Corticotropin releasing factor stimulates cAMP formation in pituitary corticotropic tumor cells

Addition of corticotropin-releasing factor (CRF) to membranes from two ACTH-secreting pituitary tumors strikingly increased in a dose-dependent fashion adenylate cyclase (AC) activity. Significant stimulation was already apparent at 10(-9)M CRF. Stimulation of AC activity by CRF in membranes from non-tumoral tissue adjacent to tumoral corticotrophs was considerably lower, and was lacking in membranes from a growth hormone secreting tumor. These data correlated well with in vivo pre-surgery and post-surgery ACTH responsiveness to CRF of the tumor bearing patients. Basal AC activity was higher in pituitary adenomas than in non-tumoral adjacent tissue. It is concluded that 1) a CRF-sensitive AC exists in ACTH-secreting tumor cells and, 2) increased sensitivity to CRF, as evidenced by greater stimulation of AC activity, may be responsible for the increased ACTH output of tumoral corticotrophs.     

Molecular mechanism of the desensitization of beta-adrenergic receptors and adenilate cyclase by hypoxia in human endothelial cells

Long-term oxygen deficiency in vivo leads to the progressive blunting of responsiveness to sympathetic stimulation and blood catecholamines in many human and animal tissues. In order to better understand the molecular processes that underlie this phenomenon we examined the effect of hypobaric hypoxia (290 mm Hg, pO2 = 40 mM Hg) on the--beta-adrenoreceptor (beta-AR) density and the activity of adenylate cyclase (AC) and phosphoinositide turnover (PI-turnover) in cultures of human pulmonary artery and umbilical vein cells. We discovered that 30 min of hypobaric hypoxia increased basal levels of inositol mono-, bis- and tris-phosphate, products of PI-turnover in endothelial cells (EC). After 60 min of hypoxia their content amounted to 250-300% of the basal level. Desensitization of PI-turnover to histamine stimulation in EC was observed after 60 min of hypoxia. Basal and isoproterenol (beta-AR-agonist)-stimulated AC activities therewith were markedly reduced. beta-AR-density was decreased in EC membranes after 2-3 hrs of hypoxia. Similar desensitization of beta-AR and AC occurred after 1-2 hrs treatment of EC with histamine and platelet activating factor (stimulators of PI-turnover) and with phorbol myristate acetate (PK C activator). Neither hyproxia nor phorbol myristate acetate influenced beta-AR density or AC activity in protein kinase C-deficient EC (72 hrs treatment with phorbol myristate acetate). The data suggest that hypoxia-induced desensitization of beta-AR and AC in endothelial cells is mediated via hypozia-stimulated turnover and subsequent protein kinase C activation.     

Cardiac adenylate cyclase activity in streptozotocin-treated rats after 4 months of diabetes: impairment of epinephrine and glucagon stimulation

Adenylate cyclase (AC) activities of cardiac membranes prepared (a) from rats that had been made diabetic 4 months previously by a single i.v. injection (50 mg/kg) of streptozotocin (STZ), and (b) from diabetic rats which had been treated during the same period by a daily dose of long-acting insulin (2-4 U/animal), were compared with the AC activity of cardiac membranes prepared from age-matched control animals. Basal (Mg++-dependent) and Mn++ (7 mM)-dependent activities, as well as Gpp(NH)p (3 X 10(-7) M) stimulation and Ca++ (pCa = 3.9) inhibition of cardiac AC were not significantly different in the three groups. At the EC50 concentration epinephrine (5 X 10(-7) M) stimulation of AC was reduced in diabetic animals but no change was observed at higher concentrations (10(-4) M). Glucagon stimulation was impaired at both the EC50 concentration (10(-7) M) and at higher concentrations (10(-5) M). Insulin treatment of the diabetic animals partially prevented the impairment of hormone stimulation. These results confirm observations that alterations of cardiac AC activity in STZ-treated rats are indeed due to diabetes and not to STZ-toxicity and suggest that AC-coupled receptors are altered either by an diabetes-induced alteration of cardiac sarcolemma.     

Differential response to gonadotropins and prostaglandin E2 in ovarian tissue during prenatal and postnatal development in cattle.

In cattle, growing follicles are present in fetal ovaries during the last part of gestation. This study examines the extent of changes in basal and hormone-stimulated adenylyl cyclase (AC) activity in ovaries of the bovine fetus when the first follicles begin to grow. The first growing follicles appeared in fetal ovaries around Day 180 and consisted mainly of primary and secondary follicles; few antral follicles were present before Day 220 of gestation. Basal AC activity in ovarian membranes increased simultaneously with the beginning of follicle growth in the fetus (5.8 +/- 0.9 vs. 9.3 +/- 1.3 pmol cAMP/mg protein/min at 130-180 and 180-210 days of gestation, respectively p less than 0.05). During the same time period, there was a significant increase in both the absolute (16.1 +/- 1.2 to 39.9 +/- 1.4 pmol cAMP/mg protein/min) and the relative (2.8 +/- 0.1 to 4.3 +/- 0.3 times the basal level, p less than 0.05) effects of guanosine triphosphate (GTP). After birth, basal and GTP-stimulated AC activities (pmol cAMP/mg protein/min) increased markedly in ovarian membranes of 1-wk-old calves and then decreased with age; the lowest levels were measured in mature cyclic cows. However, the relative effect of GTP (times the basal level) did not show this age-related variation. Prostaglandin E2 (PGE2) stimulation of AC in ovarian membranes from fetuses was high even on Day 120 (2.1 +/- 0.3 times the control level).(ABSTRACT TRUNCATED AT 250 WORDS)     

The olfactory G protein Gαolf possesses a lower GDP-affinity and deactivates more rapidly than Gsαshort: consequences for receptor-coupling and adenylyl cyclase activation

The olfactory G protein G(alphaolf) differs from the short splice variant of G(salpha) (G(salphaS)) in 80 amino acids, but little is known about biochemical differences between G(alphaolf) and G(salphaS). We addressed this question by analyzing fusion proteins of the beta2-adrenoceptor (beta2AR) and G(alphaolf) and G(salphaS), respectively, using Sf9 insect cells as expression system. The fusion ensured defined receptor/G protein stoichiometry and efficient coupling. High-affinity agonist binding studies showed that G(alphaolf) possesses a lower GDP-affinity than G(salphaS) As a result, the agonist-free beta2AR and the beta2AR occupied by partial agonists were more efficient at promoting GDP-dissociation from G(alphaolf) than from G(salphaS) a assessed by guanosine 5'-O-(3-thiotriphosphate) binding, adenylyl cyclase (AC) activity and GTP hydrolysis. Basal AC activity in the absence of GTP was almost sixfold lower in membranes expressing beta2AR-G(alphaolf) than in membranes expressing beta2AR-G(salphaS) at similar levels, reflecting the lower abundance of G(alphaolf-GDP) relative to G(salphaS-GDP). The maximum agonist-stimulated AC activity with beta2AR-G(salphaS) was more than twofold higher than with beta2AR-G(alphaolf), but the relative agonist-stimulation of AC with beta2AR-G(alphaolf) was much greater than with beta2AR-G(salphaS). The difference in maximum AC activity can be explained by more rapid deactivation of G(alphaolf-GTP) by GTP hydrolysis and GTP dissociation relative to G(salphaS-GTP). Taken together, there are biochemical differences between G(alphaolf) and G(salphaS), supporting different roles of these G proteins in vivo.     

Hormone-stimulable adenylyl cyclase and steroid concentration of follicles of the pregnant mare's serum gonadotropin-treated hen

Pregnant mare's serum gonadotropin (PMSG) treatment of the hen disrupts the follicular hierarchy and causes cessation of ovulation. We measured serum progesterone (P4) and estradiol (E2) concentrations and follicular steroid levels and adenylyl cyclase (AC) activity of PMSG-treated hens. Serum P4 and E2 levels were elevated (P less than 0.01 and P less than 0.05, respectively) in PMSG-treated hens compared to controls. There was no significant difference in P4 and E2 concentrations in granulosa and theca layers, respectively, between follicles from PMSG-treated hens and the largest (F1) follicles from control hens. Basal, luteinizing hormone (LH)-, and follicle-stimulating hormone (FSH)-stimulable AC activity was measured in granulosa layers of the largest follicles from PMSG-treated hens and the F1 and second largest (F2) follicles from control hens. Basal AC activity was increased in follicles from PMSG-treated hens (P less than 0.05) compared to F1 control follicles. There was no difference in LH- and FSH-stimulable AC of PMSG-treated hens compared to F1 controls. Control F2 follicles had lower LH- (P less than 0.001) and FSH-stimulable (P less than 0.005) AC activity than follicles from control F1 or PMSG-treated hens. Relative LH- and FSH-stimulable AC (hormone stimulable vs. basal) for follicles from PMSG-treated hens did not differ statistically from the relative AC activity of vehicle-injected F1 or F2 follicles. Therefore, in spite of the high serum P4 and E2 levels in the PMSG-treated hens, there was no change in the hormone-stimulable AC system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordinate regulation of membrane cAMP by Ca2+-inhibited adenylyl cyclase and phosphodiesterase activities

Activation of store-operated Ca(2+) entry inhibits type 6 adenylyl cyclase (EC; AC(6); Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712-6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC(6) and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca(2+) entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca(2+) inhibition of AC(6). Enzyme activity was assessed using PMVEC membranes, where Ca(2+) and cAMP concentrations were independently controlled. Endogenous AC(6) activity exhibited high- and low-affinity Ca(2+) inhibition, similar to that observed in C6-2B cells, which predominantly express AC(6). Ca(2+) inhibition of AC(6) in PMVEC membranes was observed after enzyme activation and inhibition of phosphodiesterase activity and was independent of the free cAMP concentration. Thus, under basal conditions, the constitutive type 4 phosphodiesterase activity rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC(6) to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition.     

Adenylate cyclase of myometrium and its regulation by cAMP-dependent protein kinase

The purpose of this study was to examine activity of adenylate cyclase (AC) and its cAMP-dependent phosphorylation by cAMP-dependent protein kinase (PKA) in the membrane of rabbit myometrium. Isoproterenol (IP) significantly increased AC activity of nonpregnant rabbits myometrium plasma membranes. However, during pregnancy AC of myometrium plasma membranes did not respond to IP. Phosphorylation of the myometrium membrane by PKA was followed by significantly decreased response of AC to IP.     

PACAP activated adenylate cyclase in human sweat glands. An ultracytochemical study

The ultracytochemical localization of adenylate cyclase (AC) was studied after stimulation with pituitary adenylate cyclase activating peptide (PACAP) in human sweat glands. PACAP stimulated AC in both eccrine and apocrine glands. In the secretory cells, enzymatic activity was associated with membranes involved in the secretory mechanism. In both glands, the cells of the excretory duct and myoepithelial cells presented AC activity. These localizations of enzymatic activity suggest a role for PACAP in regulating glandular secretion.     

Alterations in adenylate cyclase activity due to streptozotocin-induced diabetic cardiomyopathy

The effect of chronic experimental diabetes on the adenylate cyclase system (AC) in the rat heart was investigated. Rats were made diabetic by an intravenous injection of streptozotocin (65 mg/kg), hearts were removed 8 weeks later and washed cell particles were isolated. AC activity was measured in the absence and presence of different concentrations of forskolin, NaF, GTP analogue [Gpp(NH)p] or epinephrine. A significant depression in the epinephrine stimulated AC activity was observed in diabetic hearts. Basal AC activity and stimulation of AC with forskolin, NaF and Gpp(NH)p were not significantly different between control and diabetic preparations. These results indicate no apparent alterations in the regulatory or catalytic properties of AC in hearts from chronic diabetic rats. The observed depression in epinephrine stimulated AC activity may account for the depressed inotropic action of catecholamines in the diabetic cardiomyopathy.     

Adenylyl cyclase activity and function are decreased in rat cardiac fibroblasts after myocardial infarction

Myocardial infarction (MI) results in left ventricular remodeling (e.g., ventricular hypertrophy, dilatation, and fibrosis). Fibrosis contributes to increased myocardial stiffening, impaired ventricular filling and function, and reduced cardiac output. Adenylyl cyclase (AC) expression and activity are reduced in animal models of heart failure. Stimulation of AC can inhibit extracellular matrix production in isolated cardiac fibroblasts; however, a role for reduced AC expression and activity in fibrosis associated with cardiac remodeling after chronic MI has never been determined. We tested the hypothesis that AC expression and activity are reduced in cardiac fibroblasts after chronic (18 wk) MI. Rats underwent coronary artery ligation or sham surgery (control), and echocardiography was used to assess left ventricular remodeling 1, 3, 5, 7, 10, 12, and 18 wk after surgery. Cardiac fibroblasts were isolated from the noninfarcted myocardium and compared for differences in AC activity and collagen synthesis. End-diastolic dimension was increased [control: 0.76 +/- 0.02 cm and MI: 1.0 +/- 0.02 cm (means +/- SE), P < 0.001] and fractional shortening was decreased (control: 44 +/- 2% and MI: 17 +/- 2%, P < 0.001) in MI compared with control rats. Basal and forskolin-stimulated cAMP production were decreased by 90% and 93%, respectively, and AC5/6 expression was decreased 39% in fibroblasts isolated from MI rats compared with sham controls. Serum-stimulated collagen production was increased twofold and forskolin-mediated inhibition of collagen synthesis was reduced in fibroblasts from MI rats compared with controls. Our data demonstrate that AC expression and activity are reduced and collagen production is increased in cardiac fibroblasts of rats after MI.     

Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination

The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.     

GABAB受体与腺苷酸环化酶偶联环节的脱敏研究

将突触体膜与佛波脂（ＰＭＡ）,ＧＡＢＡＢ受体激动剂巴氯芬（Ｂａｃｌｏｆｅｎ,ＢＡＬ）预孵育一定时间后ＢＡＬ对腺苷酸环化酶（ＡＣ）基础活性及ｆｏｒｓｋｏｌｉｎ刺激的ＡＣ活性的抑制率显著降低,而ｆｏｒｓｋｏｌｉｎ预孵育时,ＢＡＬ对基础及ｆｏｒｓｋｏｌｉｎ刺激的ＡＣ活性的抑制率不变,表明ＧＡＢＡＢ受体与ＡＣ偶联环节的脱敏机制涉及蛋白激酶激活。而与蛋白激酶Ａ无关,脱敏时ＧＡＢＡＢ受体的Ｋｄ值增加,本 实验     

Hepatic adenosine-3',5'-monophosphate metabolism in newborn rats irradiated during the period of organogenesis]

The adenylate cylcase (AC) and phosphodiesterase (PDE) activities, as well as the 3'5'-AMP (cAMP) fund were studied in the liver of newborn rats, intact, and after irradiation on the 9th day of the embryonic development. A decrease of AC and PDE activities was noted with a dose of 50 r. The stationary level of cAMP in the tissue remained unchanged. The adrenaline-stimulated AC activity only tended to decrease in case of irradiation. As suggested, during critical periods of development, in the presence of the hormone inductor in the liver of irradiated rats, conditions can be created for an increase of the cAMP pool.     

Alterations of the VIP-stimulated cAMP pathway in rat distal colon after abdominal irradiation

Ionizing radiation induces hyporesponsiveness of rat colonic mucosa to vasoactive intestinal peptide (VIP). Possible mechanisms responsible for this hyporesponsiveness of the cAMP communication pathway in rat colon were investigated. VIP- and forskolin-stimulated short-circuit current (I(sc)) responses were studied after a 10-Gy abdominal irradiation in Ussing chambers as well as in single, isolated crypts. Adenylyl cyclase (AC) activity and VIP receptor characteristics were determined in mucosal membrane preparations. In addition, alterations in crypt morphology were studied. Impaired secretory responses to VIP and forskolin were observed 4 days after irradiation (decrease of 80%). cAMP analog-stimulated I(sc) responses were unchanged. In isolated crypts, VIP- and forskolin-stimulated cAMP accumulation was markedly reduced by 80 and 50%, respectively. VIP-stimulated AC activity and VIP receptor number were decreased in membrane preparations. No major change of cellularity was associated with these functional alterations. In conclusion, the decreased secretory responses to VIP of rat colon are associated with reduced cAMP accumulation, decreased AC activity, and diminution of VIP receptor numbers without a marked decrease of crypt cell number.     

Adenylate cyclase responses to sucrose stimulation in membranes of pig circumvallate taste papillae.

1. Typical adenylate cyclase (AC) responses to guanine nucleotides were found in membranes of pig circumvallate (CV) taste papillae. 2. Sucrose stimulated AC activity in the CV membranes and this stimulation was GTP dependent and tissue specific. 3. The stimulatory effect of sucrose in the CV membranes was dependent on the concentration of membranes used in the AC assay. 4. This study provides the first biochemical data on cellular transduction of taste in the pig, compares positively to preliminary results in cattle and supports recent suggestions for a role of cAMP in sweet taste transduction.     

Ethanol''s Effects on Cortical Adenylate Cyclase Activity

The effects of ethanol on beta-adrenergic receptor-coupled adenylate cyclase (AC) of mouse cerebral cortex were examined. The addition of ethanol (20-500 mM) to incubation mixtures containing cortical membranes demonstrated that ethanol could increase AC activity and potentiate the stimulatory effects of guanylyl-imidodiphosphate [Gpp(NH)p] on AC activity. Ethanol increased the rate of activation of AC by guanine nucleotides and concomitantly decreased the EC50 for magnesium required to achieve maximal stimulation of cortical AC. The EC50 values for Gpp(NH)p and isoproterenol stimulation of AC activity were also altered by ethanol. Ethanol was capable of stimulating AC extracted by use of digitonin. The AC activity in the digitonin extract was no longer sensitive to the addition of Gpp(NH)p or NaF, but was still stimulated by ethanol. We propose multiple sites of action for ethanol in stimulating cortical AC activity. These sites include actions at the beta-adrenergic receptor, at the G/F coupling proteins, and at the catalytic unit of cortical AC. Comparison of ethanol's actions on cortical beta receptor coupled AC activity with prior reported actions of ethanol on striatal dopamine (DA)-sensitive AC indicated differential sensitivities of these two AC systems to ethanol. These differences may be determined by specific coupling characteristics of the striatal and cortical AC systems or by differences in the plasma membranes in which striatal and cortical AC systems are located.     

Enzyme-ultracytochemical study of adenylate and guanylate cyclases in normal and pathologic human nasal mucosa

The ultracytochemical localization of adenylate cyclase (AC) and guanylate cyclase B (GC-B) and C (GC-C) activity was studied after stimulation with pituitary adenylate cyclase activating peptide, C-type natriuretic peptide and guanylin, respectively, in normal human respiratory nasal mucosa and mucosa of nasal polyps. To demonstrate these enzymatic activities, we employed enzyme-ultracytochemical methods for electron microscopy. Both normal and pathologic nasal mucosa contained AC, GC-B and GC-C activity. In the upper portion of respiratory epithelium, the enzymes were detected on ciliary and microvillar membranes. In ciliary membranes, GC-B was the predominant form expressed. In goblet cells and in glands of the lamina propria, enzymatic activities were localized mainly on plasma membranes and on membranes lining secretory granules. The results did not reveal any evident differences between the enzymatic activities in normal and pathological nasal mucosa and suggest complementary activities for these enzymes and their stimulators in the regulation of mucociliary transport and glandular secretion.     

一种改进的纯化和鉴定牛脑皮层Gs蛋白的方法

用１％胆酸钠和１５％孢和硫酸铵相结合的方法,从牛脑皮层细胞膜中抽提得到主要含激活型Ｇ－蛋白和腺苷酸环化酶两种蛋白组分的制剂,然后通过Ｓｅｐｈａｒｏｓｅ６Ｂ柱将两者分开,将含Ｇｓ高活力的级分用庚胺－Ｓｅｐｈａｒｏｓｅ４Ｂ柱进一步分离,即可获得高活力的Ｇｓ,ＳＤＳ－ＰＡＧＥ显示为分子量４５０００和３６０００的两条蛋白带,该法具简便,快速,重复性好、产率高等优点,且可同时获得无Ｇｓ污染的ＡＣ。用无Ｇｓ污     

Pertussis toxin blocks melatonin-induced inhibition of forskolin-stimulated adenylate cyclase activity in the chick brain.

The high-affinity guanine nucleotide-sensitive receptor sites for melatonin in the mammalian hypothalamus and pars tuberalis mediate inhibition of adenylate cyclase (AC) activity. Therefore, we have examined whether similar sites in the chick brain and retina also modulate AC activity. Melatonin did not alter basal or forskolin-stimulated AC activity in whole forebrain or retinal homogenates. In contrast, melatonin significantly inhibited forskolin-stimulated AC activity in forebrain synaptosomal membranes and partially purified retinal membranes in a concentration-dependent manner. Maximal inhibition (approximately 25-30%) of stimulated AC activity was observed at 10-100nM melatonin, while the concentrations (EC50's) which caused half-maximal effects were 22 +/- 6 pM and 30 +/- 5 pM in the brain and retina respectively. Pretreatment of forebrain slices with pertussis toxin abolished the inhibitory effect of melatonin on stimulated AC activity. These data provide the first evidence that melatonin suppresses AC activity in the chick CNS via a pertussis toxin-sensitive G-protein.