Forskolin-stimulated Adenylate Cyclase Research Articles

Growth hormone secretion is differently affected in genetically obese male and female rats.

Growth hormone (GH) secretion is markedly blunted in obesity. Reportedly, genetically obese Zucker rats show a reduced GH secretion due to an impaired function of hypothalamic neurons producing the GH-releasing hormone (GHRH). The aim of this work was: (1) to compare the in vitro GH responsiveness to GHRH in genetically obese female versus male Zucker rats and, (2) to evaluate the function of hypothalamic GHRH and somatostatin and of pituitary receptors for these neurohormones as assessed by the effectiveness of GHRH and somatostatin on adenylate cyclase (AC) activity. Baseline GH secretion of pituitaries obtained from male and female obese rats was not different and similar to that present in lean counterparts. Stimulation with 10(-7) M GHRH elicited a significantly lower GH secretion from the pituitaries of obese male rats but induced a similar GH secretion from the pituitaries of lean and obese female rats. In these pituitaries, GH concentration was similar in obese versus lean male and female rats [corrected]. A sex-related difference was also evidenced when plasma concentrations of somatomedin C (IGF-I) were evaluated. Obese male rats had lower IGF-I concentrations than lean counterparts, while this was not the case for obese versus lean female rats. Evaluation of AC activity following GHRH disclosed a lower activation in obese than in lean male rats, whereas in the females the enzyme activation was higher in obese than in lean animals. Conversely, the inhibitory effect of somatostatin on forskolin-stimulated AC was similar in pituitary membranes of obese and lean rats of both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y suppresses the neurogenic inflammatory response in the rabbit eye; mode of action.

Ocular injury in the rabbit causes miosis and breakdown of the blood aqueous barrier (aqueous flare response, AFR), reflecting a sensory nerve-mediated inflammatory response, elicited by the release of tachykinins and calcitonin gene-related peptide (CGRP) from C-fibers. Neuropeptide Y (NPY) occurs in sympathetic fibers in the eye. The study was designed to examine whether NPY and related peptides interfere with the inflammatory response to ocular injury in the rabbit in vivo. The isolated rabbit iris was studied with respect to NPY binding sites and second messenger coupling. The AFR and the miotic response to a standardized injury (infrared irradiation (IR) of the iris) were suppressed dose-dependently by NPY (0.01-1.0 nmol) injected intravitreally 30 min prior the trauma. The treated eye was compared with the contralateral eye, which received 0.9% saline and IR. The Y1 receptor agonist [Pro34]NPY, the Y2 receptor agonist NPY 13-36 and the structurally related peptide YY (1 nmol each) suppressed the AFR in response to IR. Injection of either NPY or the Y1 and Y2 receptor agonists (0.3 nmol each) suppressed the AFR evoked by exogenously applied CGRP (0.15 nmol). Saturation studies with 125I-NPY revealed both high and 'moderate' affinity binding sites in the iris. The Bmax values were 26 and 321 fmol/mg protein, respectively. NPY suppressed the forskolin-stimulated adenylate cyclase activity (IC50 value 19 nM). NPY did not affect basal or noradrenaline-induced accumulation of inositol phosphates in the iris. In conclusion, the rabbit iris seems to be rich in NPY receptors linked to inhibition of adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro Detection of Somatostatin Receptors in Human Tumors

Somatostatin receptors (SS-R) have been identified in membrane homogenates or tissue sections from several hundred human tumors. SS-R have been found in most neuroendocrine tumors, i.e. GH- and TSH-producing pituitary tumors, endocrine gastroenteropancreatic (GEP) tumors, paragangliomas, pheochromocytomas, medullary thyroid carcinomas (MTC) and small cell lung carcinomas. SS-R have also been found in the majority of malignant lymphomas, in several brain tumors (all meningiomas, most astrocytomas) and in breast tumors. The majority of tumors expressing SS-R are rather differentiated, e.g. astrocytomas in contrast to glioblastomas, but exceptions exist such as high grade malignant lymphomas. An inverse relationship exists between SS-R and receptors for epidermal growth factor in lung tumors, glial tumors and most breast tumors, whereas meningiomas express both receptors simultaneously. A minority of tumors such as ovarian tumors, MTC and insulinomas, express a subtype of SS-R, characterized by low affinity for the octapeptide SS analog octreotide. The function of SS-R in human tumors differs according to tumor type, SS-R in pituitary and GEP tumors mediate hormone secretion inhibition, and have possibly some antiproliferative effects. In meningiomas, however, activation of SS-R inhibits forskolin-stimulated adenylate cyclase activity, and weakly stimulates proliferation. Although SS-R seem to mediate antiproliferative effects in animal models and cell lines of lymphomas, breast and lung tumors, such an effect has not yet been convincingly documented in human primary tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the regulatory properties of striatal and cortical adenylate cyclase

—Biochemical properties of adenylate cyclase in striatal and cortical membranes have been analyzed in parallel with their regulation by cholinergic compounds. Striatal adenylate cyclase is more sensitive to forskolin, while the cortical enzyme is more stimulated by GTP. In the presence of GTP, more inhibition by acetylcholine is seen in the cortex than in the striatum. Acetylcholine inhibits striatal adenylate cyclase activity equally in the presence or absence of forskolin but has a diminished ability to inhibit forskolin-stimulated adenylate cyclase in the cortex. The greater sensitivity of cortical muscarinic receptor-coupled adenylate cyclase to EGTA and calcium indicates predominant involvement of the calcium/calmodulin-dependent subtype of the enzyme. The relative effectiveness of antagonists, demonstrating an order of potency of atropine > amitriptyline > pirenzepine > gallamine in reversing the inhibition of adenylate cyclase by acetylcholine for both brain regions, suggests predominantly m4 receptor-mediated responses. These results suggest an m4-type receptor may be coupled to subtypes of adenylate cyclase in the striatum and cortex which differ in their biochemical properties.

Role of altered G-protein expression in the regulation of myocardial adenylate cyclase activity and force of contraction in spontaneous hypertensive cardiomyopathy in rats.

The question of this study was whether alterations in the inhibitory guanine-nucleotide binding protein alpha-subunits (G(i)alpha) contribute to alterations in adenylate cyclase regulation in hypertensive cardiomyopathy of spontaneously hypertensive rats (SHR). G(i)alpha was measured by pertussis toxin-catalysed 32P-adenosine 5'-pyrophosphate (ADP)-ribosylation and radioimmunochemically by competition of rat myocardial membrane extracts to DS 4 antiserum binding to the 125I-radiolabelled C terminus of retinal transducin alpha (125I-KENLKDCGLF). Cardiac beta-adrenoceptors, m-cholinoceptors as well as isoprenaline-, guanine-nucleotide [Gpp(NH)p]- and forskolin-stimulated adenylate cyclase activity and inotropic responses to isoprenaline and carbachol were studied in SHR and age-matched Wistar-Kyoto (WKY, control) rats. In native membranes of SHR there was an increase in pertussis toxin substrates, but a larger increase in the presence of non-ionic detergent Lubrol PX. The radioimmunological quantification of G(i)alpha revealed an increase in membrane extracts of SHR. In addition, myocardial beta-adrenoceptors and myocardial m-cholinoceptors were reduced in SHR compared with in WKY rats. Basal adenylate cyclase, isoprenaline-, Gpp(NH)p- and forskolin-stimulated adenylate cyclase activities were also reduced. However, in the presence of 5 mmol/l MnCl2 no differences in adenylate cyclase activities between SHR and WKY rats were detected under either condition. The present study shows that the amount of G(i)alpha-proteins and not only pertussis toxin substrates are increased in membranes of hypertrophic hearts from SHR without heart failure. The results obtained with pertussis-labelling depended strongly on the substrate quality of G(i)alpha. Increased G(i)alpha expression and reduced beta-adrenoceptor number might have functional relevance in the regulation of adenylate cyclase activity and force of contraction in SHR. An increase in G(i)alpha expression might play a pathophysiological role, not only in terminal heart failure, but also in hypertrophic cardiomyopathy.

Ethanol increases receptor-dependent cyclic AMP production in cultured hepatocytes by decreasing G(i)-mediated inhibition.

Increasing evidence suggests that ethanol-induced changes in cyclic AMP (cAMP) signal transduction play a critical role in the acute and chronic effects of ethanol. Here we have investigated the effects of ethanol on cAMP signal transduction in primary cultures of rat hepatocytes. Acute exposure to ethanol had a biphasic effect on glucagon-receptor-dependent cAMP production in intact cells: 25-50 mM-ethanol decreased cAMP, whereas treatment with 100-200 mM-ethanol increased cAMP. After chronic exposure to 50-200 mM-ethanol for 48 h in culture, glucagon-receptor-dependent cAMP levels were increased, but no change in glucagon receptor number was observed. These effects of ethanol were independent of ethanol oxidation. Chronic ethanol treatment also increased adenosine-receptor- and forskolin-stimulated cAMP production. Increased cAMP production was also observed upon stimulation of adenylate cyclase with glucagon, forskolin and F- in membranes isolated from cells cultured with 100 mM-ethanol for 48 h. However, no differences were observed in basal and MnCl2-stimulated adenylate cyclase activity. The quantity of alpha i protein was decreased by 35% after chronic ethanol treatment, but no change in the quantity of alpha s protein was detected. Decreased alpha i protein was associated with a decrease in G(i) function, as assessed by the ability of 0.1 nM-guanosine 5'-[beta gamma-imido]triphosphate and 1 microM-somatostatin to inhibit forskolin-stimulated adenylate cyclase activity. Taken together, these results suggest that chronic exposure to ethanol increases receptor-dependent cAMP production in hepatocytes by decreasing the quantity of alpha i protein at the plasma membrane and thereby decreasing the inhibitory effects of G(i) on adenylate cyclase activity.

Somatostatin receptors in human cancer: Incidence, characteristics, functional correlates and clinical implications

Somatostatin receptors (SS-R) have been identified in membrane homogenates or tissue sections from several hundred tumors. SS-R were found in most neuroendocrine tumors, i.e. GH and TSH producing pituitary tumors, endocrine gastroenteropancreatic (GEP) tumors, paragangliomas, pheochromocytomas, medullary thyroid carcinomas (MTC) and small cell lung carcinomas. SS-R were also expressed in a majority of malignant lymphomas, in several brain tumors (all meningiomas, most astrocytomas) and in breast tumors. The majority of tumors expressing SS-R are rather differentiated (i.e. astrocytomas vs glioblastomas), but exceptions exist (high grade malignant lymphomas). An inverse relationship exists between SS-R and receptors for epidermal growth factor (EGF-R) incidence in lung tumors, glial tumors and most breast tumors, whereas meningiomas express simultaneously both receptors. A minority of tumors (ovarian tumors, MTC, insulinomas) express a subtype of SS-R, characterized by low affinity for the octapeptide SS analog octreotide. The function mediated by SS-R in human tumors may differ according to the tumor type. SS-R in pituitary and GEP tumor mediate hormone secretion inhibition with, in addition, possibly some antiproliferative effects. In meningiomas, however, activation of SS-R inhibits forskolin-stimulated adenylate cyclase activity, and weakly stimulates proliferation. Whereas SS-R seem to mediate antiproliferative effects in animal models and cell lines of lymphomas, breast and lung tumors, such an effect has not yet been convincingly documented in human primary tumors. The clinical implications of the presence of SS-R in tumors are manyfold: (1) as a predictive marker for efficient therapy with octreotide in pituitary and GEP tumors; (2) as a diagnostic marker: for pathobiochemical classification of tumors, using in vitro detection methods; for clinical evaluation using in vivo scanning techniques; (3) as a prognostic marker; and (4) as a potential radiotherapeutic target.

Cytokine regulation of adenylate cyclase activity in LLC-PK1 cells

Although several cytokines have been demonstrated to exert pleiotropic responses, there is little information on cytokine regulation of renal tubular epithelial cell function. In the present studies, we find that both T cell-derived (tumor necrosis factor-beta and interleukins 2 and 3) and monocyte/macrophage derived (tumor necrosis factor alpha and interleukin 1 beta) cytokines promote basal, arginine vasopressin- and forskolin-stimulated adenylate cyclase activity in cultured LLC-PK1 cells. No effect of TNF, IL-1 beta, and IL-2 to stimulate protein kinase C activity was observed. TNF-beta, IL-1 beta and IL-2 also modestly stimulated 3H release from 3H-arachidonic acid labeled cells. Mepacrine, a phospholipase A inhibitor, prevented TNF-beta stimulation of 3H release from 3H-arachidonic acid labeled cells and TNF-beta potentiation of adenylate cyclase activity. TNF-beta potentiation of adenylate cyclase activity and stimulation of 3H release from 3H arachidonic acid labeled cells was not prevented by pertussis toxin. These results demonstrate that several cytokines can stimulate adenylate cyclase activity while not affecting protein kinase C activity in cultured renal tubular epithelial cells. The effect of TNF-beta to stimulate adenylate cyclase appears to occur independent of pertussis toxin-sensitive substrate and may involve activation of phospholipase A.

Prolonged activation of inhibitory somatostatin receptors increases adenylate cyclase activity in wild-type and G sα-deficient (cyc −) S49 mouse lymphoma cells

Many cells develop enhanced adenylate cylcase activity after prolonged exposure to drugs that acutely inhibit the enzyme and it has been suggested that this adaptation may be due to an increase in G sα. We have treated wild-type and G sα-deficient cyc − S49 mouse lymphoma cells with a stable analogue (SMS 201–995) of the inhibitory agonist somatostatin. After incubation with SMS for 24 h, the forskolin-stimulated cAMP synthetic rate in intact cyc − cells was increased by 76%, similar to the increase found in the wild-type cells. Forskolin-stimulated adenylate cyclase activity in the presence of Mn 2+ was also increase in membranes prepared from SMS-treated cyc −cells; however, guanine nucleotide-mediated inhibition of adenylate cyclase activity was not changed despite a small increase in inhibitory G iα subunits detected by immunoblotting. Pretreatment of cyc − cells with pertussis toxin prevented SMS from inducing the enhancement of forskolin-stimulated cAMP accumulation in intact cells. After chronic incubation of cyc − cells with SMS, exposure to N-ethylmaleimide, which abolished receptor-mediated inhibition of cAMP accumulation, did not attenuate the enhanced rate of forskolin-stimulated cAMP synthesis compared to N-ethymaleimide-treated controls. These results with cyc − cells demonstrate that an adaptive increase in adenylate cyclase activity induced by chronic treatment with an inhibitory drug can occur in the absence of expression of G sα.

In vitro detection of somatostatin receptors in human tumors

Somatostatin receptors (SSR) have been identified in membrane homogenates or tissue sections from several hundred human tumors. SSR have been found in most neuroendocrine tumors, ie, growth hormone (GH)- and thyrotropin (TSH)-producing pituitary tumors, endocrine gastroenteropancreatic (GEP) tumors, paragangliomas, pheochromocytomas, medullary thyroid carcinomas (MTC), and small-cell lung carcinomas. SSR have also been found in the majority of malignant lymphomas, in several brain tumors (all meningiomas, most astrocytomas), and in breast tumors. The majority of tumors expressing SSR are rather differentiated, eg, astrocytomas in contrast to glioblastomas, but exceptions such as high-grade malignant lymphomas do exist. An inverse relationship exists between SSR and receptors for epidermal growth factor in lung tumors, glial tumors, and most breast tumors, whereas meningiomas express both receptors simulataneously. A minority of tumors such as ovarian tumors, MTC, and insulinomas express a subtype of SSR characterized by low affinity for the octapeptide SS analogue, octreotide. The function of SSR in human tumors differs according to tumor type; SSR in pituitary and GEP tumors mediate hormone secretion inhibition and possibly have some antiproliferative effects. However, in meningiomas, activation of SSR inhibits forskolin-stimulated adenylate cyclase activity and weakly stimulates proliferation. Although SSR seem to mediate antiproliferative effects in animal models and cell lines of lymphomas and breast and lung tumors, such an effect has not yet been convincingly documented in human primary tumors. The clinical implications of the presence of SSR in tumors are as follows: as a predictive marker for efficient therapy with octreotide in pituitary and GEP tumors; as a diagnostic marker for pathobiochemical classification of tumors using in vitro detection methods; as a diagnostic marker for clinical evaluation using in vivo scanning techniques; as a prognostic marker; and as a potential target for radiotherapy.

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.

Serotonin inhibits adenylate cyclase in human platelet membranes

Serotonin (5-HT), the non-specific 5-HT 1 receptor agonist methysergide, and the 5-HT 1B receptor agonist CGS 12066 dose-dependently inhibited forskolin-stimulated adenylate cyclase activity membranes derived from human platelets. Buspirone and ipsapirone showed partial agonist activity. The effect of 5-HT to inhibit forskolin-stimulated activity was partially antagonised by spiperone, yohimbine and pindolol, with each of these compounds showing some partial agonist activity. It is concluded that human platelet membranes possess receptors for 5-HT coupled negatively to adenylate cyclase. The relationship of these receptors to other binding sites which have been shown for 5-HT on platelet membranes remains to be finally elucidated.

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.

Desensitization of adenylate cyclase and increase of Gi alpha in cardiac hypertrophy due to acquired hypertension.

The present study investigated whether reduced adenylate cyclase activity and an increase in inhibitory guanine nucleotide binding proteins (Gi alpha), which have been observed in the failing human heart, already occur in myocardial hypertrophy before the stage of heart failure. In membranes of hypertrophic hearts from rats with different forms of experimentally induced hypertension without heart failure (one-kidney, one clip rats, deoxycorticosterone-treated rats, and rats with reduced renal mass), basal as well as isoprenaline-, 5'-guanylylimidodiphosphate-, and forskolin-stimulated adenylate cyclase activity was reduced. The activity of the catalyst was depressed in deoxycorticosterone but unchanged in one-kidney, one clip and reduced renal mass compared with controls. The number of beta-adrenergic receptors was similar in all groups. Radioimmunological quantification of Gi alpha proteins revealed an increase by 73% in one-kidney, one clip, 67% in reduced renal mass, but only 20% in deoxycorticosterone compared with sham-operated, age-matched control rats. The increase of Gi alpha was accompanied by smaller changes of pertussis toxin-induced [32P]ADP-ribosylation of a 40-kd membrane protein. It is concluded that Gi alpha contributes to the reduced adenylate cyclase activity in cardiac hypertrophy in one-kidney, one clip and reduced renal mass and to a smaller extent in deoxycorticosterone. It is suggested that an enhanced expression of Gi alpha could occur not only in severe heart failure but also in cardiac hypertrophy and could, therefore, contribute to myocardial depression and progression of disease in heart failure. In addition, Gi alpha might represent an important regulatory mechanism for cardiac adenylate cyclase activity and thus, might play an important role in various cardiac diseases.

B-HT 920 activates dopamine D2 receptors coupled to inhibition of adenylate cyclase activity.

In homogenates of female rat anterior pituitary, the azepine derivative B-HT 920 inhibited the forskolin-stimulated adenylate cyclase activity with an EC50 value of 0.35 microM. In male rat anterior pituitary, B-HT 920 curtailed the stimulation of adenylate cyclase activity by vasoactive intestinal peptide with an EC50 of 0.20 microM. In synaptic plasma membranes of rat striatum, B-HT 920 significantly reduced basal adenylate cyclase activity with an EC50 of 0.68 microM. Both in pituitary and striatum, the B-HT 920 inhibition was counteracted by the dopamine (DA) D2 receptor antagonist 1-sulpiride, but not by the alpha 2-adrenergic antagonist yohimbine. These results indicate that B-HT 920 is capable of activating DA D2 receptors negatively coupled to adenylate cyclase activity.

Opioid receptor-mediated inhibition of 3H-dopamine and 14C-acetylcholine release from rat nucleus accumbens slices. A study on the possible involvement of K+ channels and adenylate cyclase.

The release of 14C-ACh from rat nucleus accumbens slices, induced by 15 mM [K+], was inhibited by the mu- and delta-opioid agonists DAMGO and DPDPE, respectively, whereas only the kappa agonist U50,488 reduced the release of 3H-DA. The opioid receptors involved appear to be localized on nerve terminals, since blockade of action potential propagation by 1 microM TTX did not diminish the inhibitory effects of DAMGO, DPDPE or U50,488. Enhancement of the potassium concentration in the superfusion medium to 56 mM with simultaneous reduction of the Ca2+ concentration from 1.2 mM to 0.12 mM induced a release similar to that caused by 15 mM K+ and 1.2 mM Ca+. Under this conditions, the inhibitory effects of both DAMGO and DPDPE on stimulated 14C-ACh release were reduced, whereas the inhibition of evoked 3H-DA release caused by U50,488 was not affected. Activation of mu- as well as delta-opioid receptors by DAMGO and DPDPE, respectively, inhibited forskolin-stimulated adenylate cyclase activity. However, increasing the intracellular cAMP levels with 0.3 mM 8-bromo-cAMP affected neither the depolarization-induced release of 14C-ACh or 3H-DA from accumbens slices nor the inhibitory effects of opioid receptor activation thereon. The results indicate that the mechanism by which functional mu and delta receptors presynaptically inhibit the depolarization-induced 14C-ACh release from nucleus accumbens slices is likely to involve an increase of potassium channel conductance. In contrast, activation of kappa-opioid receptors, which inhibits depolarization-evoked 3H-DA release, apparently does not result in a hyperpolarization of (dopaminergic) nerve terminals. In none of these inhibitory effects presynaptic adenylate cyclase appears to be involved.

Activation of G proteins by (Rp) and (Sp) diastereomers of guanosine 5'-[beta-thio]triphosphate in hamster fibroblasts. Differential stereospecificity of Gi, Gs and Gp.

The effects of guanosine 5'-[beta-thio]triphosphate (GTP beta[S]) on G proteins have been examined in Chinese hamster lung fibroblasts (CCL39 line) permeabilized with alpha-toxin from Staphylococcus aureus. Although much less effective than guanosine 5'-[gamma-thio]triphosphate (GTP gamma[S]), both (Rp) and (Sp) diastereomers of GTP beta[S] were found to activate three G protein-mediated pathways: inhibition of forskolin-stimulated adenylate cyclase (mediated by Gi), potentiation of receptor-mediated activation of adenylate cyclase (mediated by Gs), and activation of phosphoinositide breakdown (mediated by Gp). Activation of Gi and Gs occurred above 3 microM-GTP beta[S], but activation of Gp only occurred above 100 microM-GTP beta[S]. Moreover, the order of effectiveness of the two diastereomers was not the same for the three G protein-mediated processes. Whereas both Gi and Gs were more effectively activated (about 5-fold) by (Sp)-GTP beta[S] than by (Rp)-GTP beta[S], Gp showed a marked preference for the (Rp) isomer. Indeed, (Rp)-GTP beta[S] induced the formation of inositol phosphates with a shorter latency and was a better competitor of GDP for binding to Gp than the (Sp) isomer. These results point to different guanine nucleotide-binding properties for Gi and Gs on the one hand and Gp on the other. At least two distinct Gp proteins, differing by their sensitivity to pertussis toxin, are present in CCL39 cells. Since pretreatment of cells with pertussis toxin completely suppressed the effects of (Rp)-GTP beta[S] on Gi, while only slightly attenuating its effects on Gp, we believe that it is the pertussis toxin-insensitive Gp which prefers the (Rp) isomer. Therefore (Rp)-GTP beta[S] may be a valuable tool for the selective activation and the biochemical characterization of this pertussis toxin-insensitive Gp.

Proenkephalin a derivatives in lacrimal gland: Occurrence and regulation of lacrimal function

The derivatives of proenkephalin A were measured in acid extracts of rat lacrimal glands by specific radioimmunoassay. Glands from adult male rats contained all four derivatives of the opiate precursor. The content in the gland of proenkephalin A-derived peptides Met 5-enkephalin, Leu 5-enkephalin, Met 5-enkephalin Arg 6-Phe 7 and Met 5-enkephalin Arg 6-Gly 7-Leu 8 indicates tissue specific processing with an enhancement of the heptapeptide. The effect of enkephalins on the activity of adenylate cyclase in lacrimal membranes was measured and compared with the effect of the synthetic enkephalin analogue d-ala 2-methionine enkephalinamide (DALA) that inhibits both lacrimal protein secretion and lacrimal adenylate cyclase. In the presence of the peptidase inhibitors thiorphan and bestatin, the inhibition of forskolin-stimulated adenylate cyclase activity by Met 5-enk, Leu 5-enk, Met 5-enk Arg 6-Phe 7 and DALA were identical. Maximum inhibition was approximately 35% at a dose of 50μ m enkephalin. Addition of the octapeptide, Met 5-enk Arg 6-Gly 7-Leu 8 resulted in decreased adenylate cyclase activity; however, the effect was not statistically significant. Activation of δ opioid receptors by the endogenous enkephalins is indicated by the reversal of adenylate cyclase inhibition in the presence of the δ-receptor antagonist ICI 174864. The data support the physiological significance of in vitro inhibition of lacrimal secretion by DALA and indicate a possible role for endogenous enkephalins in lacrimal function.

Involvement of multiple sulfhydryl groups in melatonin signal transduction in chick brain

To gain insight into the molecular mechanism underlying melatonin binding and signal transduction in the chick brain, we have investigated the role of -SH groups, using a sulfhydryl alkylating reagent N-ethylmaleimide (NEM). At least two -SH groups are involved in the formation of the receptor-G protein complex: one is sensitive to and the other relatively insensitive to NEM. Alkylation of the sensitive group selectively abolishes high affinity binding of 2-[ 125I]iodomelatonin ([ 125I]MEL), similar to the effect induced by GTP, thus leading to a complete loss of sensitivity to nucleotides. Modification of both groups causes a marked reduction in binding capacity. Agonists with high affinity, but not other compounds with low affinity for the melatonin receptor, protect against alkylation by NEM. GTPγs does not significantly alter the reactivity of -SH groups towards NEM, but agonist-protected receptors remain sensitive to this nucleotide. Moreover, NEM pretreatment blocks the inhibitory effect of melatonin on forskolin-stimulated adenylate cyclase activity in chick brain. These data suggest that the -SH group modulating agonist affinity may lie within the coupling domain between the receptor and G protein but outside of the GTP binding site. In addition, sulfhydryl groups are essential for melatonin binding and signal transduction in chick brain.

Expression of nanomolar-affinity binding sites for melatonin in Syrian hamster RPMI 1846 melanoma cells

Pharmacological studies indicate that Syrian hamster melanoma (RPMI 1846) cells posses a melatonin binding site similar to that found in normal hamster cells. A high correlation was observed for a series of compounds between the K i in hamster hypothalamic membranes vs. RPMI 1846 membranes ( r = 0.94, slope = 0.93, P < 0.01, n = 14). Scatchard analysis of saturation binding of 2-[ 125I]-iodomelatonin to membranes (at 0°C) indicated: K d = 0.89 ± 0.08 nM, B max = 6.2 ± 2.9 fmol/mg protein ( n = 3). Melatonin did not alter basal or forskolin-stimulated adenylate cyclase activity in RPMI 1846 membranes or intact cells. Therefore, in contrast to the picomolar-affinity receptor for melatonin in the mammalian hypothalamus and pars tuberalis, the putative nanomolar-affinity receptor is not coupled to adenylate cyclase. The RPMI 1846 cell line provides a useful model system for further studies of signal transduction via the nanomolar-affinity site for melatonin.