Gi with antisera prepared against synthetic peptides.
Adenylate cyclase in liver plasma membranes from streptozotocin-diabetic (STZ) or BB/Wor spontaneously diabetic rats showed increased responsiveness to GTP, glucagon, fluoroaluminate, and cholera toxin. Basal or forskolin-stimulated activity was unchanged in STZ rats, but increased in BB/Wor rats. No change in the alpha-subunit of Gi (alpha i) was observed in STZ or BB/Wor rats using pertussis toxin-stimulated [32P]ADP-ribosylation. Immunodetection using antibodies against the COOH-terminal decapeptides of alpha T and alpha i-3 showed no change in alpha i in STZ rats and a slight decrease in BB/Wor rats. Angiotensin II inhibition of hepatic adenylate cyclase was not altered in either diabetic rat. In both models of diabetes, Gs alpha-subunits were increased as measured by cholera toxin-stimulated [32P]-ADP-ribosylation of 43-47.5-kD peptides, reconstitution with membranes from S49 cyc- cells or immunoreactivity using antibodies against the COOH-terminal decapeptide of alpha s. These data indicate that STZ-diabetes increases hepatic Gs but does not change Gi or adenylate cyclase catalytic activity. In contrast, BB/Wor rats show increased hepatic Gs and adenylate cyclase. These changes could explain the increase in hepatic cAMP and related dysfunctions observed in diabetes.
31272204
Role of adenosine 3',5'-monophosphate in the effects of insulin and anti-insulin serum on liver metabolism.
Abstract Injection of guinea pig anti-insulin serum into rats decreased the concentration of glycogen in the liver and increased the level of nonesterified fatty acid and glucose in the blood and the concentration of citrate in the heart. The uptake of glucose was reduced in hearts removed from antiserum-treated rats and perfused in vitro. Glucose production by perfused livers from rats treated with antiserum in vivo was increased as a result of accelerated glycogenolysis and gluconeogenesis. Urea production and K+ efflux were also increased. The rates of these processes could be reduced to or below control values by addition of insulin to the perfusion medium. Insulin added in vitro also decreased these processes and the loss of inorganic phosphate in livers from normal rats. Antiserum added to the medium perfusing normal hearts or livers was without effect except that it abolished the action of insulin. Administration of anti-insulin serum in vivo caused a progressive rise in the liver cyclic AMP concentration. Diabetes induced by alloxan also resulted in an increase in liver cyclic AMP, which was rapidly abolished by insulin treatment. Insulin added to the medium perfusing livers from normal or antiserum-treated rats caused a small decrease in liver cyclic AMP. Insulin antagonized the effects of epinephrine and glucagon on glucose output by the perfused liver and reduced the level of cyclic AMP in the presence of glucagon. The hypothesis is advanced that changes in cyclic AMP levels may be partly responsible for the alterations in liver metabolism caused by insulin and diabetes.
777739
Cyclic guanosine 3',5'-monophosphate in mammalian tissues and urine.
Abstract The occurrence of cyclic guanosine 3',5'-monophosphate (3',5'-cGMP) as a natural constituent of mammalian tissues was established, and details of a method for its quantitative determination in milligram amounts of liver, kidney, and brain or in microliter samples of urine are presented. In the analytical procedure 3',5'-cGMP partially purified from tissue extracts by thin layer chromatography or in urine samples (not subjected to any purification) was converted to 5'-GMP with the aid of cyclic nucleotide 3',5'-phosphodiesterase and then to GTP by the combined actions of ATPGMP phosphotransferase and pyruvate kinase. An enzymic cycling system composed of succinate thiokinase and pyruvate kinase was then used to generate proportionate concentrations of pyruvate equivalent to several thousand times the guanine nucleotide present. By measuring the pyruvate fluorometrically it was possible to detect 10-13 to 10-14 mole of 3',5'-cGMP in biological samples. Results are presented showing that 3',5'-cGMP excretion rates were constant in dogs during the infusion of hypertonic and hypotonic NaCl solution in spite of the fact that urinary concentrations of the cyclic nucleotide and urine volume varied almost 100-fold. Exogenous 3',5'-cGMP administered intravenously was rapidly cleared by the kidney and recoverable in the urine. Tissue levels of 3',5'-cGMP ranged from 4 x 10-8 mole per kg, wet weight, in rat kidney and liver to 1 to 2 x 10-7 mole per kg in cerebral tissue. Theophylline treatment of animals led to a 2-fold elevation of renal 3',5'-cGMP but produced no measurable alteration of hepatic levels. Glucagon, epinephrine, and alloxan-induced diabetes elevated concentrations of cyclic 3',5'-adenylate (3',5'-cAMP) in liver but had no effect on those of 3',5'-cGMP. Insulin administration (6 min) also had no influence. It was concluded that the formation of 3',5'-cAMP and 3',5'-cGMP is under separate hormonal and metabolic control and that specific enzymes (cyclases) are probably involved in their biosynthesis.
7546002
Effects of glucagon on cyclic AMP and carbohydrate metabolism in livers from diabetic rats.
Abstract Cyclic AMP metabolism in normal and diabetic livers was studied using the isolated perfused rat liver and plasma membrane and supernatat fractions from rat liver. Liver from alloxan- or streptozotocin-diabetic rats had increased tissue levels of cyclic AMP and showed increased release of cyclic AMP during perfusion. Addition of glucagon or cyclic AMP to the medium produced little or no increase in the high rates of glucose production and lactate gluconeogenesis in diabetic livers. Low concentrations of glucagon (2·10 −10 M or less) did not increase tissue accumulation or release of cyclic AMP in livers from diabetic rats but were effective in normal livers. Higher concentrations of the hormone produced normal responses in diabetic livers. Basal or fluoride-stimulated adenylate cyclase activity in plasma membranes isolated from diabetic livers was not altered, but the enzyme was subnormally normally responsive to concentrations of glucagon within the range 10 −10 −10 −8 M. Insulin (10 −11 −10 −6 M) added in vitro was without effect on basal or glucagon-stimulated plasma membrane adenylate cyclase. Neither modulation of the Mg 2+ concentration, nor addition of Ca 2+ , GTP, theophylline or ouabain caused the emergence of an insulin effect. Rat liver plasma membranes contained phosphodiesterase activity with two apparent K m values of about 0.5 and 70 μM. The activity of the low K m enzyme was decreased in plasma membranes from diabetic rats and was increased by insulin treatment of the rats in vivo. Both low and high K m activities were also decreased in supernatant fractions from livers of diabetic rats. Insulin at 10 −9 or 10 −7 M concentration had no effect in vitro on plasma membrane phosphodiesterase activities.
3013259
Hepatic carbohydrate metabolism in the spontaneously diabetic Bio-Breeding Worcester rat.
The effects of diabetes on hepatic carbohydrate metabolism were investigated in spontaneously diabetic Bio-Breeding Worcester (BB/W) rats. The juvenile-onset-type syndrome displayed by these animals is characterized by beta-cell destruction with subsequent ketosis-prone insulinopenia. Livers from diabetic animals demonstrated increased adenosine 3',5'-cyclic monophosphate levels but subnormal total protein and glycogen content. Isolated perfused livers of diabetic BB/W rats demonstrated an increased rate of glucose production from [14C]lactate and an impaired rate of glycogen synthesis. These data were consonant with hepatic enzyme studies demonstrating markedly increased activities of component gluconeogenic (glucose-6-phosphatase, fructose-1,6-diphosphatase, phosphoenolpyruvate carboxykinase) and glycogenolytic (glycogen phosphorylase) enzymes with decreased activities of glycolytic (hexokinase, pyruvate kinase) and glycogenic (glycogen synthase) enzymes. These findings agree with previous studies using alloxan- and streptozotocin-induced diabetic animals and suggest that accelerated hepatic gluconeogenesis and impaired glucose utilization are pathognomonic of all insulin-deficient diabetic syndromes.
9739588
Glucagon-induced heterologous desensitization of the MDCK cell adenylyl cyclase. Increases in the apparent levels of the inhibitory regulator (Ni).
Treatment of MDCK cells with glucagon results in decreases in glucagon, NaF and prostaglandin E1-stimulated adenylyl cyclase activities, indicating the occurrence of a heterologous desensitization process. The extent of desensitization was time and glucagon concentration dependent. Maximal desensitization (30-50% decrease in stimulation by various effectors) was obtained by 4 h at 100 nM glucagon. Glucagon also induced homologous desensitization since after treatment, the Kact of glucagon was specifically increased. Treatment of cells with 10 microM 8-bromoadenosine 3':5'-monophosphate or 10 microM forskolin resulted in decreased hormonal (glucagon and prostaglandin E1) stimulation without any decrease in the stimulation by nonhormonal effectors (NaF, forskolin, and guanyl-5'-yl imidodiphosphate). The stimulatory regulator (Ns) of the adenylyl cyclase system was analyzed after desensitization with glucagon and no measurable changes in the apparent levels of the alpha s subunits of Ns or the activity of Ns as assessed by reconstitution of the cyc- S49 cell membrane adenylyl cyclase were detected. Levels of the alpha i subunit of the inhibitory regulator (Ni) were monitored by labeling with [32P]NAD and pertussis toxin. Membranes of glucagon-treated cells showed a 2-fold increase in the amount of alpha i labeled. Addition of pure Ns to glucagon-treated MDCK cell membranes restored full stimulation by NaF but did not restore stimulation by prostaglandin E1 or glucagon. It is concluded that glucagon induces heterologous and homologous desensitization of the MDCK cell adenylyl cyclase. The locus of the heterologous desensitization is at the level of the regulatory components. Decreased stimulation is thought to occur due to either an increase in the levels of Ni or due to altered interactions between the subunits of Ni.
35271347
Increased insulin binding by hepatic plasma membranes from diabetic rats: normalization by insulin therapy.
Hepatic plasma membranes prepared from rats rendered diabetic by streptozotocin bound approximately twice as much insulin per 50 mug protein as control membranes. Glucagon binding of diabetic and control membranes was virtually identical. This increased insulin binding was not due to a nonspecific effect of streptozotocin, decreased degradation of insulin slower dissociation from its receptor, or a selective higher yield of membranes prepared from the diabetic livers. Diabetic and control membranes both showed negative cooperativity. Scatchard analysis suggested that the difference in binding was due to an enhanced binding capacity of the diabetic membranes rather than increased affinity of the binding sites. Increased insulin binding of diabetic membranes was returned to normal by insulin treatment. These data are consistent with the postulate that there is an inverse relationship between circulating insulin levels and insulin binding and that insulin may modulate its own receptor. However, since it has been reported that fat, muscle, and hepatic tissue from rats made diabetic by alloxan administration are insensitive to insulin, the capacity for binding can not be the sole factor determining the response to insulin in diabetes mellitus. Therefore, sensitivity of the diabetic liver to insulin is determined, at least in part, by events subsequent to the binding of insulin to its receptor.
17626604
Decreased glucagon receptors in diabetic rat hepatocytes. Evidence for regulation of glucagon receptors by hyperglucagonemia.
The effects of endogenous and exogenous hyperglucagonemia on the specific binding of glucagon to hepatocyte receptors was studied, as was the response of cAMP to glucagon. In streptozotocin diabetic rats, blood glucose and plasma glucagon increased and plasma insulin decreased as compared with controls. Insulin treatment in diabetic rats restored blood glucose and plasma glucagon toward normal and elevated plasma insulin. Specific binding of (125)I-glucagon to isolated hepatocytes (10(6) cells) decreased in diabetic rats (8.17+/-0.38%) compared to controls (14.05+/-0.87%) and was restored by insulin treatment (12.25+/-0.93%). Specific binding of (125)I-insulin in controls was 7.30+/-10.16%; it increased in diabetic rats to 12.50+/-0.86%, and decreased in diabetic rats after insulin treatment (9.08+/-0.87%). Scatchard analysis and the competition plots of the data indicate that decreased glucagon binding and increased insulin binding in diabetes were due to change in the number of receptors rather than a change in their affinity. Hepatocyte cAMP response to glucagon (0.25-5.0 ng/ml) was almost abolished in diabetic rats and was restored with insulin treatment. Specific glucagon binding by hepatocytes from chronically hyperglucagonemic (glucagon injected) rats was decreased (P < 0.005) to 8.76+/-0.61% compared with controls (13.20+/-0.74%) and acutely hyperglucagonemic animals (13.53+/-1.33%). The decreased binding was associated with a 70% decrease in hepatocyte cAMP response to glucagon compared with a normal response in acutely hyperglucagonemic rats.These data appear to support the concept of receptor regulation by ambient hormone level. In both endogenous and exogenous hyperglucagonemia, however, there was a disproportionately large decrease in cAMP response to glucagon compared to the decrease in glucagon binding.
31299714
Increased dose-response relationship of liver plasma membrane adenylate cyclase to glucagon stimulation in diabetic rats. A possible role of the guanyl nucleotide-binding regulatory protein
SummaryIn view of controversial findings regarding the mechanism for the increased intracellular hepatic cyclic 3′:5′ adenosine monophosphate levels in diabetic rats, we studied the dose-response relationship of the adenylate cyclase to glucagon stimulation in severely diabetic and in diabetic, insulin-treated rats. An enhanced response to glucagon and an additional augmenting effect of guanosine triphosphate on hormonal stimulation of the adenylate cyclase activity were found in diabetes which were reversible with insulin treatment. The results suggest a role of the regulatory guanyl nucleotide-binding protein in diabetes leading to an increased dose response relationship of the hepatic adenylate cyclase system to glucagon.
35485892
Changes in adenylate cyclase and 5′-nucleotidase activities in liver membranes from alloxan diabetic rats
Liver membrane adenylate cyclase activity was significantly higher and 5′-nucleotidase activity significantly lower in alloxan diabetic rats compared with normal rats.
39912841
Assessment of effects of vasopressin, angiotensin II, and glucagon on Ca2+ fluxes and phosphorylase activity in liver.
Publisher Summary Vasopressin, angiotensin II, and α 1 -adrenergic angonists exert their effects on liver and certain other tissues by raising the Ca 2+ concentration in the cytosol. Glucagon exerts its effects on liver by raising cyclic adenosine monophosphate (cAMP) although it can increase cytosolic Ca 2+ at high concentrations. The alterations in liver cell Ca 2+ induced by these agents can be monitored by measuring Ca 2+ fluxes across the plasma membrane, changes in the Ca 2+ content of intracellular organelles, or changes in cytosolic Ca 2+ . The intracellular enzyme routinely used to monitor cytosolic Ca 2+ is phosphorylase b kinase, which phosphorylates and activates phosphorylase. Phosphorylase b kinase is sensitive to Ca 2+ in the range normally encountered in the cytosol and produces a stable change in phosphorylase activity, which can be assayed in cell homogenates providing certain precautions are taken. Through this mechanism glucagon and β 2 -adrenergic agonists activate phosphorylase in liver. Thus, in utilizing phosphorylase as an index of cytosolic Ca 2+ in liver cells, it is crucial to exclude possible changes in cAMP and vice versa.
34818904
Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution.
The sensitivity of radioimmunoassays for cyclic AMP and cyclic GMP has been markedly improved to readily detect femtomole (10-15) amounts in tissue extracts by acetylating the cyclic nucleotides at the 2'0 position with acetic anhydride. Acetylation of cyclic nucleotides by acetic anhydride in aqueous solution proceeds more rapidly than the hydrolysis of acetic anhydride to acetic acid thus yielding 100% acetylated cyclic nucleotide. 2'0 substituted cyclic nucleotides have greater affinity for the antibody than the parent cyclic nucleotides because the antibody has been made to a protein conjugate coupled at the 2'0 position. This simple acetylation technique makes it possible to measure cyclic AMP and cyclic GMP in minute quantities of tissue without purification or concentration of the sample.
9437852
Reconstitution of catecholamine-sensitive adenylate cyclase activity: interactions of solubilized components with receptor-replete membranes.
Membranes of mouse L cells that contain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] but lack beta-adrenergic receptors have been solubilized with Lubrol 12A9. Addition of such adenylate cyclase-containing extracts to beta-adrenergic receptor-replete membranes from adenylate cyclase-deficient S49 lymphoma cells results in the production of a catecholamine-sensitive adenylate cyclase system. The effects of beta-adrenergic agonists and antagonists on the reconstituted system reproduce those that are characteristic of the wild-type S49 lymphoma cell. The uncoupled variant of the S49lymphoma contains adenylate cyclase, but donor extracts from this clone fail to reconstitute the hormone-sensitive enzyme activity when added to adenylate cyclase-deficient membranes. Thus, the uncoupled and adenylate cyclase-deficient variants of the S49 cell are not complementary.
39259089
Resolution of some components of adenylate cyclase necessary for catalytic activity.
Adenylate cyclase activity in a Lubrol 12A9 extract of wild type S49 lymphoma plasma membranes is completely inactivated by incubation at 37 degrees for 20 min. Activity is restored by mixing this heated extract of wild type membranes with an unheated detergent extract of membranes from a variant clone that lacks measureable adenylate cyclase activity (AC-). The factor(s) donated by the AC- extract is labile to heating at 30 degrees (t1/2 = 3 min) or to treatment with N-ethylmaleimide or trypsin. The factor(s) donated by the heated wild type extract is also sensitive to proteases or N-ethylmaleimide. This extract displays more complex inactivation kinetics at 50 degrees, consistent with the existence of separate factors necessary for the stimulatory effects of NaF and guanyl-5'-yl-imidodiphosphate. We suggest that at least two proteins are necessary for adenylate cyclase activity and that one of these is retained in the phenotypically adenylate cyclase-deficient variant.
90261
Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium.
Biochemical studies in vertebrate olfactory tissue indicate that certain odorants stimulate adenylyl cyclase in a GTP-dependent manner. Additionally, immunochemical and toxin-labeling studies demonstrate the presence of several GTP-binding protein (G-protein) species in vertebrate olfactory epithelium. To identify the G-protein(s) responsible for olfactory signal transduction, we screened a rat olfactory cDNA library with an oligonucleotide probe and isolated 32 recombinant clones encoding five distinct types of G-protein alpha subunits. The majority of the clones encoded G alpha s, while the remaining clones encoded G alpha o, G alpha i1, G alpha i2, and a novel species, G alpha i3. Messenger RNA corresponding to each G alpha was detectable in all tissues examined; however, the levels for a given G alpha varied in a tissue-specific manner. In olfactory tissue, G alpha s was the most abundant of these messages and in combination with the biochemical studies suggests that G alpha s is the G-protein component of the olfactory signal transduction cascade.
34966836
ADP-ribosylation of membrane components by pertussis and cholera toxin.
Publisher Summary Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (Ns) and inhibitory (Ni) regulatory components of adenylyl cyclise. Cholera toxin acts on Ns by ADP-ribosylating its αs subunit. It uses NAD+ as a cosubstrate. ADP-ribosylation of Ns alters its properties so that its guanosine triphosphate (GTP) hydrolyzing capacity is inhibited and the action of GTP is potentiated. In intact cells, this leads to the increases in cyclic adenosine monophosphate (AMP) levels. Both pertussis and cholera toxin are hexameric multisubunit molecules. Cholera toxin is composed of one A and 5 B subunits; pertussis toxin is formed of one S1, one S2, one S3, two S4, and one S5 subunits. This chapter presents a set of protocols, as developed in the laboratory, that can be used to study simultaneously and comparatively the susceptibility of Ns and Ni to be ADP-ribosylated by cholera and pertussis toxin.
37668482
The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production.
Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.
38860978
Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex.
ADP-ribosylation by pertussis toxin has been used to identify the alpha subunit of Ni, the guanine nucleotide-binding protein which mediates hormone and GTP inhibition of adenylate cyclase. Two proteins have been purified from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin, a 41-kDa protein (alpha 41) and a 39-kDa protein (alpha 39). The 41-kDa protein is very similar to the subunit of Ni purified from other tissues while the function of the 39-kDa protein is unknown (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229; Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813). We now show that the purified alpha 39 protein from bovine brain is a relatively hydrophilic protein which associates with a hydrophobic beta gamma component. The complex can be dissociated by guanosine 5'-(3-O-thio)triphosphate. The alpha 39 component binds guanosine 5'-(3-O-thio)triphosphate with a KD of 27 nM. We have developed polyclonal antibodies to alpha 39 and beta. The antibodies to alpha 39 cross-react weakly with alpha 41 in an immunoblot assay indicating some homology between the two proteins but making it unlikely that alpha 39 is derived from alpha 41. Using the antibodies for quantitation we found that alpha 39 is 0.5% and beta is 0.7% of membrane proteins. While the antibodies cross-react with alpha 39 and beta proteins in many different species, central nervous system tissues always have more immunoreactivity than membranes from peripheral organs. Anti-beta antibody recognizes the beta subunit when it is associated with alpha 39 or alpha 41 and can immunoprecipitate both alpha . beta gamma trimers. The guanine nucleotide-dependent dissociation of the alpha 39 . beta gamma trimer suggests that the complex could inhibit adenylate cyclase by liberating free beta gamma units. The function of alpha 39 may not, however, be exclusively to regulate adenylate cyclase but may include coupling hormone receptors to other effectors. Antibodies specific for alpha 39 and beta will be useful tools in determining the functions of alpha 39 and beta in hormone-responsive cells.
11493305
Antibody specific to the alpha subunit of the guanine nucleotide-binding regulatory protein Go: developmental appearance and immunocytochemical localization in brain.
A polyclonal rabbit antibody (9120) against the alpha subunit of the "other" guanine nucleotide-binding protein Go (alpha o) was raised against a synthetic alpha o peptide fragment (Asp-Gly-Ile-Ser-Ala-Ala-Lys-Asp-Val) attached to a branched core system. Antiserum 9120, at a final dilution of 1:400, can detect alpha o in as little as 0.2 microgram of Go on immunoblots, and, at a final dilution of 1:20,000, can detect alpha o in 1 microgram of Go on immunoblots. Antiserum and affinity-purified antibody are specific to alpha o. No cross-reactivity was detected to the alpha subunits of the stimulatory or inhibitory guanine nucleotide-binding regulatory proteins or of transducin (alpha s, alpha i, or alpha T) or to the beta or gamma subunits. Immunoblots revealed a high density of alpha o in rat brain and lung membrane preparations, but other tissues (such as adipose tissue, heart, erythrocytes, and liver) have no detectable alpha o. Developmental studies showed that alpha o in rat brain was low before birth, increased after birth, and reached the full adult level at 4 weeks of age. In contrast, ADP-ribosylation of 40-kDa proteins increased for up to 1 week and then decreased. Immunocytochemistry revealed that alpha o was localized to somatic and synaptic membranes in rat brain, whereas little or no alpha o was detected in the cytoplasm of neuronal cell bodies. Our observations suggest that Go in brain might have a role in membrane signal transduction at synaptic and extrasynaptic sites.