Catalytic Unit Of Adenylate Cyclase Research Articles

Differential regulation of the β-adrenoceptor density and cyclic AMP level with age and sex in turkey cardiac chambers

Decreased responses of the heart to β-adrenoceptor stimulation with aging have been shown to occur merely in selected heart chambers in relation to increased catecholamine levels. However, there are no systematic studies that investigate all cardiac chambers with regard to receptor density and cAMP (adenosine 3′, 5′-cyclic monophosphate) responses. We used meat-type turkey poults (British United Turkey (B.U.T.) Big 6) with increasing age because their heart seems to decrease in weight in relation to body weight and they are often used as an animal model for heart failure. The receptor density and distribution were quantified by radioligand binding analysis using (-)-[125I]-iodocyanopindolol and β-adrenoceptor subtype-specific antagonists (ICI 118.551 and CGP 20712 A) in membranes of four cardiac chambers (right and left atria and ventricles) of 6-week-, 12-week-, 16/21-week-, and 57-week-old B.U.T. BIG 6 turkeys. Receptor function was determined by measuring basal and stimulated cAMP production. In both sexes, the β-adrenoceptor density decreased significantly in all chambers with age without altered β-adrenoceptor subtype distribution. The receptor affinity (KD) to the radioligand was similar in hearts of all age groups. β-adrenoceptor-(isoproterenol and guanosine 5′-triphosphate), G-protein-(NaF) and catalytic unit of adenylate cyclase (forskolin, Mn2+) mediated cAMP responses were not chamber-dependent. Indeed, the cAMP level was significantly lower in 57-week-old hearts than in 6-week-, 12-week-, 16/21-week-old hearts. These data suggest that with increasing age and body weight, the β-adrenoceptor signal transduction pathway was highly blunted in all cardiac chambers, occurring by decreased receptor density and cAMP responses.

Increased Gsα within blood cell membrane lipid microdomains in some depressive disorders: An exploratory study

The stimulatory guanine nucleotide binding protein Gs couples many cellular receptors to adenylate cyclase, and the Gsα subunit activates all 9 isoforms of the adenylate cyclase catalytic unit to produce the enzyme product cyclicAMP or cAMP. In prefrontal cortex and cerebellum of unipolar depressive suicides, Rasenick and colleagues have found increased concentrations of Gsα in membrane lipid microdomains (Donati et al., 2008), where the ensconced Gsα is less likely to activate adenylate cyclase by receptor and postreceptor pathways (Allen et al., 2005, 2009).We report that a group of 7 depressed patients (DP-1) had (1) reduced activation of platelet receptor-stimulated adenylate cyclase by both prostaglandins E2 and D2 compared to controls, and (2) reduced postreceptor stimulation of adenylate cyclase by aluminum fluoride ion in both platelets and mononuclear leukocytes when compared to both another group of depressed patients (DP-2, n = 17) and to controls (n = 21). Our observations in the blood cells of the group DP-1 support the findings of Donati et al. (2008), and they reflect the importance of this interaction between the activated Gsα subunit and membrane lipid microdomains in the pathophysiology and treatment of some major depressive disorders.

Association of adenylate cyclase with an actin-like protein in the human myometrium

Regulation of muscle contraction by second messengers such as cAMP and regulation of the adenylate cyclase enzyme by the cytoskeleton have been previously described. However, the physical association of both effector and structural elements is still unknown. In this context, we have copurified a human myometrial adenylate cyclase with an actin-like protein in a two-step purification protocol. The adenylate cyclase catalytic unit was solubilized with Lubrol-PX, submitted to anionic exchange chromatography and purified about 7-fold. The eluate was applied to a forskolin-agarose column obtaining an adenylate cyclase extract enriched 257-fold (enzymatic activity of 1390 pmol/30 min per mg protein) that co-eluted with a 74.6-kDa protein that possessed the 18–27 amino-acid fragment from the N-terminal region of human actin. Under non-reducing conditions, the apparent molecular weight of this protein decreased to 54 kDa, which has been previously described for arthrin. These results provide the first demonstration of the physical association of human myometrial adenylate cyclase with a cytoskeleton-related protein, supporting the hypothesis that adenylate cyclase is regulated by mechanical stimuli.

Functional effects of chronic electroconvulsive shock on serotonergic 5-HT 1A and 5-HT 1B receptor activity in rat hippocampus and hypothalamus

The aims of this work were to determine the influence of chronic electroconvulsive shock (ECS) on presynaptic 5-HT 1A receptor function, postsynaptic 5-HT 1A receptor function in hippocampus and hypothalamus, and presynaptic 5-HT 1B receptor function in hippocampus and hypothalamus. This represents part of an on-going study of the effects of ECS on serotonergic receptor activity in selected brain areas which may be relevant to the effects of electroconvulsive therapy (ECT) in humans. Chronic ECS reduced the ability of the 5-HT 1A receptor agonist 8-hydroxy-2(di- n-propylamino)tetraline (8-OH-DPAT) (0.2 mg/kg s.c.) to decrease 5-HT levels in hypothalamus as shown by in vivo microdialysis, indicative of a reduction in sensitivity of presynaptic 5-HT 1A autoreceptors. The ability of the 5-HT 1B receptor antagonist GR 127935 (5 mg/kg s.c.) to increase 5-HT levels in both hippocampus and hypothalamus was unaffected by chronic ECS. 8-OH-DPAT (0.2 mg/kg s.c.) increased cyclic AMP levels in hippocampus measured by in vivo microdialysis approximately 2-fold. The degree of stimulation of cyclic AMP formation was not altered by chronic ECS. However the cyclic AMP response to forskolin (50 μM) administered via the microdialysis probe, which was approximately 4-fold of basal in sham-treated rats, was almost completely abolished in ECS-treated rats. Since this indicates that either adenylate cyclase catalytic unit activity or Gs protein activity is reduced in the hippocampus after chronic ECS, the lack of change in 8-OH-DPAT-induced cyclic AMP formation may be taken as possible evidence of an increase in sensitivity of postsynaptic 5-HT 1A receptors in the hippocampus by chronic ECS. Chronic ECS increased basal plasma levels of corticosterone, ACTH and oxytocin. The ACTH response to s.c. injections of 0.2 mg/kg or 0.5 mg/kg 8-OH-DPAT was reduced by chronic ECS. Postsynaptic 5-HT 1A receptor activity in the hypothalamus, in contrast to the hippocampus, thus appears to be desensitized after chronic ECS. We conclude that chronic ECS has regionally specific effects on both pre- and post-synaptic 5-HT 1A receptors, but, in contrast to some antidepressant drugs, does not affect presynaptic 5-HT 1B receptor activity.

Involvement of κ‐Opioids in the Mouse Cerebellar Adenosinergic Modulation of Ethanol‐induced Motor Incoordination

Using rotorod performance as the test response, possible modulation and co-modulation of ethanol-induced motor incoordination by the cerebellar kappa-opioid and adenosine A1 receptors was studied. A dose-related accentuation of ethanol-induced motor incoordination was observed after direct cerebellar microinfusion of three kappa-opioid receptor agonists: U-50488, U-62066, and bremazocine. On the contrary, significant and dose-related attenuation of ethanol's motor impairment was produced by intracerebellar nor-binaltorphimine, a kappa-opioid receptor antagonist. Furthermore, the accentuation by kappa-agonists was virtually abolished by kappa-antagonist nor-binaltorphimine. Therefore, the accentuation and attenuation by kappa-opioid receptor agonists/antagonist, respectively, was through specific kappa-opioid receptors. Pretreatment with the intracerebellar adenosine A1-selective agonist, N6-cyclohexyladenosine, further enhanced the ethanol-induced motor incoordination and its accentuation by intracerebellar kappa-opioid receptor agonists. Ethanol-induced motor incoordination was markedly attenuated by intracerebellar pertussis toxin (PTX) pretreatment, suggesting an involvement of PTX-sensitive G protein in the expression of motor incoordinating effect of ethanol. Additionally, the intracerebellar PTX also markedly attenuated the accentuation by kappa-opioid agonists of ethanol-induced motor impairment, suggesting participation of PTX-sensitive GTP-binding G protein (Gi, Go) in the kappa-opioid modulation of ethanol's motor impairment. It also confirms that kappa-opioid receptors are linked to PTX-sensitive G protein. The functional similarity between kappa-opioid and adenosine A1 receptors in increasing ethanol's motor incoordination, together with their anatomical co-localization primarily on the axons and axonal terminals of the cerebellar granule cells, suggests a possible common catalytic unit of adenylate cyclase as the basis of modulation of ethanol-induced motor incoordination by both receptor mechanisms.

Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors.

Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8+/-0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38-42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-alpha, PKC-betaII and PKC-epsilon) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase.

Abnormal beta-adrenergic transmembrane signaling in rabbits with adriamycin-induced cardiomyopathy.

We investigated alterations in the beta-adrenergic receptor-adenylate cyclase system in rabbits with congestive heart failure induced by adriamycin cardiotoxicity. A dose of 24 mg/kg adriamycin was administered over 16 weeks in 16 rabbits. Five of them died and 4 of them could not tolerate the full dose of adriamycin. Complete data were obtained in the remaining 7 rabbits. Another 7 rabbits received physiological saline for the same period and served as controls. Plasma norepinephrine concentration increased in adriamycin-treated rabbits, but not in the control rabbits. Cardiac output was lower in the adriamycin-treated group than in the control group. Both the left and right ventricular end-diastolic pressure were higher in the adriamycin-treated group. The density of myocardial beta-adrenergic receptors and the norepinephrine content were reduced in both ventricles in the adriamycin-treated group. Basal and isoproterenol-, sodium fluoride- and forskolin-stimulated adenylate cyclase activities were lower in the adriamycin-treated group. Thus, alterations in beta-adrenergic signaling occurred in both ventricles in animals with chronic biventricular failure induced by adriamycin. These may be the result of post-receptor abnormalities, including abnormalities of guanine nucleotide-binding proteins or of the catalytic unit of adenylate cyclase.

Mouse Cerebellar GABAB Participation in the Expression of Acute Ethanol-Induced Ataxia and in Its Modulation by the Cerebellar Adenosinergic A1 System

The possible modulation and of co-modulation by the cerebellar GABAB and adenosine A1 receptors of ethanol-induced motor impairment were investigated in the mice using rotorod performance as the test response. Direct cerebellar microinfusion of GABAB agonist, baclofen, and antagonist, phaclofen, into the permanently cannulated mice, produced a dose-dependent accentuation and attenuation, respectively, of ethanol-induced motor impairment. The baclofen and phaclofen exhibited accentuation and attenuation, respectively, via GABAB receptors linked to pertussis toxin-sensitive G protein. A co-modulation by the cerebellar adenosine A1 receptors was also observed because intracerebellar microinfusion of adenosine agonists N6-cyclohexyladenosine (CHA), 5′-N-ethylcarboxamidoadenosine (NECA), and 2-p-(2-carboxyethyl)-phenyl-ethylamino-5′-N-ethylcarbox-amidoadenosine (CGS-21680), and antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), also accentuated and attenuated, respectively, ethanol-induced motor impairment. The accentuation of ethanol-induced motor impairment by baclofen was further enhanced after the intracerebellar microinfusion of CHA, suggesting a co-modulation by the co-localized adenosine A1 receptors. A similar response was observed after the intracerebellar microinfusion of adenosine A1 = A2 agonist NECA and the several-fold higher dose of adenosine A2-selective agonist CGS-21680. Ethanol-induced motor impairment was markedly blocked by intracerebellar A1-selective antagonist, DPCPX, as well as by the intracerebellar pertussis toxin pretreatment suggesting again a co-modulation by the adenosine A1 receptors and the involvement of pertussis toxin-sensitive G protein, respectively. The almost 25-fold higher dose of CGS-21680 to accentuate and DPCPX to attenuate, respectively, ethanol-induced motor impairment together with the reported cerebellar localization of adenosine A1 subtype only, suggested A1 receptor activation by NECA and CGS-21680. The functional similarity between GABAB and adenosine A1, receptors associated with their anatomical co-localization on the cerebellar granule cells, mainly axons and axonal terminals, may suggest a possible common adenylate cyclase catalytic unit as the basis of modulation of ethanol's motor impairment by these two receptor mechanisms.

Mouse cerebellar GABA B participation in the expression of acute ethanol-induced ataxia and in its modulation by the cerebellar adenosinergic A 1 system

The possible modulation and of co-modulation by the cerebellar GABA B and adenosine A 1 receptors of ethanol-induced motor impairment were investigated in the mice using rotorod performance as the test response. Direct cerebellar microinfusion of GABA B agonist, baclofen, and antagonist, phaclofen, into the permanently cannulated mice, produced a dose-dependent accentuation and attenuation, respectively, of ethanol-induced motor impairment. The baclofen and phaclofen exhibited accentuation and attenuation, respectively, via GABA B receptors linked to pertussis toxin-sensitive G protein. A co-modulation by the cerebellar adenosine A 1 receptors was also observed because intracerebellar microinfusion of adenosine agonists N 6-cyclohexyladenosine (CHA), 5′-N-ethylcarboxamidoadenosine (NECA), and 2-p-(2-carboxyethyl)-phenyl-ethylamino-5′-N-ethylcarbox-amidoadenosine (CGS-21680), and antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), also accentuated and attenuated, respectively, ethanol-induced motor impairment. The accentuation of ethanol-induced motor impairment by baclofen was further enhanced after the intracerebellar microinfusion of CHA, suggesting a co-modulation by the co-localized adenosine A 1 receptors. A similar response was observed after the intracerebellar microinfusion of adenosine A 1 = A 2 agonist NECA and the several-fold higher dose of adenosine A 2-selective agonist CGS-21680. Ethanol-induced motor impairment was markedly blocked by intracerebellar A 1-selective antagonist, DPCPX, as well as by the intracerebellar pertussis toxin pretreatment suggesting again a co-modulation by the adenosine A 1 receptors and the involvement of pertussis toxin-sensitive G protein, respectively. The almost 25-fold higher dose of CGS-21680 to accentuate and DPCPX to attenuate, respectively, ethanol-induced motor impairment together with the reported cerebellar localization of adenosine A 1 subtype only, suggested A 1 receptor activation by NECA and CGS-21680. The functional similarity between GABA B and adenosine A 1, receptors associated with their anatomical co-localization on the cerebellar granule cells, mainly axons and axonal terminals, may suggest a possible common adenylate cyclase catalytic unit as the basis of modulation of ethanol's motor impairment by these two receptor mechanisms.

Regulation of basal adenylate cyclase activity in neuroblastoma x glioma hybrid, NG108-15, cells transfected to express the human beta 2 adrenoceptor: evidence for empty receptor stimulation of the adenylate cyclase cascade.

Clones of neuroblastoma x glioma hybrid, NH108-15, cells expressing differing levels of the human beta 2 adrenoceptor were isolated. Two clones were examined in detail, beta N22 which expressed some 4000 fmol/mg of membrane protein and clone beta N17 which expressed approx. 300 fmol/mg of membrane protein of the receptor. In beta N22 cells 'basal' adenylate cyclase activity measured in the presence of Mg2+ was significantly greater than that in wild-type NG108-15 or beta N17 cells. Both isoprenaline and iloprost were able to stimulate adenylate cyclase activity in each of beta N22 and beta N17 membranes. However, the EC50 for isoprenaline stimulation of adenylate cyclase in membranes of beta N22 cells (6 nM) was significantly lower than that in membranes of beta N17 cells (80 nM), whereas the EC50 for iloprost stimulation of adenylate cyclase (approx. 25 nM) was the same in the two clones and in parental NG108-15 cells. The high basal adenylate cyclase activity of beta N22 cell membranes was not a reflection of higher levels of expression of the adenylate cyclase catalytic unit, as adenylate cyclase activity measured in the presence of Mn2+ was equivalent in membranes of each of wild-type NG108-15 cells and clones beta N22 and beta N17. Basal adenylate cyclase activity measured in the presence of Mg2+ in clone beta N22 was significantly reduced, however, by the beta-receptor antagonist propranolol, whereas this agent was without effect on basal adenylate cyclase activity in membranes of wild-type NG108-15 cells. These data indicate that the elevated basal adenylate cyclase cascade in NG108-15 cells expressing high levels of the beta 2 adrenoceptor represents empty receptor activation of the signalling cascade.

Actions of lithium on the cyclic AMP signalling system in various regions of the brain--possible relations to its psychotropic actions. A study on the adenylate cyclase in rat cerebral cortex, corpus striatum and hippocampus.

It has been estimated that in most industrialized countries 1 person out of every 1000 in the population is undergoing lithium treatment to stabilize their episodic mood disturbances due to manic-depressive illness. Lithium may stabilize mood swings by altering the action of certain neurotransmitters at the synaptic level in the brain. Recent research suggests that lithium alters neurotransmission by affecting neurotransmitter-coupled second messenger systems. A major second messenger system is the adenylate cyclase, which generates intracellular cAMP from ATP. The adenylate cyclases (type I-IV) are regulated by stimulatory and inhibitory receptors, which either stimulate or inhibit the adenylate cyclase activity. The stimulatory and inhibitory neurotransmitter-receptor signals are transferred to the catalytic unit of the adenylate cyclase by Gs and Gi, respectively. The activated receptor induces GTP stimulation of the heterotrimeric G protein, leading to a dissociation of the protein into the active alpha*GTP and the beta gamma complex. The former stimulates the catalytic unit of adenylate cyclase. The stimulation is terminated by a GTPase located on the alpha subunit that converts GTP to inactive GDP. At present, G proteins are known to play a central role in coupling receptors to effector proteins. In addition to extracellular regulation due to neurotransmitters, some adenylate cyclases (type I, III) are regulated by CaM as a consequence of enhanced intracellular concentrations of free Ca2+. The Ca(2+)-dependent stimulation of adenylate cyclase by CaM is assumed to occur by a direct effect on the catalytic unit. The catalytic units sensitive to Ca(2+)-CaM are also subjected to regulation by stimulatory and inhibitory neurotransmitter stimuli. Magnesium is essential for adenylate cyclase activity, since MgATP2- is the enzyme substrate. Furthermore, one Mg2+ site located on the G protein regulates both the receptor agonist affinity and the dissociation of the G protein during the activation cycle. A second Mg2+ site on the catalytic unit is responsible for Mg2+ regulation of the catalytic activity. The present work aimed at investigating the mechanisms by which lithium in vitro and after chronic treatment (ex vivo) affects adenylate cyclase activities in various regions of the rat brain. Lithium in vitro and ex vivo inhibited the selective stimulation of adenylate cyclase by Ca(2+)-CaM in the cerebral cortex. Furthermore, lithium in vitro interacted directly with the catalytic unit of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional properties of polyclonal antibodies raised against the N-terminus region (residues 9-30) of the follicle-stimulating hormone (FSH) receptor: significance of this receptor region in FSH recognition and signal transduction.

In this study, we raised polyclonal antibodies in rabbits against a synthetic peptide corresponding to a unique region of the FSH receptor, residues 9-30, with no sequence homology to receptors for LH and TSH, and examined their characteristics relevant to receptor function. Binding of [125I]human (h) FSH to membrane-bound receptors was inhibited in a concentration-dependent manner by the anti-FSH receptor-(9-30) peptide antibody. Preimmune serum had no effect. Lineweaver-Burke plot analysis of [125I]hFSH binding to membrane receptors in the presence or absence of antireceptor peptide antibody indicated that the antibody effectively competed with FSH at a hormone-binding site on the receptor. Also, antireceptor peptide antibody, but not preimmune serum, inhibited the ability of FSH to stimulate the conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulation of estradiol synthesis by Sertoli cells caused by cholera toxin or forskolin (which are known to act through the Gs-protein and catalytic unit of adenylate cyclase, respectively) was not inhibited by antireceptor peptide antibody. Indirect immunofluorescence staining of cultured rat Sertoli cells showed binding of antibody to plasma membrane receptor. No fluorescent staining of receptor was observed when cells were incubated with preimmune serum or antireceptor peptide antibody in the presence of excess receptor-(9-30) peptide or hFSH. These results were consistent with specific labeling of membrane-bound FSH receptors by anti-receptor-(9-30) peptide antibody. When detergent-solubilized membrane preparations from rat Sertoli cells were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and then subjected to Western blot analysis, antireceptor peptide antibody, but not preimmune rabbit serum, specifically recognized intact FSH receptor. Although the antireceptor peptide antibody occupied the N-terminus 9-30 epitope region in the FSH receptor, it did not induce postbinding events, such as receptor patching (aggregation), as shown by indirect immunofluorescence staining of rat Sertoli cells and the estradiol response. In contrast, a polyclonal antibody against the FSH holoreceptor capable of interacting with multiple epitopes on the receptor could induce FSH-like effects, such as receptor patching and estradiol response in Sertoli cells. In conclusion, antibody raised against the N-terminus region (9-30) of the FSH receptor recognized intact FSH receptor, inhibited FSH binding, and behaved as an antagonist, suggesting that this N-terminus epitope region of the receptor is involved in hormone binding.(ABSTRACT TRUNCATED AT 400 WORDS)

The GTP-binding regulatory proteins, Gs and G(i), are altered in erythrocyte membranes of patients with ischemic heart disease resulting from coronary atherosclerosis.

Acute ischemic heart disease is associated with alterations in the cardiac adenylate cyclase system response, although the specificity and mechanism of these events are unknown. We studied the characteristics of inhibitory (G(i)) and stimulatory (Gs) GTP-binding regulatory proteins (G proteins) of adenylate cyclase in erythrocyte membranes of patients (n = 16) with nonacute ischemic heart disease resulting from coronary atherosclerosis. Gs was measured by reconstitution with the resolved catalytic unit of adenylate cyclase and by cholera toxin-catalyzed ADP-ribosylation of a 42-kD protein; G(i) was tested as a 41-kD substrate of pertussis toxin-catalyzed ADP-ribosylation. Gs activity was decreased by 27 +/- 2% in the cholate extract and by 25 +/- 3% in the supernatant of guanosine 5'-(gamma-thio)triphosphate-treated membranes. The amount of cholera toxin substrate was decreased by 33 +/- 3%, and the pertussis toxin substrate was increased by 27 +/- 5% compared with healthy subjects (n = 10). All changes in G-protein characteristics appear to be specific relative to other erythrocyte membrane proteins and hemoglobin. Those patients who have a decreased Gs possess approximately normal Gi, and those with increased G(i) showed no change in Gs. Patients with increased G(i) (normal Gs) exhibited more severe deterioration of their coronary arteries than did patients with decreased Gs (normal G(i)) (P < .05), but these two groups did not differ significantly in serum lipids, hormones, drug therapy, historical data, or baseline assessment (P < 0.05).

Altered development of basal and forskolin-stimulated adenylate cyclase activity in brain regions of rats exposed to nicotine prenatally

Exposure of the fetus to nicotine is known to affect cellular development, synaptogenesis and synaptic activity of a wide variety of neurotransmitter pathways in the central nervous system. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipumps. After birth, offspring of the nicotine infused dams displayed marked alterations in membrane-associated adenylate cyclase activity; the regional selectivity correlated both with nicotinic cholinergic receptor concentration and the maturational timetable of each region. In the midbrain and brainstem, which display relatively high receptor concentrations and earliest cell development, basal adenylate cyclase activity in the nicotine group was elevated in the immediate period postpartum, returned to normal by the end of the first month, but then became subnormal in young adulthood. The initial promotion of basal activity was mirrored by forskolin-stimulated activity, suggesting that in this phase, the alterations were occuring at the level of the adenylate cyclase catalytic unit itself. The lack of effect on forskolin stimulation in the later phase, where basal activity was subnormal in the nicotine group, suggests that some alterations in regulatory subunits are responsible for the maturational switch in nicotine's effects on adenylate cyclase. In the cerebellum, where cell replication occurs primarily after birth and receptor concentrations are low, basal adenylate cyclase showed only a deficit in the nicotine group; again, although forskolin stimulation was significantly affected, the actions on basal activity were much more prominent, suggesting defects at the level of G-proteins. The forebrain, with intermediate profiles for receptor concentration and cell maturation, also displayed effects on adenylate cyclase midway between those of the other two regions. Thus, prenatal nicotine exposure disrupts basic cellular transduction systems shared by numerous trophic factors and neurotransmitters. These results can explain, in part, why neurobehavioral teratology attributable to nicotine is expressed across multiple neuronal pathways, rather than being confined to cholinergic systems.

Effect of somatostatin on adenylate cyclase activity in normal and neoplastic thyroid tissue.

Thyroid stimulating hormone (TSH) and other substances increase adenylate cyclase (AC) activity and growth of normal and neoplastic thyroid tissue. Factors that inhibit cAMP may provide targeted therapy to tumors dependent on cAMP for growth. Somatostatin has been reported to inhibit the growth of gastrinomas and carcinoid tumors. We therefore studied the effects of somatostatin on basal, TSH, pertussis toxin, and forskolin stimulated adenylate cyclase activity in normal and neoplastic thyroid tissue from 19 patients. Adenylate cyclase (AC) activity was determined by the conversion of alpha 32P-ATP to 32P-cAMP in pmoles/mg protein/30 minutes in an 8000 x g particulate fraction rich in thyroid plasma membranes. TSH (300 mU/ml) and forskolin (100 mM) (a diterpine that directly stimulates the catalytic unit of AC) increased AC activity in normal and neoplastic thyroid tissue. The AC stimulation was greater in the neoplasms (p less than 0.01). Somatostatin (5 x 10(-6)M) decreased basal and TSH stimulated AC activity below basal levels in both normal and neoplastic thyroid tissue (including papillary, follicular, and medullary carcinomas). The inhibition of AC by somatostatin was greater in neoplastic tissue (p less than 0.025). Pertussis toxin (which blocks the inhibitory guanyl nucleotide regulatory protein) was able to partially reverse the effect of somatostatin. Somatostatin partially inhibited forskolin stimulated AC activity. Somatostatin inhibits basal and TSH stimulated AC activity in both normal and neoplastic human thyroid tissue, with a greater effect on neoplasms. These studies establish that somatostatin blocks a major regulator of thyroid growth and provides the rationale for the use of somatostatin analogs in the treatment of thyroid cancers.

EFFECTS OF CAPTOPRIL ON THE CARDIAC β-ADRENOCEPTOR PATHWAY IN CHRONIC MYOCARDIAL INFARCTION

Effects of captopril and hydralazine on the cardiac β-adrenoceptor pathway in chronic heart failure were investigated. After myocardial infarction (MI) had been experimentally induced in rats, captopril (Cap) or hydralazine (Hyd) was administrated orally for four weeks. Then β-adrenoceptor density (Bmax) and cAMP yield, by either isoproterenol (ISO) -or forskolin-stimulation, were determined. Bmax was significantly increased in MI (N) rats (rats with MI without treatment) as compared with sham-operated rats (63.0 ± 4.6 vs. 38.8±4.4fmol/mg prot. : p<0.05). Cap-treatment of MI had no effect on receptor density (61.8±5.1fmol/mg prot.). On the other hand, Hyd-treatment of MI significantly increased Bmax (92.8 ± 4.6 : p< 0.01). The cAMP yield of MI (N) significantly declined as compared with that of sham operated rats. Both treatments, Cap or Hyd, increased cAMP yield following ISO-stimulation, but cAMP remained unchanged following forskolin-stimulation. Thus, in chronic heart failure due to myocardial infarction, the reactivity of myocytes to catecholamine deteriorate in spite of the increase in β-adrenergic receptor density. This is due to neither increasing receptor density nor improving the activity of the catalytic unit of adenylate cyclase. Captopril seems to improve the activity of G-protein. This regulatory effect of captopril in heart failure is different from that of hydralazine.

μ-Opioid receptor-regulated adenylate cyclase activity in primary cultures of rat striatal neurons upon chronic morphine exposure

Rat striatal neurons cultured in serum-free, hormone-supplemented medium, were exposed to 10 μM morphine for several hours or days before intracellular cyclic AMP production was measured. Dopamine D 1 receptor- and β-adrenoceptor-stimulated cyclic AMP production were prefoundly increased upon morphine exposure (up to 150% of control). In contrast, cyclic AMP production induced by direct activation of the catalytic unit of adenylate cyclase with forskolin remained unaffected. Interestingly, the relative inhibitory effect of the μ-opioid receptor agonist [D-Ala 2,MePhe 4,Gly-ol 5]enkephalin (DAGO) on dopamine D 1 receptor-stimulated cyclic AMP production was unchanged after exposure to morphine. On the other hand, unlike μ-opioid receptors chronically exposed to morphine, β-adrenoceptors mediating activation of adenylate cyclase were rapidly desensitized upon prolonged exposure of the neurons to isoprenaline. It is suggested that tolerance to morphine may be caused by the fact that morphine is acting against up-regulated signal transduction mechanisms rather than by μ receptor desensitization. Moreover, this enhanced effector system function may be involved in opioid dependence. The adaptive changes following morphine treatment appear to be independent of possible alterations at the level of dopaminergic or noradrenergic nerve terminals which are not present in primary cultures of rat striatum.

Effect of chronic prenatal morphine treatment on μ-opioid receptor-regulated adenylate cyclase activity and neurotransmitter release in rat brain slices

Timed-pregnant ratsreceived a semisynthetic diet with or without morphine (0.5–1 mg/g) for 2 weeks. After 21 days of gestation the morphine-dependent dams were decapitated and the foetal brains were dissected. Chronic morphine administration caused a profound increase of adenylate cyclase activity stimulated by postsynaptic D 1 dopamine receptors in striatal slices. The relative inhibitory effect of [D-Ala 2,MePhe 4,Gly-ol 5]enkephalin (DAGO) on D 1-stimulated cyclic AMP (cAMP) production was unaffected. In contrast, cAMP production induced via direct activation of the catalytic unit of adenylate cyclase with forskolin was not changed upon long-term morphine treatment, although DAGO strongly inhibited the effect of forskolin. The electrically evoked release of [ 3H]noradrenaline (NA) from superfused neocortical slices was strongly enhanced upon morphine treatment, whereas release induced by the calcium ionophore A23187, bypassing voltage-sensitive calcium channels, was unchanged. Again, the inhibitory effect of the μ receptor agonist DAGO was unaffected in neocortical slices from morphine-treated rats. It is suggested that tolerance to morphine may be caused by the fact that the opiate is acting against up-regulated signal transduction mechanisms, rather than by desensitization of central μ-opioid receptors. The pre- and postsynaptic changes may include an enhanced expression and/or biochemical modification of D 1 receptors, G s proteins and calcium channels in central neurons on which μ-opioid receptors are present. At the same time, these adaptive changes may underlie morphine withdrawal phenomena.

Treatment of intact hepatocytes with synthetic diacyl glycerols mimics the ability of glucagon to cause the desensitization of adenylate cyclase

Incubation of intact hepatocytes with either of the synthetic diacyl glycerols 1-oleoyl-2-acetyl glycerol (OAG) or dihexanoyl glycerol (DHG) caused the transient uncoupling of the ability of glucagon to stimulate adenylate cyclase in membranes prepared from those cells. No change occurred in either the activity of the catalytic unit of adenylate cyclase or the coupling of G 5 to adenylate cyclase. Diacyl glycerol action appeared to mimic glucagon-mediated desensitization of adenylate cyclase, suggesting that protein kinase C activation may provide the molecular trigger for glucagon desensitization.

The effect of PGF 2α on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E 1

The effect of prostaglandin F2 alpha (PGF2 alpha) was investigated in MC3T3E1 cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2 alpha increased the membrane-associated protein kinase C (PKC) activity, indicating the activation of this enzyme. The effect of PTH to increase cAMP production was enhanced by pretreatment with PGF2 alpha. Phorbol 12-myristate 13-acetate also enhanced cAMP production stimulated by PTH, and PKC inhibitor H7 attenuated the enhancement of PGF2 alpha. A23187 did not reproduce the PGF2 alpha effect, and this effect was not antagonized by the calmodulin antagonist W7. PGF2 alpha did not change the ED50 nor the maximally responsive dose of PTH in stimulating cAMP production. The effect of PGF2 alpha was not affected by pertussis toxin, and PGF2 alpha also enhanced cholera toxin- or forskolin-stimulated cAMP production. In accordance with the response of cAMP to PTH, the resorption of mouse limb bones stimulated submaximally by PTH was enhanced by the concomitant presence of PGF2 alpha. These results indicate that PGF2 alpha modulates cAMP response through the activation of PKC, the target of which might be the catalytic unit of adenylate cyclase. Such interaction between signal transduction systems may have significance in modulating the effect of PTH on bone, i.e., bone resorption.