Characterization of Dopamine and β-Adrenergic Receptors Linked to Cyclic AMP Formation in Intact Cells of the Clone D384 Derived from a Human Astrocytoma

3,4-Dihydroxyphenylethylamine (dopamine) and beta-adrenergic receptor agonists and antagonists were assessed for their effects on cyclic AMP accumulation in human astrocytoma derived clone D384 cells. Dopamine, SKF 38393, and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene increased cyclic AMP content with Ka values of 2.0, 0.2, and 1.6 microM. The D1-selective antagonists SCH 23390 (Ki, 1.2 nM) and SKF 83566 (Ki, 0.8 nM) were over 5,000-fold more potent than the D2-selective antagonist domperidone (Ki, 6.7 microM) at inhibiting dopamine stimulation of cyclic AMP formation. SCH 23388 (Ki, 560 nM; the S-enantiomer of SCH 23390) was 400-fold less potent than SCH 23390. Isoprenaline, adrenaline, salbutamol, and noradrenaline increased cyclic AMP content with Ka values of 0.13, 0.12, 0.22, and 7.60 microM. The beta 2-selective antagonist ICI 118,551 (Ki,0.8 nM) was almost 8,000-fold more potent than the beta 1-selective antagonist practolol (Ki, 5.9 microM) at inhibiting isoprenaline stimulated cyclic AMP accumulation. These results demonstrate that D384 cells express D1-dopamine and beta 2-adrenergic receptors linked to adenylate cyclase. Furthermore, the dopamine receptor expressed by D384 cells exhibits a pharmacological profile typical of a mammalian striatal D1-receptor and therefore the use of this clone represents another approach to studying central D1-receptors.     

Regulation of Cyclic AMP Formation in Cultures of Human Foetal Astrocytes by β2-Adrenergic and Adenosine Receptors

Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline greater than adrenaline greater than salbutamol much greater than noradrenaline, which is consistent with the presence of beta 2-adrenergic receptors. This study reports that the beta 2-adrenergic-selective antagonist ICI 118,551 is approximately 1,000 times more potent at inhibiting isoprenaline stimulation of cyclic AMP (cAMP) formation in both NEP2 and NEM2 than the beta 1-adrenergic-selective antagonist practolol. This observation confirms the presence of beta 2-adrenergic receptors in these cell cultures. The formation of cAMP in NEP2 is also stimulated by 5'-(N-ethylcarboxamido)adenosine (NECA) more potently than by either adenosine or N6-(L-phenylisopropyl)adenosine (L-PIA), which suggests that this foetal astrocyte expresses adenosine A2 receptors. Furthermore, L-PIA and NECA inhibit isoprenaline stimulation of cAMP formation, a result suggesting the presence of adenosine A1 receptors on NEP2. The presence of A1 receptors is confirmed by the observation that the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine reverses the inhibition of isoprenaline stimulation of cAMP formation by L-PIA and NECA. Additional evidence that NEP2 expresses adenosine receptors linked to the adenylate cyclase-inhibitory GTP-binding protein is provided by the finding that pretreatment of these cells with pertussis toxin reverses the adenosine inhibition of cAMP formation stimulated by either isoprenaline or forskolin.     

Cyclic AMP Concentrations in Rat Neocortex and Hippocampus During and Following Incomplete Ischemia: Effects of Central Noradrenergic Neurons, Prostaglandins, and Adenosine

The concentrations of cyclic AMP, noradrenaline, glycogen, glucose, lactate, pyruvate, labile phosphate compounds, and free fatty acids were investigated in the rat neocortex and hippocampus during and following cerebral ischemia. An incomplete ischemia of 5 and 15 min duration was induced by bilateral carotid clamping combined with hypotension. The postischemic events were studied after 5, 15, and 60 min of recirculation. Five minutes of ischemia did not significantly alter the neocortical or hippocampal concentrations of cyclic AMP. After 15 min of ischemia the neocortical levels decreased significantly below control values. In the recirculation period following ischemia a significant elevation of the cyclic AMP concentrations was observed. Following 5 min of recirculation after 5 min of ischemia the levels increased from 2.53 +/- 0.21 nmol X g-1 to 5.18 +/- 0.09 nmol X g-1 in the neocortex and from 2.14 +/- 0.16 nmol X g-1 to 3.52 +/- 0.35 nmol X g-1 in the hippocampus. Five minutes of recirculation following 15 min of ischemia led to a significant increase in the levels of cyclic AMP, to 12.86 +/- 1.43 nmol X g-1 in the neocortex to 5.58 +/- 0.57 nmol X g-1 in the hippocampus. With longer recirculation periods the cyclic AMP levels progressively decreased and were similar to control values after 60 min. Depletion of cortical noradrenaline by at least 95% was performed by injections of 6-hydroxydopamine into the ascending axon bundles from the locus ceruleus. The lesion did not significantly change the ischemic or post-ischemic neocortical and hippocampal levels of cyclic AMP, glycogen, or free fatty acids including arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes induced in adrenergic lipolysis by the administration of diethylstilboestrol, oestradiol and clomiphene

The authors studied changes in adrenergic lipolysis in the epididymal adipose tissue of rats to which diethylstilboestrol and oestradiol combined with the anti-oestrogen clomiphene were administered. The maximum lipid-mobilizing effect of isoprenaline was increased not only by subcutaneously administered diethylstilboestrol, but also by the highest dose of the antioestrogen clomiphene used (p.o., 200 micrograms.kg-1 b.w.). Under the given experimental conditions, with 4 1/2 h incubation of adipose tissue, clomiphene was also effective when added in vitro. Its own oestrogenic effect probably stimulated the lipid-mobilizing action of isoprenaline. On combining the administration of increasing doses of clomiphene (p.o., 1-5 days) with a constant dose of oestradiol (200 micrograms.kg-1, s.c. on the 8th day, i.e. 24 h before the actual experiment), changes in isoprenaline lipolysis depended on the dose of clomiphene. In low doses clomiphene inhibited the stimulating effect of subsequently administered oestradiol on isoprenaline-induced lipolysis, but in large doses (100 and 200 micrograms.kg-1 daily) it potentiated, together with oestradiol, the lipid-mobilizing effect of isoprenaline. The results show that the non-steroid oestrogen diethylstilboestrol and the antioestrogen clomiphene may be included among the hormones capable of altering the response of adipose tissue to sympathomimetics (isoprenaline). We attribute the fact that clomiphene acted either as an antagonist or as an agonist of oestradiol to its combined oestrogenic and anti-oestrogenic effects.     

IN VIVO CHANGES OF CEREBRAL CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE INDUCED BY BIOGENIC AMINES: ASSOCIATION WITH PHOSPHORYLASE ACTIVATION

—The intravenous injection of adrenaline, isoprenaline and histamine to 4-6-day-old chicks resulted in a rapid increase in the cyclic AMP content of cerebral hemispheres that had been removed and frozen within 0·5 s using a freeze-blowing technique. Noradrenaline, dopamine, adenosine, 5-HT and acetylcholine did not significantly alter the nucleotide concentration in vivo. Addition of adrenaline, isoprenaline and histamine to incubated chick cerebral cortex slices also increased the cyclic AMP content of the tissue. Noradrenaline was considerably less potent than these amines and adenosine was ineffective. Low phosphorylase a levels (16 per cent of total activity) were observed in instantaneously frozen cerebral hemispheres of untreated chicks. The injection of adrenaline, isoprenaline and histamine resulted in a rapid conversion of phosphorylase b to a and a significant fall in tissue glycogen. Administration of noradrenaline was without effect on the relative forms of phosphorylase and also failed to influence cerebral glycogen. Phosphorylase activation was not observed in chick cerebral slices under conditions producing large increases in cyclic AMP. It is suggested that in vivo phosphorylase activation and subsequent glycogenolysis may occur, at least in part, in glia and that these cells may be damaged during preparation of cerebral slices. The results provide evidence of a metabolic role for cyclic AMP in cerebral tissue.     

Estradiol-17 beta inhibition of lutropin and dibutyryl cyclic AMP induced steroidogenesis in intact bovine luteal cells. Absence of effect on induced protein phosphorylations

Estradiol-17 beta is known to inhibit in a dose dependent manner the lutropin-induced stimulation of progesterone synthesis in luteal cells without affecting the intracellular cyclic AMP increase produced by the hormone. The hypothesis that this inhibitory action could involve an inhibition of the cyclic AMP dependent phosphorylation of cytosolic proteins was investigated by using incubations of selected small bovine luteal cells. Doses of 10 and 100 micrograms/ml of estradiol-17 beta inhibited respectively 60 and 90% of the progesterone synthesis induced by lutropin as well as by dibutyryl cyclic AMP in small bovine luteal cells. At the concentrations of 10 and 100 micrograms/ml, estradiol-17 beta was unable to affect the cyclic AMP dependent protein kinase activation induced by lutropin. At the concentration of 10 micrograms/ml the steroid was without effect on the lutropin or dibutyryl cyclic AMP induced protein phosphorylations. However 100 micrograms/ml of estradiol-17 beta seemed to produce a slight inhibition of the induced protein phosphorylations.     

Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.     

Hypoxia Increases the Cyclic AMP Content of the Cat Carotid Body In Vitro

The cyclic AMP content of cat carotid bodies in vitro measured with a radioimmunoassay under control conditions (PO2: 230 torr) was 0.79 +/- 0.10 pmol/carotid body (n = 10). Lowering medium PO2 to 20 torr for 2 min significantly increased cyclic AMP content to 1.13 +/- 0.14 pmol/carotid body (n = 10). This increase was inhibited neither by propranolol (34 microM) nor by propranolol plus haloperidol (27 microM). Inhibition of the cyclic nucleotide phosphodiesterase with 1-methyl-3-isobutylxanthine (0.8 mM) provoked a fast and large increase in cyclic AMP during both control and hypoxic conditions. The cyclic AMP increase induced by hypoxia was still observed when extracellular Ca2+ was absent. Inhibition of the adenylate cyclase by N-(cis-2-phenylcyclopentyl)azacyclotridecan-2-imine hydrochloride (MDL 12330A; 20-1,000 microM) under zero-Ca2+ conditions irreversibly inhibited the cyclic AMP increase produced by hypoxia. Similarly, inhibition of the Ca2(+)-calmodulin complex by trifluoperazine (0.2 mM) or calmidazolium (R 24571; 50-200 microM) prevented the cyclic AMP response. These results suggest that cyclic AMP may be involved in the PO2-sensing mechanism of the carotid body. Hypoxia appears to activate adenylate cyclase directly and independent of any hormone-receptor interactions.     

Characterization of beta-adrenoceptors responsible for venom production in the venom gland of the snake Bothrops jararaca

We have shown that the stimulation of beta-adrenoceptors is an important step in venom production in the Bothrops jararaca venom gland. In the present study, the pharmacological profile of the beta-adrenoceptor present in Bothrops jararaca venom gland was characterized by radioligand binding assay and by the ability of isoprenaline to promote accumulation of cyclic AMP in dispersed secretory cells. In both cases, the venom glands were obtained from non-extracted snakes (quiescent stage) or from snakes which venom was extracted 4 days before sacrifice (venom production stimulated stage). [125I]-iodocyanopindolol ([125I]-ICYP) bound to extracted gland membranes in a concentration-dependent and saturable manner, but with low affinity. Propranolol, beta1- or beta2-selective adrenoceptors ligands displaced the [125I]-ICYP binding with low affinity, while selective beta3-adrenoceptor ligands did not displace the [125I]-ICYP binding. The displacement of [125I]-ICYP by propranolol was similar in non-extracted and extracted glands, showing the presence of beta-adrenoceptors in both stages. In dispersed secretory cells of non-extracted glands, isoprenaline (1 microM) increased the cyclic AMP production and propranolol (10 microM) was able to block this effect. On the other hand, in extracted glands, isoprenaline had no effect. The results suggest that the beta-adrenoceptors present in the Bothrops jararaca venom glands are different from those (beta1, beta2 or beta3) described in mammals, but are coupled to the Gs protein, like the known beta-adrenoceptor subtypes. Moreover, previous in vivo stimulation of venom production desensitizes the beta-adrenoceptors system and, although the receptors could be detected by binding studies, they are not coupled to the Gs protein, indicating that beta-adrenoceptors stimulation contributes to the initial steps of venom synthesis.     

Mannose, glucosamine and inositol monophosphate inhibit the effects of insulin on lipogenesis. Further evidence for a role for inositol phosphate-oligosaccharides in insulin action.

The mechanism of insulin signalling is not yet understood in detail. Recently, a role for inositol phosphate (IP)-oligosaccharides as second messengers transmitting the insulin signal at the post-kinase level was proposed. To evaluate this hypothesis further, we studied whether IP-oligosaccharides isolated from 'haemodialysate' have insulin-like activity. We found that these compounds mimic, in a dose-dependent fashion, the following effects of insulin in adipocytes. (1) Lipogenesis. Incorporation of [3H]glucose into lipids (expressed in nmol/min per 10(6) cells): basal, 0.74 +/- 0.05; insulin (1 mu unit/ml), 4.43 +/- 0.21; IP-oligosaccharide (2 micrograms/ml), 4.07 +/- 0.19. (2) Inhibition of isoprenaline (isoproterenol) (1 microM)-stimulated cyclic AMP levels and lipolysis. Cyclic AMP (pmol/10(5) cells): basal 0.84 +/- 0.05; isoprenaline, 4.03 +/- 0.19; isoprenaline + insulin (200 mu units/ml), 2.06 +/- 0.7; isoprenaline + IP-oligosaccharides (2 micrograms/ml), 2.4 +/- 0.29. Inhibition of lipolysis (mumol of glycerol/mg of protein): isoprenaline (1 microM), 166 +/- 11; isoprenaline+insulin (150 mu units/ml), 53 +/- 3.5; isoprenaline+IP-oligosaccharides (2 micrograms/ml), 58 +/- 5. (3) Stimulation of 3-O-methylglucose transport; basal, 9 +/- 3%; insulin (1 mu unit/ml), 67 +/- 4%, IP-oligosaccharides (2 micrograms/ml), 54 +/- 2%. To identify the active molecules of the IP-oligosaccharide fraction, competition experiments were performed. IP-oligosaccharide effects on lipogenesis were blocked by inositol monophosphate, glucosamine and mannose. In contrast, these compounds did not inhibit IP-oligosaccharide effects on membrane-mediated functions (3-O-methylglucose transport, cyclic AMP levels, lipolysis). We also found that the effect of insulin on lipogenesis was blocked by mannose, glucosamine and inositol monophosphate, whereas the insulin effects on 3-O-methylglucose, cyclic AMP and lipolysis were unaffected. The following conclusions were reached. (1) IP-oligosaccharides mimic the major metabolic effects of insulin in adipocytes. This is consistent with the proposed role of IP-oligosaccharides as second messengers of certain insulin effects. (2) Mannose and glucosamine are functionally important sugar residues for the effect of IP-oligosaccharide on lipogenesis. (3) The observation that mannose, inositol monophosphate and glucosamine block the action of insulin of on lipogenesis supports a role of mannose- and glucosamine-containing IP-oligosaccharides as second messengers for this insulin effect.     

Esmolol: effects on isoprenaline- and exercise-induced cardiovascular stimulation in conscious dogs

Esmolol, a recently developed ultra-short acting beta-adrenoceptor blocking agent, was evaluated in 12 conscious chronically instrumented dogs with intact autonomic reflexes. The significance of its beta 1-adrenoceptor selectivity was examined at various cardiovascular activation levels established by either incremental isoprenaline infusion or graded treadmill exercise. The observed parameters were heart rate, systolic and diastolic arterial blood pressure, left ventricular dp/dtmax, and left ventricular end-diastolic pressure. Intravenous infusion of esmolol (25 and 250 micrograms.kg-1.min-1) led to a dose-dependent reduction of the isoprenaline-induced increase in positive dp/dtmax. The concomitant increase in heart rate was suppressed to a lesser extent. Characteristically of a beta 1-selective agent, esmolol had only a slight effect on the isoprenaline-induced reduction in diastolic blood pressure. The impact of esmolol on exercise-induced hemodynamic activation was much smaller. Exercise-induced increase in positive dp/dtmax was more sensitive to beta-adrenoceptor blockade than the concomitant increase in heart rate. Diastolic blood pressure was not influenced significantly. beta-Adrenoceptor blockade was virtually reversed within 20 min of discontinuation of esmolol infusion.     

Beta-adrenergic receptor subtypes and subcellular compartmentation of cyclic AMP and cyclic AMP-dependent protein kinase in rabbit cardiomyocytes

In purified ventricular myocytes from adult rabbit, beta-adrenergic stimulation causes cyclic AMP accumulation and cyclic AMP-protein kinase activation in both particulate and soluble fractions of the cell, whereas prostaglandin E1 elevates cyclic AMP and cyclic AMP-protein kinase activity in the soluble fraction exclusively. Only activation of particulate cyclic AMP-protein kinase activity results in phosphorylase b----a conversion. Using radioligand binding technics, we have determined whether beta 1- and beta 2-receptor subtypes mediate beta-adrenergic effects in particulate and soluble subcellular compartments, respectively. The non-selective antagonist [125I]iodocyanopindolol binds to intact ventricular myocytes with KD of 25 pM and a Bmax of 2.6 X 10(5) receptors/myocyte. Competition for [125I]iodocyanopindolol binding to intact myocytes by the beta-receptor subtype-specific antagonists practolol (beta 1) and zinterol (beta 2) results in monophasic curves with antagonist KD values of 1 microM and 1.5 microM, respectively. We conclude that adult rabbit cardiac myocytes do not possess detectable beta 2 receptors. Further, the ability of isoproterenol to cause elevation of cyclic AMP in two functionally distinct regions within the myocyte must pertain to the actions of a single subtype of beta-receptor, the beta 1-receptor.     

Cyclic nucleotides, calcium, bicarbonate, and protein secretion in canine pancreatic juice in response to cholecystokinin-pancreozymin.

The secretion of cyclic AMP, cyclic GMP, protein, calcium, and bicarbonate in the pancreatic juice of three nonanesthetized dogs with chronic gastric and duodenal Thomas cannulae has been studied. Intravenous infusions of increasing doses of cholecystokinin-pancreozymin (CCK) (1.5, 3, 6, 12, 24 Crick Harper-Raper (CHR) U kg-1 h-1) were administered together with a continuous submaximal dose of secretin (1 clinical unit (CU) kg-1 h-1). Doubling CCK doses every 45 min induced a parallel increase in the output of both cyclic nucleotides. Cyclic AMP output peaked at between 15 and 30 min for 3 and 6 U kg-1 h-1 of CCK and later for 12 and 24 U kg-1 h-1 of CCK whereas cyclic GMP output increased more constantly. Calcium output followed a pattern similar to that of cyclic GMP secretion. Flow rate and protein output attained their peaks at between 30 and 45 min. A strong linear correlation was found between the quantities of cyclic AMP, cyclic GMP, and the quantities of protein secreted in response to each CCK dose. This study demonstrates the presence of cyclic GMP in the canine pancreatic juice and the dose-dependent stimulation of the secretion of cyclic GMP and cyclic AMP by CCK in the presence of secretin.     

Dopamine-stimulated cyclic AMP and binding of [3H] dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H] dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1.10(-8) M and was half-maximal at 7.9 +/- 3.4 10(-7) M. The increase at 1.10(-5) M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1.10(-9) M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1.10(-5) M dopamine was 2.3 +/- 0.9.10(-6) M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H] Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37 degrees C, it was rapid, reversible, saturable and stereospecific. The Kd value for high affinity binding sites was 0.43 +/- 0.1.10(-7) M and 4.7 +/- 1.6.10(-7) M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2 +/- 0.4.10(-7) M epinine, 4.1 +/- 1.8.10(-6) M fluphenazine, 8.0 +/- 1.6.10(-6) M haloperidol, 4.2 +/- 1.2.10(-6) M cis-flupenthixol, 2.7 +/- 4.0.10(-5) M trans-flupenthixol, less than 1.10(-5) M apomorphine, sulpiride, naloxone and isoproterenol.     

Evidence for a D2 dopamine receptor in frog retina that decreases cyclic AMP accumulation and serotonin N-acetyltransferase activity

The regulation of serotonin N-acetyltransferase (NAT) activity and cyclic AMP accumulation in the retina of the African clawed frog (Xenopus laevis) was studied using an in vitro eye cup preparation. Retinal NAT, a key enzyme in the synthesis of melatonin, is expressed as a circadian rhythm with peak activity at night. The increase of NAT activity at night appears to be mediated by cyclic AMP and is suppressed by light. Dopamine inhibits the nocturnal increase of retinal NAT activity; approximately 80% inhibition was observed with 1 microM dopamine. Dopamine at 1 microM did not stimulate retinal cyclic AMP accumulation. The effect of dopamine on NAT activity was antagonized by the D2-selective receptor antagonists spiperone and metoclopramide, but not by the putative D1 selective antagonist SCH 23390. The nocturnal rise in NAT activity was inhibited by LY 171555, a putative D2 selective agonist, but not by SKF 38393, a putative D1 selective agonist. LY 171555 also decreased cyclic AMP accumulation in eye cups incubated under similar conditions. Dopamine inhibited the stimulation of NAT activity in light by 3-isobutylmethylxanthine, but not that by dibutyryl cyclic AMP, suggesting that dopamine acts by decreasing cyclic AMP formation in the NAT-containing cells. Thus, the effects of dopamine on NAT activity may be mediated by a receptor with the pharmacological and biochemical characteristics of a D2 receptor.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Cardiac effects of prostaglandins E1 and F1 alpha

The effects of prostaglandin E1 (PGE1) and prostaglandin F1 alpha (PGF1 alpha) were studied on perfused rat hearts and isolated rat atria. Both PGE1 and PGF1 alpha produced dose-dependent increases in right atrial rate but had no effect on left atrial tension development. PGE1 (10(-4) M) increased right atrial cyclic AMP content without changing phosphorylase a activity. PGF1 alpha (10(-4) M) did not change right atrial cyclic AMP or cyclic GMP content. Both prostaglandins had no effect on left atrial cyclic nucleotide content. When infused at a rate of 1 microgram/min, PGE1 produced a time-dependent increase in cyclic AMP content in the Langendorff perfused hearts but did not alter contractile force development or phosphorylase a activity. An infusion of PGF1 alpha produced a dose-dependent increase in tension development which was secondary to a negative chronotropic effect. PGF1 alpha (1 microgram/min) did not produce any changes in cyclic nucleotide levels or phosphorylase a activity in the Langendorff perfused hearts. These results show that PGE1 can selectively increase myocardial cyclic AMP content without altering contractile force or phosphorylase activity and that PGF1 alpha does not increase rat cardiac AMP levels.     

Sites of interactions of surfactants with beta-adrenergic responses in trout (Salmo gairdneri) gills

Summary The aims of theis study were to investigate the effects of surfactants on the -adrenergic responses of the arterio-arterial (lamellar) vascular pathway of isolated gills from the troutSalmo gairdneri. The resistance of the arterio-arterial vasculature was monitored by measuring the input pressure of isolated gills perfused at a constant flow and at constant efferent pressure. Specific -adrenergic stimulation with isoprenaline produced a dose-dependant reduction in vascular resistance which was mimicked by the presence of cyclic 8-(4-chlorophenylthio) adenosine-3: 5-monophosphate (8-cpt cyclic AMP). These results suggest that cyclic AMP is involved as an intracellular second messenger of -adrenergic regulatory responses of branchial haemodynamics.Surfactants in the perfusate at nominal concentrations of 1.0 mol l^–1 (0.3–0.5 mg l^–1) inhibited the response of the branchial tissue to isoprenaline but did not affect the response to 8-cpt cyclic AMP. This suggests that the surfactant effects are the result of events occurring within the cell membrane. Furthermore because the surfactants used affected the efficacy, rather than the affinity, of the isoprenaline response then the mechanism of action is unlikely to be an effect on agonist/receptor interaction but may be further along the stimulus response chain, perhaps affecting receptor/regulatory nucleotide/adenylate cyclase interaction or via direct inhibition of the cyclase itself.     

The receptors responsible for heat production in brown adipose tissue in the young rabbit

It is known that adrenergic agonists stimulate thermogenesis in the brown fat of the young rabbit but the receptors responsible for mediating the response have not been identified. The infusion of either noradrenaline or isoproterenol (1-2 micrograms . kg-1 X min-1) produced an increase in subcutaneous temperature (0.93 +/- 0.15 and 1.22 +/- 0.10 degrees C, respectively over the interscapular brown fat. At low doses (0.4 microgram . kg-1 X min-1) only isoproterenol was effective. The thermogenic response to isoproterenol was blocked by atenolol, a beta 1-adrenergic antagonist. Neither salbutamol or terbutaline, both beta 2-agonists, produced a temperature increase. Collectively, these data suggest that stimulation of beta 1-adrenoceptor is primarily responsible for the thermogenic activity of brown fat in the rabbit. However, it was found that 53% of the increase in temperature could be blocked by prazosin, an alpha 1-antagonist. Phentolamine was not effective as a blocker. Although a maximal brown fat thermogenic response can be achieved by stimulating the beta-adrenoceptors, the alpha-adrenoceptors appears to play at least an auxiliary role in young rabbit.     

The A1 adenosine receptor antagonist 1,3, dipropyl-8-cyclopentylxanthine (DPCPX) displays adenosine agonist properties in the FRTL5 thyroid cell line

We have evaluated whether the type I adenosine receptor mediates adenosine's ability to inhibit thyrotropin-stimulated cyclic AMP generation and DNA synthesis in FRTL5 cells. The xanthine derivative 1,3-dipropyl-8-cyclopentylxanthine, a selective antagonist for the type 1 adenosine receptor, binds to FRTL5 with high affinity and specificity. 1,3-Dipropyl-8-cyclopentylxanthine does not alter basal cyclic AMP levels but does reverse adenosine's ability to inhibit thyrotropin-stimulated cyclic AMP generation. 1,3-Dipropyl-8-cyclopentylxanthine also potently inhibits thyrotropin-stimulated and dibutyryl cyclic AMP-stimulated [3H]-thymidine incorporation into DNA in FRTL5 cells. Thus, in FRTL5 cells, 1,3-dipropyl-8-cyclopentylxanthine displays both adenosine antagonist and adenosine agonist properties, the latter occurring at a site distal to cyclic AMP generation.