Inhibition Of Adenylate Cyclase Activity Research Articles

Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1

The guanine nucleotide-binding regulatory proteins (G proteins) in myocardium with ischemia.

Guanine nucleotide-binding regulatory proteins (G proteins) play a major role in the regulation of a number of physiological processes, such as stimulation or Inhibition of adenylate cyclase activity or gaiting of ionic channels. Myocardial ischemia could induce the changes in receptor-G protein signal transduction system in the heart. Therefore, this article will focus on the role and alterations of G proteins (especially, Gs and Gi) in myocardial ischemia. The Gi protein rapidly loses functional activity during very early myocardial ischemia. In contrast to Gi protein, the function of Gs protein during this phase has not been evaluated. Moreover, the changes in Gs protein after 30 min of ischemia are contradictory. However, the sensitization of the adenylate cyclase activity in the very early phase of acute ischemia is gradually replaced by a decrease in adenylate cyclase activity with prolonged ischemia. The decrease in the function and amount of Gs protein may be one of the factors that induce these changes. The function of Gs protein was also decreased in the canine hearts with ischemia and reperfusion. In contrast to ischemia and reperfusion, there are no significant alterations in G proteins and modulation of adenylate cyclase in the stunned myocardium. It has become increasingly evident that Gi protein may play an important role in the cardioprotective effects of preconditioning. When beta-adrenoreceptor densities are reduced in chronic myocardial ischemia, decreased in the amount and function of Gi protein and increased amount of Gs protein may play the role in preservation of the adenylate cyclase activity. These alterations in G proteins may play the important role in the myocardial function during myocardial ischemia.

Novel N6-(substituted-phenylcarbamoyl)adenosine-5'-uronamides as potent agonists for A3 adenosine receptors.

A series of adenosine-5'-uronamide derivatives bearing N6-phenylurea groups have been synthesized and tested for their affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells. Some N6-arylcarbamoyl derivatives, N6-((2-chlorophenyl)carbamoyl)-, N6-((3-chlorophenyl)carbamoyl)-, and N6-((4-methoxyphenyl)carbamoyl)adenosine-5'-ethyluronamide (4l-n), were found to have affinity at A3 receptors in the low nanomolar range (Ki values < 10 nM). In CHO cells stably transfected with the rat A3 receptor, compound 4n was found to be a full agonist in inhibiting adenylate cyclase activity. The present study represents the first example of N6-acyl-substituted adenosine analogs having high affinity at adenosine receptors and, in particular, at the A3 receptor subtype.

5-HT 4, 5-HT 6, 5-HT 7; molecular pharmacology of adenylate cyclase stimulating receptors

Throughout the nervous system the receptors for the neurotransmitter serotonin form an unusually diverse set. Receptor-mediated responses to serotonin utilize a large collection of biochemical second messenger pathways, such as the stimulation or inhibition of adenylate cyclase activity, the hydrolysis of phosphoinositides, the mobilization of calcium and direct gating of ion channels. This diversity of structure and activity has been substantiated by the application of molecular cloning techniques which have now yielded at least 15 distinct molecular entities. The most recent subset of receptors for serotonin to be cloned are those that couple to the stimulation of adenylate cyclase activity. These subtypes: 5-HT 4, 5-HT 6 and 5-HT 7, although sharing a common signal transduction pathway, are remarkably divergent in their amino acid sequences, distribution in the brain and pharmacological properties. This digression from the expected relationships of receptor subtypes based on other serotonin receptors, as well as other biogenic amino receptor families, is unexpected and raises many questions about the extreme diversity of this signaling system.

Alpha‐Adrenergic inhibition of the beta‐adrenoceptor‐dependent chloride current in guinea‐pig ventricular myocytes.

1. alpha 1-Adrenoceptor-mediated inhibition of the beta-adrenoceptor-dependent Cl- current was investigated in guinea-pig ventricular myocytes using the patch clamp technique. The Cl- conductance activated by noradrenaline (0.1-10 microM) with an alpha 1-blocker (prazosin, 5 microM) was significantly greater than that activated by noradrenaline alone. Phenylephrine and methoxamine, alpha 1-agonists, exerted an inhibitory effect on the Cl- conductance activated by isoprenaline. The dose-response relationship for isoprenaline and the Cl- current activation was shifted to higher doses in the presence of phenylephrine (30 microM). 2. The interaction of alpha 1- and beta-agonists on Cl- current was also observed on the single channel level; in some of the outside-out membrane patches, phenylephrine (50 microM) depressed the activity of the single Cl- channel which was induced by 5 microM adrenaline. 3. Phenylephrine had no effect on the Cl- conductance induced by forskolin (0.5-5 microM), an activator of adenylate cyclase. The Cl- conductance activated persistently by isoprenaline in GTP gamma S-loaded cells was also insensitive to phenylephrine. The results suggest that the observed alpha 1-adrenergic attenuation of the beta-adrenergic response is not primarily due to inhibition of adenylate cyclase activity. The alpha 1-adrenergic action may interfere with the processes leading to enzyme activation in the beta-adrenergic pathway.

Chromosomal Mapping of the Mouse A3 Adenosine Receptor Gene,Adora3

A3 adenosine receptor is a member of Gi protein-coupled receptors that mediate inhibition of adenylate cyclase activity upon binding to the ligand. We determined the chromosome localization of the mouse A3 gene for future genetic studies, utilizing an interspecific backcross panel formed from the cross (C57BL/6J ×Mus spretus)F1×M. spretus.Genomic DNAs from 94 individuals in the backcross were analyzed by Southern hybridization with murine A3 receptor cDNA probe. Unique map positions were determined by haplotype analysis with 1388 previously mapped loci in the mouse backcross. The mouse A3 receptor gene (Adora3) mapped to chromosome 3, in tight linkage with DNA markerD3Bir15.

Psycholeine, a natural alkaloid extracted from Psychotria oleoides, acts as a weak antagonist of somatostatin

The effects of psycholeine, a plant alkaloid, were investigated on binding of radiolabelled somatostatin ([ 125I] N-Tyr-SRIF) and on somatostatin (SRIF)-induced inhibition of adenylate cyclase activity and growth hormone (GH) secretion by rat anterior pituitary cells. Psycholeine was shown to displace specific binding of [ 125I] N-Tyr-SRIF to pituitary membrane preparations, with an IC 50 of 10 −5 M. At this concentration, psycholeine was also effective in significantly reducing the SRIF-induced inhibition of adenylate cyclase activity previously stimulated by growth hormone releasing factor (GRF). In parallel, it reduced the SRIF-induced inhibition of GH release stimulated by GRF in primary pituitary cell cultures in a dose-dependent manner. At a moderate concentration, the alkaloid affected neither adenylate cyclase activity nor GH release when applied in the absence of SRIF. These data suggest that psycholeine has antagonistic properties at the SRIF receptor. Quadrigemine C, a precursor of psycholeine, has a similar action.

Refractory hypothalamic adenylate cyclase in anorectic tumor-bearing rats: implications for NPY-induced feeding

Although isoproterenol stimulated adenylate cyclase activity in hypothalamic membranes taken from freely-feeding, food-restricted or nonanorectic tumor-bearing rats, the response was greatly reduced in anorectic tumor-bearing rats. The addition of NPY to the membrane preparation inhibited adenylate cyclase activity in hypothalamus taken from freely-feeding and food-restricted rats, but NPY-inhibitory activity was significantly reduced in both groups of tumor-bearing rats. These results suggest that cyclic AMP formation is refractory in anorectic tumor-bearing rats, and that NPY-induced inhibition of hypothalamic adenylate cyclase is reduced in tumor-bearing rats prior to the onset of significant anorexia. Therefore, NPY-induced feeding may be reduced in tumor-bearing organisms due to a dysfunction in the cyclic AMP second messenger system.

Decreased sensitivity of adenylate cyclase to sodium ions in lymphocytes of patients with arterial hypertension

Patients with arterial hypertension showed decreased sensitivity of isoproterenol-stimulated adenylate cyclase of mononuclear lymphocytes toward sodium ions compared with normotensive donors. Ouabain, an inhibitor of Na,K-ATPase, increased the half-maximal inhibition of adenylate cyclase activity by sodium ions in the cells of healthy subjects and did not change enzyme sensitivity to sodium in the lymphocytes of hypertensive patients.

Insulin inhibits the phosphorylation of α-Gi-2 in intact hepatocytes

Challenge of intact hepatocytes with insulin reduced the level of phosphorylated alpha-Gi-2 found under basal (resting) conditions. At maximally effective concentrations of insulin the steady-state labelling of alpha-Gi-2 was reduced by approximately 21%. Insulin achieved this in a time- and dose-dependent fashion, exhibiting an IC50 value of 109 +/- 22 pM. The increased labelling of alpha-Gi-2 seen after challenge of cells with phorbol 12-myristate 13-acetate was also attenuated by insulin. Treatment of hepatocytes with the protein phosphatase inhibitor okadaic acid increased the labelling of alpha-Gi-2 in a fashion which was insensitive to the action of insulin. It is suggested that insulin may reduce the level of phosphorylation of alpha-Gi-2 by stimulating intracellular protein phosphatase activity and that this action may offer a molecular explanation for the ability of insulin to inhibit adenylate cyclase activity in hepatocytes by increasing the level of non-phosphorylated alpha-Gi-2.

Rolipram, a selective c-AMP phosphodiesterase inhibitor suppresses oro-facial dyskinetic movements in rats

Since striatal dopamine D2 receptor supersensitivity in the etiology of tardive dyskinesia has been suggested and dopamine D2 receptors are known to inhibit adenylate cyclase activity resulting in a decrease of cyclic adenosine 3′,5′-monophosphate (cAMP) levels, we hypothesized that an increase in cAMP levels ameliorates the condition. In the present study, 21-day haloperidol treatment (1.5 mg/kg I.P.) in rats resulted in an increase in striatal [ 3H]-spiperone (D2) binding whereas [ 3H] SCH23390 (D1) binding was unaltered. This haloperidol treatment also induced a significantly increase in the frequency of involuntary chewing movements and tongue protrusions, which are considered as a model of tardive dyskinesia. These dyskinetic movements were suppressed by administration of rolipram (0.5 and 1.0 mg/kg I.P.), an inhibitor of the cAMP phosphodiesterase type IV. The present results suggest that selective cAMP phosphodiesterase type IV inhibitors could be putative therapeutic drugs for tardive dyskinesia.

Adenylate cyclase, cyclic AMP and extracellular-signal-regulated kinase-2 in airway smooth muscle: modulation by protein kinase C and growth serum.

Bradykinin and phorbol 12-myristate 13-acetate stimulate adenylate cyclase activity in serum-depleted cultured airway smooth muscle via a protein kinase C (PKC)-dependent pathway. The probable target is the type II adenylate cyclase, which can integrate coincident signals from both PKC and Gs. Therefore, activation of Gs (by cholera-toxin pre-treatment) amplified the bradykinin-stimulated cyclic AMP signal and concurrently attenuated the partial activation of extracellular-signal-regulated kinase-2 (ERK-2) by bradykinin. We have previously demonstrated that, in order to induce full activation of ERK-2 with bradykinin, it is necessary to obliterate PKC-stimulated cyclic AMP formation. We concluded that the cyclic AMP signal limits the magnitude of ERK-2 activation [Pyne, Moughal, Stevens, Tolan and Pyne (1994) Biochem. J. 304, 611-616]. The present study indicates that the bradykinin-stimulated ERK-2 pathway is entirely cyclic AMP-sensitive, and suggests that coincident signal detection by adenylate cyclase may be an important physiological route for the modulation of early mitogenic signalling. Furthermore, the direct inhibition of adenylate cyclase activity enables bradykinin to induce DNA synthesis, indicating that the PKC-dependent activation of adenylate cyclase limits entry of cells into the cell cycle. These studies suggest that the mitogenicity of an agonist may be governed, in part, by its ability to stimulate an inhibitory cyclic AMP signal pathway in the cell. The activation of adenylate cyclase by PKC appears to be downstream of phospholipase D. However, in cells that were maintained in growth serum (i.e. were not growth-arrested), bradykinin was unable to elicit a PKC-stimulated cyclic AMP response. The lesion in the signal-response coupling was not at the level of either the receptor or phospholipase D, which remain functionally operative and suggests modification occurs at either PKC or adenylate cyclase itself. These studies are discussed with respect to the cell signal regulation of mitogenesis in airway smooth muscle.

Molecular pharmacology of somatostatin receptors.

Somatostatin was discovered for its ability to inhibit growth hormone (GH) secretion. Later, it was found to be widely distributed in other brain regions, in which it fulfills a neuromodulatory role, and in several organs of the gastrointestinal tract where it can act as a paracrine factor or as a true circulating factor. In mammals, two molecules of 14 (somatostatin 14) and 28 (somatostatin 28) amino acids are the only biologically active members of the family. They originate from a single gene which gives rise to a single propeptide alternately cleaved in different tissues. In 1992, a major breakthrough in our understanding of somatostatin functions was made with the cloning of five different receptor genes (sstr1 to sstr5) which belong to the seven transmembrane domain receptor family. Their closer relatives are opioid receptors. In first approximation, the tissular expression of the sstrs matches quite well with the distribution of somatostatin binding sites in the "classical" targets of the peptide ie brain, pituitary pancreatic islets and adrenals. The pharmacology of GH inhibition is very close to sstr2 binding but other actions of somatostatins have not yet been attributed clearly to a single receptor subtype. All clinically relevant agonists tested so far (octreotide, lanreotide and vapreotide) are selective of sstr2 being less potent on sstr3 and inactive for sstr1 and sstr4. Surprisingly, rat sstr5 displays nanomolar affinities for octreotide and vapreotide while these agonists are only active at much higher concentrations on human sstr5. All five receptors can be more or less efficiently coupled to inhibition of adenylate cyclase activity in transfected cell systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of adenylate cyclase activity in rat ventral and dorsal hippocampus by rat galanin

Rat galanin inhibits basal as well as forskolin-stimulated adenylate cyclase activity in rat ventral and dorsal hippocampus. The inhibition of adenylate cyclase activity, both basal and forskolin-stimulated, is characterised by IC 50 values being 250-fold lower in ventral hippocampus (IC 50 = 1.1 nM) compared to the dorsal hippocampus (IC 50 = 270 nM). The maximal inhibition of basal and forskolin-stimulated adenylate cyclase activity in both ventral and dorsal hippocampus in the presence of 10 μM rat galanin is 34–45%. The analysis of the binding data obtained with 125I-labelled Tyr 26-porcine galanin as a tracer reveals similar binding constants for rat galanin in both ventral and dorsal hippocampus with 4.8-fold higher concentration of galanin receptors in the ventral hippocampus. Putative galanin receptor subtype differences between the ventral and dorsal hippocampus have been noted by Hedlund et al. (Eur. J. Pharmacol., 224 (1992) 203–205). This study yields further confirmation for the existence of different galanin receptor subtypes or for differential coupling of galanin receptors to the adenylate cyclase in the dorsal versus ventral hippocampus.

Effect of somatostatin-14 on duodenal mucosal bicarbonate secretion in guinea pigs

The role of somatostatin-14 in duodenal mucosal HCO3- secretion was investigated in anesthetized, indomethacin-treated guinea pigs. Net HCO3- output from the isolated, perfused (24 mM NaHCO3 + 130 mM NaCl) proximal duodenum was measured during intravenous infusion (alone or in combination) of somatostatin-14, carbachol, vasoactive intestinal peptide (VIP), and prostaglandin E2 (PGE2). In homogenates of duodenal enterocytes, the effect of these agents on adenylate cyclase activity was studied. Basal duodenal HCO3- secretion (3.5 +/- 0.2 mumol/cm/10 min) was reduced dose dependently by somatostatin-14 (10(-11) mol/kg, 10(-9) mol/kg, and 10(-7) mol/kg). Carbachol, VIP, and PGE2 (all 10(-8) mol/kg) increased basal duodenal HCO3- secretion two- to threefold. Somatostatin-14 (10(-7) mol/kg) abolished the stimulatory effect of carbachol and VIP, but not that of PGE2. Basal adenylate cyclase activity in isolated duodenal enterocytes (9.4 +/- 1.0 pmol cAMP/mg protein/min) was unaltered by somatostatin (10(-6) mol/liter) or carbachol (10(-3) mol/liter). VIP (10(-8) mol/liter) and PGE2 (10(-7) mol/liter) increased adenylate cyclase activity two- to threefold, and these effects were unchanged by somatostatin-14 (10(-6) mol/liter). In conclusion, somatostatin-14 inhibits basal and carbachol- and VIP-stimulated duodenal HCO3- secretion, and its mechanism of action is not via inhibition of adenylate cyclase activity in duodenal enterocytes.

Pharmacological identity between somatostatin SS-2 binding sites and SSTR-1 receptors

Somatostatin (SRIF) SS-2 binding sites were originally defined in rat brain cerebral cortex membranes using [ 125I]Tyr 11-SRIF-14 in the presence of 120 mM NaCl. These sites were characterized by their high affinity for SRIF-14 and SRIF-28, but very low affinity for cyclic peptides such as octreotide (SMS 201-995) and seglitide (MK 678). The characteristics of SS-2 sites are reminiscent of 125I]CGP 23996-labelled sites in rat brain which have been termed SRIF-2 sites. In the present study, the pharmacological profile of SS-2 sites was determined in radioligand binding studies performed in rat cortex membranes using [ 125I]SRIF-14 in the presence of 120 mM NaCl and compared to that of human SSTR-1 receptors expressed in human embryonic kidney (HEK 293) cells, using [ 125I]SRIF-14. The rank orders of affinity of a variety of SRIF analogues and synthetic peptides for SS-2 binding sites and recombinant human SSTR-1 receptors were very similar and correlated highly significantly ( r = 0.99). However, SS-2 binding correlated also with binding to recombinant SSTR-4 receptors ( r = 0.91). Autoradiographic studies were performed using the radioligand [ 125I]CGP 23996 which has been claimed to label selectively SRIF-2 binding sites and compared with the distribution of SSTR-1 receptor mRNA determined using in situ hydridization in rat brain. Although some overlap was observed between the distribution of SSTR-1 mRNA and [ 125I]CGP 23996 binding sites, the latter were clearly more widespread, suggesting this ligand to label SSTR-1 and other sites. In addition, inhibition of forskolin-stimulated adenylate cyclase was investigated in HEK 293 cells transfected with human SSTR-1 receptors; a variety of SRIF analogues and short synthetic peptides behaved as agonists at adenylate cyclase and displayed a rank order of potency highly similar to that observed for these compounds at SS-2 binding sites. Seglitide acted as an antagonist at SSTR-1 receptor mediated inhibition of adenylate cyclase activity with a pK B of 4.42. It is concluded that the pharmacological profile of SS-2 binding sites resembles most closely that of SSTR-1 receptors (although similarities with SSTR-4 receptors were observed), that [ 125I]CGP 23996 labels presumably several SRIF receptors in rat brain, and that SSTR-1 receptors are negatively and efficiently coupled to adenylate cyclase activity.

Mechanism of action of gamma-aminobutyric acid on frog melanotrophs.

We have previously demonstrated that gamma-aminobutyric acid (GABA) is a potent regulator of secretory and electrical activity in melanotrophs of the frog pituitary. The aim of the present study was to investigate the intracellular events which mediate the response of melanotrophs to GABA. We first observed that GABA (1-100 microM) inhibited both basal and forskolin-stimulated cyclic AMP (cAMP) formation. The inhibitory effect of GABA on cAMP levels was mimicked by the GABAB receptor agonist baclofen (100 microM) and totally abolished by a 4-h pretreatment with pertussis toxin (0.1 microgram/ml). In contrast, the specific GABAA agonist 3-aminopropane sulphonic acid (3APS) did not affect cAMP production. Both GABA and 3APS (100 microM each) induced a biphasic effect on alpha-MSH release from perifused frog neurointermediate lobes, i.e. a transient stimulation followed by an inhibition of alpha-MSH secretion. Administration of forskolin (10 microM) prolonged the stimulatory phase and attenuated the inhibitory phase evoked by GABA and 3APS, indicating that cAMP modulates the response of melanotrophs to GABAA agonists. Ejection of 3APS (1 microM) in the vicinity of cultured melanotrophs caused a massive increase in intracellular calcium concentration ([Ca2+]i). The stimulatory effect of 3APS on [Ca2+]i was abolished when the cells were incubated in a chloride-free medium. The formation of inositol trisphosphate was not affected by 3APS, suggesting that the increase in [Ca2+]i cannot be ascribed to mobilization of intracellular calcium stores. omega-Conotoxin did not alter the secretory response of frog neurointermediate lobes to 3APS, while nifedipine blocked the stimulation of alpha-MSH secretion induced by 3APS. In conclusion, the present data indicate that, in frog pituitary melanotrophs, (i) the stimulatory phase evoked by GABAA agonists can be accounted for by an influx of calcium through L-type calcium channels, (ii) the inhibitory effect evoked by GABAB agonists can be ascribed to inhibition of adenylate cyclase activity and (iii) cAMP attenuates the inhibitory phase evoked by GABAA agonists. Taken together, these data suggest that activation of GABAB receptors may modulate GABAA receptor function.

Modulation of adenosine signalling in sheep adipose tissue by growth hormone

Adenosine is a paracrine/autocrine antilipolytic agent that binds to A, receptors of adipocyte membranes, thereby activating GTP-binding proteins, G,, which inhibit adenylate cyclase activity and hence lipolysis. The mechanism whereby GH facilitates lipolysis is not fully explained, however it is possible that GH exerts its effects by modulating the acute regulation of this process by other hormones rather than having a direct effect on lipolysis itself. Initially studies focused on modulation of response of adipocytes to catecholamines, but very recent studies [ I , 21 suggest that the G,-mediated antilipolytic system is a major target of GH action. We have investigated this further by (a) treating sheep with recombinant bovine growth hormone, (Monsanto) 10 mg per day for 7 days in vivo and (b) by maintaining sheep adipose tissue explants in culture with GH (metabolic activities and other processes are particularly well maintained in sheep adipose tissue during culture [3]. For culture, adipose tissue explants were preincubated for 24h with no hormones and then GH (100ng/ml) was added for the next 24h. In both cases, response to adenosine in vitro was determined using N6-phenylisopropyladenosine (PIA) as described previously [2] (in essence lipolysis was activated with 100 nM isoprenaline and inhibited with various concentrations Treatment of sheep in vivo with GH decreased maximum inhibition of isoproterenol-stimulated lipolysis by 100 nM PIA from 68.8 f 11.4 to 27.8 f 9.2% (P<0.05, results means f SEM of 5 observations). This effect of GH was not accompanied by any discernable change in the number of adenosine receptors or amounts of the various isoforms of G, (measured as described previously, [2]). This suggested that a decrease in G, activity was occumng (GH also decreased response to PGE, which activates G, but via a distinct receptor). Maintenance of sheep adipose tissue in culture for up to 48h in the absence of added hormones had no effect on response to PIA. Addition of GH for 24h again decreased (P < 0.05) maximum response to adenosine from 72.8 to 40.2% inhibition of isoprenaline-stimulated lipolysis (results are means of 4 observations, SED = 9.8). Again there was no change in either the number of adenosine receptors or amounts of G, isoforms (not shown). As there is evidence that G,2a can be phosphorylated by PKC and at least one other kinase [4] we tested the effect of protein kinase and phosphatase inhibitors in the tissue culture system. Addition of 100 pM H7 (a protein serine kinase inhibitor) completely abolished the effects of GH on response to PIA (Table 1) while having no apparent effect on control tissue. This is consistent with a kinase mediating the effects of GH on the G, system. Further evidence for this was obtained using the protein-serine phosphatase inhibitor, okadaic acid. Addition of this agent to the culture medium mimicked the effect of GH; effects of the two agents were additive (Table 1). Table 1. Effect of H7 (100 p M ) and okadacic acid (10 nM) on growth hormone induced inhibition of PIA action on isoprenaline-stimulated lipolysis Tissue was cultured for 24h without additions after which GH, H7 and okadaic acid were added, singly or in combination, for the next 24h. Lipolysis was measured in the presence of isoprenaline (100 nM) and adenosine deaminase (0.8 pg/ml) PIA (100 nM). Results are means of 4 observations; SED was 7.2 and 7.3 for the H7 and okadaic acid experiments respectively.

Ischaemic preconditioning in the rat heart: the role of G-proteins and adrenergic stimulation.

Since recent findings indicate the involvement of G-proteins in the mechanism of ischaemic preconditioning (PC), the present study was aimed to investigate the role of adrenergic mechanisms, such as G-proteins and stimulation of adrenergic receptors, in this phenomenon. For this purpose, isolated Langendorff-perfused rat hearts were subjected to regional ischaemia (30 min occlusion of LAD) followed by reperfusion. The effect of PC (a single 5 min occlusion/reperfusion before a long occlusion) on ischaemia- and reperfusion induced arrhythmias was studied in conjunction with an assessment of G-proteins in the myocardial tissue by means of Western blotting and ADP-ribosylation with bacterial toxins. To follow the link between G-proteins and adrenergic receptors, their stimulation by exogenous norepinephrine (NE) was applied to test whether it can mimic the effect of PC on arrhythmias. Thirty min ischaemia and subsequent reperfusion induced high incidence of ventricular tachycardia (VT) and fibrillation (VF). PC significantly reduced a total number of extrasystoles, incidence of VT and abolished VF. It was, however, insufficient to suppress reperfusion-induced sustained VF. Measurement of G-proteins revealed that PC led to a reduction of stimulatory Gs proteins, whereas inhibitory Gi proteins were increased. NE (50 nmol) introduced a manner of similar to PC (5 min infusion, 10 min normal reperfusion) reduced ischaemic arrhythmias in the same way, as PC. In addition, in NE-pretreated hearts reperfusion induced mostly transient VF, which was spontaneously reverted to normal sinus rhythm. A transient increase in heart rate and perfusion pressure during NE infusion completely waned before the onset of ischaemia, indicating that antiarrhythmic effect was not related to haemodynamic changes and to conditions of myocardial perfusion. antiarrhythmic effect of PC may be mediated by a stimulation of adrenergic receptors coupled to appropriate G-proteins. Consequently, the inhibition of adenylate cyclase activity and reduction in cAMP level, as well as the activation of protein kinase C may be considered as two possible pathways leading to a final response.

Alteration of Phosphoinositide Metabolism by Attenuation of cAMP Resulting from Expression of the H- ras Oncogene

Most studies characterizing H- ras have been conducted in constitutively expressing cell lines. To explore the early interaction between H- ras p21 and signal transduction systems we have utilized an NIH3T3 fibroblast line transfected with a steroid inducible MMTV H- ras vector. Exposure to dexamethasone resulted in transcription of H- ras accompanied by an increase in Pl turnover. Addition of cAMP analogs restored Pl metabolism to control level. We postulate that these effects are due to the regulatory action of PKA on PLC and that H- ras may interfere with cAMP metabolism, negating its regulatory effect on PLC. Therefore, we investigated the role of p21 in cAMP metabolism and PLC activity. We demonstrated that after p21 reached maximal level of expression, cAMP synthesis was reduced to 45% of the control. Radioimmunoassay of cAMP also indicated H- ras acts to inhibit adenylate cyclase activity. Further, we found a 4-fold increase in PLC activity in H- ras expressing cells that could be reversed by elevation of cAMP. Incubation with the PKA inhibitor, KT5720, resulted in activity similar to that observed in H- ras expressing cells. Additionally, we have shown no correlation between H- ras expression and GTPγs stimulated Pl metabolism, indicating that H- ras is not functioning as a G protein in PLC activation. These results imply that H- ras functions, in this system, to decrease levels of cAMP, thus negating the regulatory effect of PKA on PLC.