Increase In Cyclic AMP Production Research Articles

A timely characterization of vasopressin-sensitive adenylyl cyclase isoforms in the mouse inner medullary collecting duct

second messenger signaling in the kidney mediates several of the organ's basic physiological functions including the regulation of blood pressure, control of fluid volume, and maintenance of the body's water homeostasis in times of duress. Tight regulation of the second messenger cAMP is critical

Calmodulin Is Required for Vasopressin-stimulated Increase in Cyclic AMP Production in Inner Medullary Collecting Duct

Calmodulin plays a critical role in regulation of renal collecting duct water permeability by vasopressin. However, specific targets for calmodulin action have not been thoroughly addressed. In the present study, we investigated whether Ca2+/calmodulin regulates adenylyl cyclase activity in the renal inner medullary collecting duct. Rat inner medullary collecting duct suspensions were incubated in the presence or absence of 0.1 nM vasopressin and the calmodulin inhibitors, monodansylcadaverine, W-7, and trifluoperazine, followed by measurement of cAMP. Vasopressin-stimulated cAMP elevation was significantly attenuated in the presence of calmodulin inhibitors. Analysis of transglutaminase 2 knock-out mice confirmed that these compounds were not acting through inhibition of transglutaminase 2 activity. Calmodulin inhibitors also blocked both cholera toxin- and forskolin-stimulated cAMP accumulation. In isolated perfused tubules, W-7 reversibly blocked vasopressin-stimulated urea permeability, a process that requires a rise in intracellular cAMP but does not appear to involve protein trafficking to the apical plasma membrane. These results suggest that calmodulin is required for vasopressin-stimulated adenylyl cyclase activity in the intact inner medullary collecting duct. Reverse transcription-PCR, immunoblotting, and immunohistochemistry revealed the presence of the calmodulin-sensitive adenylyl cyclase type 3 in the rat collecting duct, an isoform previously not known to be expressed in the collecting duct. Long-term treatment of Brattleboro rats with a vasopressin analog markedly decreased adenylyl cyclase type 3 protein abundance, providing an explanation for long-term down-regulation of vasopressin response in the collecting duct. These studies demonstrate the importance of calmodulin in the regulation of collecting duct adenylyl cyclase activity and transport function.

The role of the 8-18 helix of CGRP8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions.

1. The role of individual residues in the 8-18 helix of CGRP(8-37) in promoting high-affinity binding to CGRP(1) receptors expressed on rat L6 and human SK-N-MC cells has been examined. The relative potencies of various derivatives were estimated from their ability to inhibit the human alphaCGRP-mediated increase in cyclic AMP production and the binding of [(125)I]-human alphaCGRP. 2. Arg(11) and Arg(18) were replaced by serines to give [Ser(11,18)]CGRP(8-37). These bound with pKi values <6 to SK-N-MC cells and had apparent pA(2) values of 5.81+/-0.04 and 5.31+/-0.11 on SK-N-MC and L6 cells. CGRP(8-37) had a pKi of 8.22 on SK-N-MC cells and pK(b) values on the above cell lines of 8.95+/-0.04 and 8.76+/-0.04. 3. The arginines were replaced with glutamic acid residues. [Glu(11)]CGRP(8-37) had a pK(b) of 7.14+/-0.14 on SK-N-MC cells (pKi=7.05+/-0.05) and 6.99+/-0.08 on L6 cells. [Glu(18)]CGRP(8-37) had a pK(b) of 7.10+/-0.0.08 on SK-N-MC cells (pKi=6.91+/-0.23) and 7.12+/-0.09 on L6 cells. 4. Leu(12), Leu(15) and Leu(16) were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA(2) values of [bpa(12)]-, [bpa(15)]- and [bpa(16)]CGRP(8-37) were respectively 7.43+/-0.23, 8.34+/-0.11 and 5.66+/-0.16 (pKi values of 7.14+/-0.17, 7.66+/-0.21 and <6): on L6 cells they were 7.96+/-0.36, 8.28+/-0.21 and 6.09+/-0.04 (all n=3). 5. It is concluded that the Arg(11) and Arg(18) are involved in specific electrostatic interactions with other residues, either on the CGRP(1) receptors or elsewhere on CGRP(8-37). Leu(16) is in a conformationally restricted site when CGRP(8-37) binds to CGRP(1) receptors, unlike Leu(12) and Leu(15).

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol x l(-1). The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl- concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol x l(-1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane.

Identification of the adenylyl cyclase-activating 5-hydroxytryptamine receptor subtypes expressed in the rat submandibular gland.

1. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to increase cyclic AMP production in dispersed cell aggregates from the major salivary glands of the rat. The goal of the present study was to identify the 5-HT receptor subtypes that mediate these effects in rat submandibular glands (SMG). 2. Among the 5-HT receptor subtypes identified in the rat, 5-HT(4(a,b)), 5-HT(6) and 5-HT(7(a,b,c)) activate adenylyl cyclase (AC). We used subtype specific primers to screen rat SMG by reverse transcription-PCR. Results indicate the presence of mRNA for 5-HT(4(b)) and 5-HT(7(a)) but not for 5-HT(4(a)), 5-HT(6) and 5-HT(7(b,c)). 3. In dispersed SMG cells, 5-carboxyamidotryptamine (5-CT), a 5-HT(7) receptor agonist, stimulated cyclic AMP synthesis with higher potency (EC(50)=27+/-5 nM) but lower efficacy than 5-HT, suggesting a 5-HT(7) component and an additional component in the response to 5-HT. The 5-HT(7) contribution was further supported by antagonism of the 5-CT effect by metergoline, a 5-HT(7) antagonist, which exhibited an affinity (K(i)=50 nM) similar to that obtained at the cloned 5-HT(7) receptor. 4. In the presence of a maximally effective concentration of 5-CT, 5-HT produced an additional increase in cyclic AMP production that was inhibited by the 5-HT(4) receptor antagonist, GR113808, suggesting that the second component of cyclic AMP production is mediated by 5-HT(4) receptors. 5. These findings indicate the presence in rat SMG of both 5-HT(4(b)) and 5-HT(7(a)) receptors positively coupled to AC.

CDNA cloning of GTH receptor family in starfish ovaries

In starfish, oocytes in the ripe ovary do not develop beyond the end of the first prophase stage of meiosis. Such immature oocytes are not fertilisable. The resumption of meiosis in these oocytes and release from the ovary are induced by the maturation-indueing hormone 1-methyladenine (1-MeAde) (Kanatani et al., 1969). 1-MeAde is produced by ovarian follicle cells after stimulation by a gonad-stimulating hormone (GSS) secreted from radial nerves. The action of GSS on 1-MeAde production in follicle cells appears to be mediated by its receptor, G-proteins and adenylyl cyclase (Mita &amp; Nagahama, 1991). It has been shown that upon incubation of follicle cells with GSS, there is a dose-related increase in cyclic AMP production, coincident with an increase in 1-MeAde production (Mita et al., 1989). Thus, it was suggested that a G-protein coupled (seven transmembrane type) receptor is involved in GSS signal transduction in a similar manner to the pituitary–gonadal axis in vertebrates. To elucidate the mechanism of starfish oocyte maturation, we focused on a receptor protein for GSS on the ovarian follicle cell surface. In this study, we cloned the cDNA with glycoprotein hormone receptor homology in a transmembrane region from ovaries of the starfish Asterina pectinifera.Using consensus sequences from glycoprotein hormone receptors, a 3953 bp cDNA was cloned from mRNA of starfish A. pectinifera ovaries. The region from nucleotide 72 to 3109 represents the open-reading frame. The seven transmembrane spanning domains were found in the region from nucleotide 1745 to 2533. The lengths of specific domains in the cDNA were 1671 bp, 789 bp and 579 bp for extracellular (ECD), transmembrane (TMD) and intracellular domains (ICD), respectively. Similar to other glycoprotein hormone receptors, the cDNA from starfish ovaries had a large ECD region. With respect to the predicted amino acid sequence, the ECD, TMD and ICD were composed of 557, 263 and 192 amino acids, respectively.

The effect of site-directed mutagenesis of two transmembrane serine residues on agonist-specific coupling of a cloned human alpha2A-adrenoceptor to adenylyl cyclase.

1. The effects of substitution of the Ser200 and Ser204 residues with alanine on the signalling properties of the cloned human alpha2A-adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine. 2. The Ser-->Ala200 or the Ser-->Ala204 mutant forms of the alpha2A-adrenoceptor, when expressed in cells in the absence of pertussis toxin pretreatment, are two orders of magnitude more sensitive to inhibition of cyclic AMP production by (+/-)-para-octopamine and (+/-)-meta-octopamine, respectively, than cells expressing the wild-type receptor. Binding studies indicate that the effects are not due to an increased agonist affinity for the mutant receptors and that they are likely to be due to agonist-mediated conformational changes in receptor structure. 3. After incubation with pertussis toxin, (+/-)-meta-octopamine (100 microM and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser-->Ala204 mutant form of the alpha2A-adrenoceptor but showed no stimulation in cells expressing the Ser-->Ala200 mutant receptor. Under these conditions (+/-)-para-octopamine did not produce any increases in cyclic AMP production in cells expressing either of the mutant receptor forms or the wild-type receptor. 4. The results emphasise the importance of the Ser200 and Ser204 residues of the alpha2A-adrenoceptor in exerting an inhibitory influence on the ability of (+/-)-para-octopamine and (+/-)-meta-octopamine respectively, to induce a receptor-agonist conformation capable of inhibiting forskolin-stimulation of cyclic AMP levels. 5. It is clear that Ser204 also prevents meta-octopamine from generating a receptor-agonist conformation that can increase cyclic AMP levels, emphasising the importance of this residue in the agonist-specific coupling of this receptor to different second messenger systems.

Long term orexigenic effect of a novel melanocortin 4 receptor selective antagonist.

1. We designed and synthesized several novel cyclic MSH analogues and tested their affinities for cells expressing the MC1, MC3, MC4 and MC5 receptors. 2. One of the substances HS028 (cyclic [AcCys11, dichloro-D-phenylalanine14, Cys18, Asp-NH2(22)]-beta-MSH11-22) showed high affinity (Ki of 0.95nM) and high (80 fold) MC4 receptor selectivity over the MC3 receptor. HS028 thus shows both higher affinity and higher selectivity for the MC4 receptor compared to the earlier first described MC4 receptor selective substance HS014. 3. HS028 antagonised a alpha-MSH induced increase in cyclic AMP production in transfected cells expressing the MC3 and MC4 receptors, whereas it seemed to be a partial agonist for the MC1 and MC5 receptors. 4. Chronic intracerebroventricularly (i.c.v.) administration of HS028 by osmotic minipumps significantly increased both food intake and body weight in a dose dependent manner without tachyphylaxis for a period of 7 days. 5. This is the first report demonstrating that an MC4 receptor antagonist can increase food intake and body weight during chronic administration providing further evidence that the MC4 receptor is an important mediator of long term weight homeostasis.

Selective inhibition of adenylyl cyclase by octopamine via a human cloned alpha 2A-adrenoceptor.

1. In this study we have compared the abilities of the enantiomers of the structural isomers of the phenolamines, octopamine and synephrine, and the catecholamines, noradrenaline and adrenaline, to couple selectively a human cloned alpha 2A-adrenoceptor, stably expressed in a Chinese hamster ovary (CHO) cell line, to G-protein linked second messenger pathways mediating an increase and a decrease in cyclic AMP production. 2. The catecholamines couple the alpha 2A-adrenoceptor to both an increase and a decrease in the rate of cyclic AMP production. In the absence of pertussis toxin pretreatment both catecholamines tested showed a dose-dependent decrease with a maximum at 100 nM. After pertussis toxin pretreatment they both produced a dose-dependent increase in cyclic AMP production with a maximum at 10 microM. 3. The phenolamines, octopamine and synephrine were only able to couple the alpha 2A-adrenoceptor to a dose-dependent decrease in cyclic AMP production at concentrations up to 1 mM, with the synephrine isomers being more potent than the corresponding octopamine isomers. The meta-isomers of both phenolamines were more potent than the corresponding para-isomers and the (-)-enantiomers were more potent than the (+)-enantiomers. Thus, (-)-meta-synephrine [(-)-phenylephrine] was the most effective isomer tested with an observable decrease occurring between 100 nM and 1 microM. 4. The effects of octopamine and the catecholamines on the decrease in cyclic AMP production were additive at submaximal concentrations, whilst octopamine reduced the stimulant effect of submaximal concentrations of noradrenaline on cyclic AMP production after pertussis toxin pretreatment. 5. The time courses of the inhibitory effects of both meta-octopamine and noradrenaline were parallel and peaked after a 1 min exposure to the agonist. In contrast, the stimulant effects of noradrenaline after pertussis toxin pretreatment were of a much slower time course with a maximum effect occurring after a 5 min incubation period. 6. Since octopamine and synephrine occur naturally in, and are co-released with catecholamines from, mammalian tissues, the results of the present study suggest that the human cloned alpha 2A-adrenoceptor can be coupled selectively by different endogenous agonists to G-protein pathways mediating the regulation of adenylyl cyclase activity.

Inhibition of stimulated cyclic AMP production by multiple neuropeptide Y receptors in the rat brainstem

Neuropeptide Y (NPY) has been shown to modulate blood pressure, heart rate and to inhibit the baroreceptor reflex at the level of nucleus tractus solitarius (NTS). The aim of this study was to examine effects of NPY and its related peptides on forskolin (1 μM)-stimulated cyclic AMP production in slices of the rat NTS. Each peptide was present at 0.3 μM. Pretreatment with NPY inhibited the stimulated increase in cyclic AMP levels in slices of rat NTS. Also [Pro 34]NPY, an analog, which activates Y1, Y3 (and Y5) receptors inhibited the stimulated increase in cyclic AMP levels. However, pretreatment with the Y1 receptor-selective antagonist BIBP3226 (3 μM) did not affect the [Pro 34]NPY-evoked inhibition of cyclic AMP levels. In addition, [Leu 31,Pro 34]NPY, an Y1 (and PP1/Y4 and Y5) receptor agonist did not inhibit the stimulated increase in cyclic AMP production. Also the Y2 receptor-selective agonist C2-NPY inhibited the stimulated elevation of cyclic AMP levels, while peptide YY, which does not recognize Y3 receptors did not significantly affect the stimulated cyclic AMP production. In conclusion, it seems that Y2 and Y3 receptors are coupled to inhibition of adenylate cyclase activity in the rat NTS.

Myotropic activity of Acheta diuretic peptide on the foregut of the house cricket, Acheta domesticus (L.)

The myotropic activity and mode of action of an endogenous corticotropin-releasing factor (CRF)-related diuretic peptide ( Acheta-DP) was investigated using isolated denervated foreguts from the house cricket, Acheta domesticus. Acheta-DP increased the frequency and amplitude of contractions of the foregut at a threshold concentration of 3.74 ± 0.002 n m. The response to the peptide was unaffected by tetrodotoxin (TTX), and is likely to be a direct action on the muscle cells. Acheta-DP caused a 10-fold increase in cyclic AMP production by isolated foreguts. The ability of IBMX to potentiate the myotropic activity of Acheta-DP suggests the peptide acts via a cyclic AMP-dependent mechanism. In support of this, forskolin produced a peptide-like response, and enhanced the effect of Acheta-DP. Myotropic activity was dependent on extracellular calcium, and was inhibited by the L-type calcium channel antagonist, nifedipine. Bay K 8644 enhanced the response of the foregut to both Acheta-DP and to dibutyryl cyclic AMP, suggesting that the physiological response arose from calcium influx through L-type calcium channels. It is proposed that Acheta-DP binds to a receptor on muscle fibre membranes activating adenylyl cyclase. The resultant increase in intracellular cyclic AMP is thought to open L-type calcium channels and thereby stimulate gut contractions.

Glucagon facilitates adrenal catecholamine release mediated by nicotinic receptors but not by muscarinic receptors in anesthetized dogs.

We examined the effects of glucagon on the release of adrenal catecholamines (CAs) in response to splanchnic nerve stimulation (SNS), acetylcholine (ACh), the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), and muscarine in anesthetized dogs. Glucagon and the cholinergic drugs were infused and injected, respectively, into the adrenal gland through the phrenicoabdominal artery. SNS (1 and 3 Hz) or injections of ACh (1.5 and 3 micrograms), DMPP (1 and 2 micrograms), and muscarine (1 and 2 micrograms) produced a frequency- or dose-dependent increase in both epinephrine (EPI) and norepinephrine (NE) output, determined from adrenal venous blood. Glucagon infusion (0.1, 0.3, and 1 microgram/min) enhanced the SNS-, ACh-, and DMPP-induced increases in EPI and NE output in a dose-dependent manner but did not affect the muscarine-induced increases in CA output. The increase in basal CA output induced by the highest dose of glucagon was only slight. Glucagon increased cyclic AMP overflow determined from adrenal venous blood. Our results indicate that glucagon has a facilitatory action on adrenal CA release mediated by nicotinic receptors but not by muscarinic receptors in dogs and suggest that an increase in cyclic AMP production in adrenal medullary cells may be responsible for its selective action.

Inhibition by amphetamine of testosterone secretion through a mechanism involving an increase of cyclic AMP production in rat testes.

1. The effect of amphetamine on the secretion of testosterone and the production of testicular adenosine 3':5'-cyclic monophosphate (cyclic AMP) in rats was studied. 2. A single intravenous injection of amphetamine decreased the basal and human chorionic gonadotropin (hCG)-stimulated levels of plasma testosterone. Plasma LH levels were not altered by the injection of amphetamine. 3. Administration of amphetamine in vitro resulted in a dose-dependent inhibition of both basal and hCG-stimulated release of testosterone. 4. Amphetamine enhanced the basal and hCG-increased levels of cyclic AMP accumulation in vitro in rat testes. 5. These results suggest that amphetamine inhibits the spontaneous and hCG-stimulated secretion of testosterone from the testes through a mechanism involving an increase in cyclic AMP production.

In vitro effect of histamine and histamine H1 and H2 receptor antagonists on cellular proliferation of human malignant melanoma cell lines.

Histamine is an established growth factor for gastric and colorectal cancer. Contradictory data for response of melanoma to histamine have been reported. Our aims were to determine the effect of histamine and H1 and H2 receptor antagonists on cell growth and cyclic AMP production. Four human melanoma cell lines were cultured with a range of concentrations of histamine, and with the H2 receptor antagonists cimetidine, ranitidine or famotidine, or the H1 receptor antagonist diphenhydramine. Cellular proliferation was measured by the uptake of [3H]-thymidine. Cyclic AMP production was also measured to determine the receptor status of the cell lines. Histamine significantly stimulated growth in two of four cell lines, with maximal stimulation at 1 x 10(-8) M. This effect was inhibited by all four antagonists in a dose-dependent manner. Histamine [10(-7) to 10(-4) M] also induced a dose-dependent increase in cyclic AMP production in the two histamine-responsive cell lines, suggesting that these cell lines express H2 receptors. We conclude that there may be a role for histamine receptor antagonists in melanoma treatment and that further investigation is warranted.

Interleukin-1 β independently stimulates production of prostaglandin E 2 and cyclic AMP from human decidual cells

Interleukin-1 β (IL-1 β) increased the production of cyclic AMP and prostaglandin E 2 (PGE 2) by cultured human decidual cells during 24 h of stimulation, but not over short incubation times (< 6 h). At concentrations of IL-1 β ranging from 1 to 100 pg/ml, there were parallel changes in cyclic AMP and PGE 2 levels, but 1000 pg of IL-1 β/ml inhibited cyclic AMP production while still stimulating PGE 2 synthesis. The possible link between cyclic AMP and PGE 2 was therefore studied further. Inhibition of IL-1β-stimulated PGE 2 synthesis by indomethacin and direct addition of PGE 2 had no effect on cyclic AMP levels, indicating that PGE 2 did not increase cyclic AMP production by human decidual cells and confirming the independent synthesis of cyclic AMP and PGE 2. The increase in cyclic AMP production induced by IL-1 β is dependent on protein synthesis, but it is not known which component of the adenylate cyclase is increased. A phosphodiesterase inhibitor potentiated the effects of IL-1β on cyclic AMP synthesis, indicating that the cytokine may increase cyclic AMP metabolism. We suggest that high concentrations of IL-1β activate phosphodiesterase activity more than adenylate cyclase, which gives rise to the low levels of cyclic AMP noted above. IL-1 β also decreased forskolin-stimulated cyclic AMP production, which again indicates increased cyclic AMP metabolism. Since most concentrations of IL-1β alone increased cyclic AMP levels, this stimulation must out-weigh the increase in metabolism apparent in the presence of forskolin, phosphodiesterase inhibitor or high levels of interleukin. It is clear that IL-1β increased decidual PGE 2 production independently of cyclic AMP, and that other second messenger must mediate the action of this cytokine.

Quantification of signalling components and amplification in the β-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes

We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5'-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.

Adenosine A1 receptors mediate chronic ethanol-induced increases in receptor-stimulated cyclic AMP in cultured hepatocytes.

Cellular responses to adenosine depend on the distribution of the two adenosine receptor subclasses. In primary cultures of rat hepatocytes, adenosine receptors were coupled to adenylate cyclase via A1 and A2 receptors which inhibit and stimulate cyclic AMP production respectively. R-(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA), the adenosine A1 receptor-selective agonist, inhibited glucagon-stimulated cyclic AMP production with an IC50 of 19 nM. This inhibition was blocked by the A1-specific antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPDX). 5'-N- Ethylcarboxamidoadenosine (NECA), an agonist which stimulates A2 receptors, increased cyclic AMP production with an EC50 of 0.6 microM. Treatment of primary cultures of rat hepatocytes with 100 mM ethanol for 48 h decreases the quantity and function of the inhibitory guanine-nucleotide regulatory protein (G(i)), resulting in a sensitization of receptor-stimulated cyclic AMP production [Nagy and deSilva (1992) Biochem. J. 286, 681-686]. When cells were cultured with 2 units/ml adenosine deaminase, to degrade extracellular adenosine, ethanol-induced increases in cyclic AMP production were completely prevented. Moreover, the specific A1-receptor antagonist, CPDX, also blocked the chronic effects of ethanol on receptor-stimulated cyclic AMP production. Treatment with adenosine deaminase or CPDX also prevented the decrease in quantity of the alpha subunit protein of G(i) observed in hepatocytes after chronic treatment with ethanol. Taken together, these results suggest that activation of adenosine A1 receptors on primary cultures of hepatocytes is involved in the development of chronic ethanol-induced sensitization of receptor-stimulated cyclic AMP production.

Adaptive changes in the number of G s- and G i-proteins underlie adenylyl cyclase sensitization in morphine-treated rat striatal neurons

In the present study we investigated the possible role of changes in the number of membrane-bound G-proteins in the sensitization of dopamine D 1 receptor-stimulated adenylyl cyclase, observed in primary cultures of rat striatal neurons chronically exposed to morphine. Whereas exposure of these neurons to 10 μM morphine for 7 days caused a profound increase in cyclic AMP production, induced by the dopamine D 1 receptor agonist SKF 38393 (1 μM), Scatchard analysis of [ 125I]SCH 23982 binding to membrane preparations revealed that neither the B max nor the K d values of dopamine D 1 receptor binding sites were affected. Interestingly, immunoblotting experiments revealed an increase (of more than 50%) in the number of stimulatory G-proteins (G αs) in neurons displaying an enhanced adenylyl cyclase activity. In morphine-treated neurons, the number of inhibitory G-proteins (G αi) appeared to be slightly reduced (by about 16%). Moreover, the observation that cholera toxin (0.1 nM)-stimulated cyclic AMP production, unlike that induced by forskolin (1 μM), was enhanced in morphine-treated neurons, indicates a causal relationship between the reciprocal changes in G-protein number and the increase of dopamine D 1 receptor-stimulated adenylyl cyclase activity. The possible role of these changes in G-protein number in the development of morphine tolerance and dependence is discussed.

Calcitonin inhibits the growth of human gastric carcinoma cell line KATO III

Calcitonin has a wide variety of actions on gastrointestinal function. In this study, we investigated the effects of calcitonin on the growth of human gastric carcinoma cell line KATO III in comparison with those of calcitonin gene-related peptide (CGRP). Calcitonin, but not CGRP, significantly and dose-dependently inhibited the growth of KATO III cells. This inhibition of cell growth was accompanied by an increase in cyclic AMP production. The proliferation of KATO III cells was also inhibited by forskolin and dibutyryl cyclic AMP, although agents which do not stimulate cyclic AMP production had no effect. Furthermore, in the presence of GTP, calcitonin stimulated adenylate cyclase activity in KATO III cell membranes, and this increase was reduced in the absence of GTP. On the other had, neither calcitonin nor CGRP enhanced the turnover of inositolphospholipid or the intracellular Ca 2+ level. In addition, 125I-labeled human calcitonin was specifically bound to KATO III cell membranes, and this binding was dose-dependently displaced by unlabeled calcitonin but not CGRP. Furthermore, the specific binding of 125I-labeled human calcitonin to KATO III cell membranes was significantly reduced by addition of GTP but not ATP. These results suggest that calcitonin inhibits the growth of human gastric carcinoma cell line KATO III by stimulating cyclic AMP production via a GTP-dependent process coupled to specific calcitonin receptors.

Dynamics of cyclic-AMP efflux in relation to α-MSH secretion from melanotrope cells of Xenopus laevis

An important factor in regulating secretion from endocrine cells is the cytoplasmic concentration of cyclic-AMP. Many regulatory substances are known to either stimulate or inhibit the production of this second messenger through activation of their receptors. In the present study, we have monitored changes in cyclic-AMP efflux from melanotrope cells of Xenopus laevis in response to established neurochemical regulators of α-MSH secretion. In vitro superfusion of neurointermediate lobes allows for a dynamic recording of cyclic-AMP production in relation to hormone secretion. Unlike α-MSH secretion, the efflux of cyclic-AMP was not dependent on the concentration of extracellular calcium, indicating that hormone release and cyclic-AMP efflux are mediated by different mechanisms. The phosphodiesterase inhibitor IBMX and the adenylate cyclase activator forskolin stimulated cyclic-AMP efflux, but had no stimulatory effect on α-MSH release. This indicates that an increase in cyclic-AMP production in melanotrope cells is not necessarily accompanied by an increase in the rate of α-MSH release. Corticotropin-releasing factor stimulated cyclic-AMP efflux with dynamics similar to that induced by the amphibian peptide sauvagine. Dopamine and the GABA B receptor agonist baclofen both inhibited cyclic-AMP efflux and α-MSH release, with similar dynamics of inhibition and similar dose-response relationships. It is proposed that an inhibition of cyclic-AMP efflux is coupled to an inhibition of α-MSH secretion.