Forskolin-stimulated Adenylate Cyclase Research Articles

Decrease in Adenylate Cyclase Activity Antecedes Neurofibrillary Tangle Formation

In previous studies, the formation of cAMP was seen to be significantly reduced in various brain regions in Alzheimer patients. Recently, a staging system was introduced in which Alzheimer-related histopathological changes were classified according to the pattern of alterations rather than their density. In this study, we examined the degree of correlation between these stages and the postmortem activity of the cAMP-generating enzyme adenylate cyclase. Our findings suggest that forskolin-stimulated adenylate cyclase activity is significantly decreased before major neurofibrillary changes develop. Early impairment of postreceptor signalling could have potentially important consequences for drug treatment and/or for the development of neurofibrillary changes.

N-[2-[(substituted chroman-8-yl)oxy]ethyl]-4-(4-methoxyphenyl)butylamines: synthesis and wide range of antagonism at the human 5-HT1A receptor.

A series of N-[2-[(substituted chroman-8-yl)oxy]ethyl]-4-(4-methoxyphenyl)butylamines was prepared and examined for their 5-HT1A receptor antagonist activity. The parent compound 3a and seven analogs bearing five kinds of substituents on the chroman ring were prepared from the corresponding 8-hydroxychroman intermediates. Radioligand binding assays proved the compounds 3a-h to have high affinity for the rat hippocampal 5-HT1A receptor with varied selectivity for adrenaline alpha1 and dopamine D2 receptors. Their antagonism was evaluated in a forskolin-stimulated adenylate cyclase assay performed with CHO cells expressing the human 5-HT1A receptor. Among the series, the C6-fluoro analog 3c showed both extremely potent affinity (Ki = 0.22 nM) and antagonism (EC50 = 13 nM) for the 5-HT1A receptor. Correlation analysis using substituent descriptors revealed a linear and negative correlation between molar refractivity of the C6-substituent and the binding affinity expressed in pKi.

Interaction between G protein-operated receptors eliciting secretion in rat adrenals: A possible role of protein kinase C

Catecholamine release induced by angiotensin II, histamine, bradykinin and methacholine from the rat adrenal gland perfused in vitro was studied under conditions in which the activity of protein kinase C (PKC) was modified. Perfusion of glands with 10 nM bradykinin abolished, in a reversible way, the secretion induced by short pulses of angiotensin II, histamine and methacholine but did not modify the release evoked by 23.6 mM KCl (high K +). Perfusion with histamine or methacholine (30 μM) inhibited the secretion induced by the other agents by 30–50%, whereas incubation with angiotensin II (100 nM) caused little or no reduction in the release evoked by the other agents. The treatment of glands with 1 nM of the PKC activator phorbol 12,13-dibutyrate (PDBu) suppressed the responses induced by angiotensin II, histamine and methacholine, did not affect those evoked by bradykinin, and potentiated the secretion evoked by high K +. The adenylate cyclase stimulator forskolin (1 μM) did not affect the basal secretion but strongly potentiated the release evoked by all secretagogues used, suggesting a role for protein kinase A (PKA) downstream of the receptor. The PKC inhibitor Ro-31-8220 partially reversed the inhibitory effect of bradykinin. Our results suggest that angiotensin II, histamine and muscarinic receptors share some common transduction mechanism that is regulated by PKC. PKC activity was enhanced by these agents PDBu ⪢ bradykinin = histamine > methacholine = angiotensin II. Bradykinin receptor transduction does not appear to be regulated by PKC.

Neurotransmission and the contraction and relaxation of penile erectile tissues

The balance between contractant and relaxant factors controls the smooth muscle of the corpus cavernosum and determines the functional state of the penis (detumescence and flaccidity versus tumescence and erection). Noradrenaline contracts both the corpus cavernosum and penile vessels, mainly via stimulation of alpha(1)-adrenoceptors. Recent investigations have demonstrated the presence of several subtypes of alpha 1-adrenoceptors (alpha(1A), alpha(1B), and alpha(1D)) in the human corpus cavernosum and also that the noradrenaline-induced contraction in this tissue is probably mediated by two or, possibly, three receptor subtypes. Even if much of the available in vitro information suggests that endothelins (ETs) may be of importance for mechanisms of detumescence and flaccidity, the role of the peptides in the control of penile smooth-muscle tone in vivo is unclear, as is the question as to whether they can contribute to erectile dysfunction. For further evaluation of the clinical importance of ETs in penile physiology and pathophysiology, clinical studies on ET-receptor antagonists would be of interest. Neurogenic nitric oxide (NO) has been considered the most important factor for relaxation of penile vessels and the corpus cavernosum, but recent studies in mice lacking neurogenic NO synthase (NOS) have shown these animals to have normal erections. This focuses interest on the role of endothelial NOS and on other agents released from nerves or endothelium. For the time being the most effective means of inducing penile erection in men involves the intracavernous administration of prostaglandin E1 (PGE1). PGE1 may act partly by increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP). Recent results obtained with the adenylate cyclase stimulator forskolin suggest that penile smooth-muscle relaxation leading to penile erection can be achieved through the cAMP pathway. Thus, transmitters and agents acting through this second-messenger system may significantly contribute to relaxation of penile smooth muscle and to erection.

NGD 94-I: A Specific Dopamine-4-Receptor Antagonist

NGD 94-1 (2-phenyl-4(5)-[4-(2-pyrimidinyl)-piperazin-1-yl)-methyl]-imidazole dimaleate) is a highly selective agent at the D4 receptor—a D4 antagonists. NGD 94-1 possesses a preclinical profile that is at once similar to clozapine and also quite different from clozapine. Its affinity for D4 receptors is 3 nM, while its affinity at D1, D2, D3, and D5 receptors is greater than 2 pM, NGD 94-1 has no significant affinity for a wide variety of monoamine or other neurotransmitter receptor or modulatory sites. The only other significant receptor activity is found at 5HT1A receptors with an affinity of 180 nM. The nonspecific agonist, quinpirole, is a specific D4 agonist and inhibits forskolin-stimulated adenylate cyclase activity in a cell line stably expressing only D4 receptors. NGD 94-1 inhibits this agonist effect of quinpirole without possessing significant agonist activity of its own. Thus, NGD 94-1 appears to be a D4 receptor antagonist in this system. In direct binding experiments, [ 3 H]NGD 94-1, prepared by the reductive tritiation of a halogenated precursor, binds to a single set of receptors in transformed cells expressing the human D4 receptor. The pharmacological binding profile of [ 3 H]NGD 94-1 is consistent with expected D4 pharmacology.

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

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

Adenosine A1 receptors in cultured cerebellar granule cells: role of endogenous adenosine.

Adenosine A1 receptors as well as other components of the adenylate cyclase system have been studied in cultured cerebellar granule cells. No significant changes in adenosine A1 receptor number, assayed by radioligand binding in intact cells, were detected from 2 days in vitro (DIV) until 7 DIV. Nevertheless, a decline in this parameter was detected at 9 DIV. The steady-state levels of alpha-Gg and alpha-Gi, detected by immunoblotting, showed similar profiles, increasing from 2 to 5 DIV and decreasing afterward. Forskolin-stimulated adenylate cyclase levels also showed an increase until 5 DIV, decreasing at 7 and 9 DIV. The adenosine A1 receptor analogue cyclopentyladenosine (CPA) was able to inhibit cyclic AMP accumulation at 2, 5, and 7 DIV but failed to do so at 9 DIV. This inhibition was prevented by the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The presence of adenosine deaminase in the culture increased adenosine A1 receptor number during the period studied and induced recovery of the inhibitory effect of CPA, lost after 7 DIV. These data suggest that functional expression of adenosine A1 receptors and the other components of the adenylate cyclase system is subjected to regulation during the maturation of cultured cerebellar granule cells and demonstrates a key role for endogenous adenosine in the process.

PK 11195 blockade of benzodiazepine‐induced inhibition of forskolin‐stimulated adenylate cyclase activity in the striatum

1. The effects of benzodiazepine receptor antagonists on the inhibition of forskolin-stimulated adenylate cyclase (AC) activity by various benzodiazepine (BZ) and indoleamine agonists in the rat striatum were investigated. 2. A biphasic inhibition of forskolin-stimulated AC activity by the peripheral-type agonist, Ro5-4864, and a multiphasic inhibition by the non-selective BZ, diazepam, was observed. One phase of AC inhibition is consistent with a Gi-coupled receptor-mediated action, whereas the other phases appear to involve a direct effect on the enzyme itself. 3. While the central-type antagonist, flumazenil, had no effect on the ability of Ro5-4864 to inhibit AC activity, the peripheral-type receptor ligand, PK 11195, abolished the first phase of inhibition. 4. PK 11195 and pertussis toxin were found to block the inhibitory effect of various BZs and the indoleamines, melatonin and 2-iodomelatonin, on induced AC activity. 5. Saturation binding studies, conducted at 30 degrees C with [3H]-diazepam revealed a single binding site in the rat striatum (KD = 19.3 +/- 0.80 nM) which significantly decreased in affinity in the presence of GTP (KD = 30.5 +/- 2.6 nM; P < 0.05). No significant change in Bmax was observed. 6. These findings indicate the presence of Gi-coupled BZ receptors in the rat striatum. Thus, suppression of cyclic AMP production may contribute to the diverse neuropharmacological effects of BZs, melatonin and related drugs.

Effect of iron on renal tubular epithelial cells

Since iron has been implicated as a potential nephrotoxin, we examined the effect of iron on several aspects of cultured renal tubular epithelial cell biology. We found that exposure to 10(-4) M of either the ferrous or ferric form of iron impaired healing of denuded areas made within confluent monolayers of LLC-PK1 cells. This impairment required 30 to 80 hours of exposure to iron to occur and was also seen in another renal tubular epithelial cell line (MDCK cells). To delineate the potential mechanism(s) of this impairment, we examined the expression of a key integrin subunit involved in cell-matrix adhesion. Exposure of LLC-PK1 cells to 10(-4) M ferric citrate for 72 hours significantly decreased expression of the beta 1 integrin subunit as determined by flow cytometry. To determine if iron impairs another process that occurs at the basolateral surface, the effects of 72 hours of exposure to iron on adenylate cyclase activity were examined. Both ferric and ferrous citrate significantly enhanced vasopressin- and forskolin-stimulated adenylate cyclase activity. To examine if iron can regulate proliferation, the effect of iron on 3H-thymidine uptake was measured. We found that ferric citrate diminished proliferation and this decrease required the presence of either serum or transferrin. To ascertain if iron affected ultrastructure, we used transmission electron microscopy and found that iron accumulation within cells was much more apparent with ferric than ferrous citrate. Ferric iron induced mild-to-moderate cytopathic changes. These results indicate that iron is capable of inducing multiple changes in renal tubular epithelial function. The effect of iron to impair wound healing may be related to diminished expression of the beta 1 integrin subunit and perhaps to impaired proliferation.

Preclinical profile of the mixed 5-HT 1A/5-HT 2A receptor antagonist S 21357

This study evaluated the pharmacological and behavioral effects of S 21357, a drug with high affinity for both 5-HT 1A and 5-HT 2A receptors. The drug behaved as antagonist at both 5-HT 1A autoreceptors and postsynaptic 5-HT 1A receptors, as it prevented the inhibitory effect of lesopitron on the electrical discharge of the dorsal raphé nucleus (DRN) 5-HT neurons and the activity of forskolin-stimulated adenylate cyclase in hippocampal homogenates. In addition, S 21357 (4 and 128 mg/kg, PO) inhibited 5-HTP-induced head-twitch responses in mice, indicating that it possesses 5-HT 2A antagonistic properties. In a test battery designed to assess defensive behaviors of Swiss-Webster mice to the presence of, or situations associated with, a natural threat stimulus (i.e., rat), S 21357 (0.12–2 mg/kg, IP) reduced contextual defense reactions after the rat was removed, risk assessment activities when the subject was chased, and finally, defensive attack behavior. These behavioral changes are consistent with fear/anxiety reduction. Furthermore, the drug strongly reduced flight reactions in response to the approaching rat. This last finding, taken together with recent results with panic-modulating drugs, suggest that S 21357 may have potential efficacy against panic attack. Finally, our results suggest that compounds sharing high affinities for both 5-HT 1A and 5-HT 2A receptors may directly or synergistically increase the range of defensive behaviors affected.

Cells retrovirally transfected with fibroblast growth factor-2 isoforms exhibit altered adenylate cyclase activity and G-protein functionality.

Basic fibroblast growth factor (FGF-2) is synthesized as different molecular mass isoforms all lacking the signal-peptide sequence. The high molecular-mass isoforms (21-24 kDa) possess a signal sequence directing their nuclear translocation. The role of each isoform is still poorly understood, however, modifications in intracellular signalling pathways could explain some effects of these peptides. In order to evaluate the role of FGF-2 isoforms on the adenylate cyclase (AC) signalling pathway, we retrovirally infected a rat pancreatic cell line (AR4-2J) with point-mutated FGF-2 cDNAs, allowing the expression of the 18 (A5 cells) or 22.5 kDa isoform (A3 cells) at a low level. In membrane preparations of A3 cells, unscheduled expression of the 22.5 kDa FGF-2 isoform induced a 2-fold decrease in both basal and forskolin-stimulated AC activity. Studies carried out on intact cells also showed decreased accumulation of cAMP in A3 cells in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Both FGF-2 peptides also induced functional modifications of G-proteins without affecting their levels. The 22.5 kDa peptide led to enhanced ADP-ribosylation of both alpha(s)-subunits in vitro, whereas the expression of the low molecular-mass 18 kDa peptide resulted in a 2-fold increase in alpha12 and alpha0 ADP-ribosylations. Furthermore, control CAT cells (AR4-2J cells transfected with the retrovirus containing the chloramphenicol acetyltransferase gene) and A5 cells were growth-inhibited by 8-Br-cAMP, in contrast to A3 cells. These data provide evidence that the expression of FGF-2 peptides could play a role in cell functions by modifying the AC signalling pathway. FGF-2 peptides are able to modulate both AC activity and the regulatory G-proteins. Finally FGF-2 expression may interfere with cAMP-regulated cell proliferation.

In vitro pharmacological profile of YM-43611, a novel D2-like receptor antagonist with high affinity and selectivity for dopamine D3 and D4 receptors.

1. We investigated some neurochemical properties of a novel benzamide, YM-43611, [(S)-N-(1-benzyl-3-pyrrolidinyl)-5-chloro-4-cyclopropylcarbonylamino+ ++-2- methoxybenzamide] in comparison with putative D2-like receptor antagonists using both rat and human cloned dopamine D2-like receptors in vitro. 2. Receptor binding studies revealed that YM-43611 had appropriately potent affinities for both rat and human D2-like receptors, with moderate selectivity for D3 receptors and high selectivity for D4 receptors over D2 receptors (Ki values (nM) for rat receptors: D2, 165; D3, 35.5; D4, 1.85, and for human receptors: D2, 42.9; D3, 11.2; D4, 2.10). 3. YM-43611 displayed weak or negligible affinity for other neurotransmitter receptors, namely D1, D5, alpha(1), alpha(2), beta, 5-HT1A, 5-HT2A, 5-HT3, H1, M1 and M2 receptors. 4. Dopamine stimulated low-Km GTPase activity on membranes from Chinese hamster ovary (CHO) cells expressing the human D2-like receptor subtype. This response to dopamine of low-Km GTPase activity was inhibited by use of putative D2-like receptor antagonists. YM-43611 showed a moderate selectivity for D3 receptors (Ki = 45.5 nM) and a high selectivity for D4 receptors (Ki = 3.28 nM) over D2 receptors (Ki = 70.6 nM). 5. Dopamine inhibited forskolin-stimulated adenylate cyclase in intact CHO cells expressing the human D2-like receptor subtype. YM-43611 shifted the inhibition curve of dopamine on respective D2-like receptor subtype-mediated cyclic AMP formation to the right in a parallel fashion, showing a pA2 value of 7.42 (38.1 nM) for D2 receptors, a pKB value of 8.06 (8.68 nM) for D3 receptors, and a pA2 value of 8.42 (3.77 nM) for D4 receptors. 6. YM-43611 but not the other D2-like receptor antagonists exhibited good selectivity with respect to dual antagonism for D3 and D4 receptors in both receptor binding and functional assays. 7. These results indicate that YM-43611 is a novel D2-like receptor antagonist with high potency and selectivity for both D3 and D4 receptors. YM-43611 is therefore expected to be valuable in exploration of the physiological role of D3 and D4 receptors.

Exposure to either gamma or a mixed neutron/gamma field irradiation modifies vasoactive intestinal peptide receptor characteristics in membranes isolated from pig jejunum.

The effect of acute whole body exposure to ionizing radiation was investigated on intestinal vasoactive intestinal peptide (VIP) receptors and adenylate cyclase activity in membranes isolated from pig jejunum. Pigs under light anaesthesia were exposed to a single dose (6 Gy) of gamma (gamma) or to mixed neutron/gamma field (ratio 1:1; neutron/gamma) irradiation. Seven days after irradiation, plasma-membranes were prepared from post mortem jejunal mucosal scrapings. Marker enzyme activities (sucrase, leucine aminopeptidase (LAP), Na,K-ATPase) were measured in each preparation. The characteristics (KD, Bmax) of VIP receptors were determined using 125I-labelled VIP. In addition VIP-sensitive adenylate cyclase activity was measured. Results showed that enzyme activities were reduced following both gamma (sucrase 67%; LAP 53%; Na/K-ATPase 29%; N = 7) and neutron/gamma (sucrase 53%; LAP 59%; Na/K-ATPase 68%; N = 5) compared with control values (N = 5). VIP receptor affinity was decreased following either type of irradiation (gamma or neutron/gamma P < 0.01) and receptor numbers increased. Both VIP- and forskolin-stimulated adenylate cyclase activities were reduced but the sensitivity of the enzyme remained the same for VIP (EC50 values (nmol dm-3)-control-1.27 +/- 0.35; gamma-2.18 +/- 0.41; neutron/gamma-1.91 +/- 0.28). In conclusion, exposure to either gamma or neutron/gamma irradiation attenuates intestinal enzyme activities and VIP receptor affinity but increases VIP receptor numbers.

Early alterations in the brown adipose tissue adenylate cyclase system of pre-obese Zucker rat fa/fa pups: decreased G-proteins and beta 3-adrenoceptor activities.

This study was undertaken to determine whether receptor and non-receptor components of the adenylate cyclase (AC) cascade were altered in brown adipose tissue (BAT) of 14-day-old pre-obese (fa/fa) rats, before endocrine status is strongly modified by fa gene expression. Activity of the AC catalytic subunit did not differ between the two genotypes. In fa/fa rats compared with control Fa/fa rats, there was a 50% decrease in the activity of alpha Gs (stimulated by NaF or guanosine 5'-[gamma-thio]triphosphate) but no change in protein content (Western blotting). alpha Gi function, assessed by the inhibitory action of low concentrations of guanosine 5'-[beta gamma-imido]triphosphate upon 10(-4) M forskolin-stimulated AC activity, was equally low in both genotypes. Analysis of dose-response curves for different beta-agonists revealed that (i) both the basal and the maximally stimulated activity of AC were 2-fold lower in fa/fa rats than in Fa/fa rats; (ii) BRL37344 and CGP12177 (beta 3 agonists) were less potent in fa/fa than in Fa/fa rats (Kact. multiplied by 2); (iii) noradrenaline and isoprenaline (Iso), at the low-affinity site (beta 3-AR), were less potent in fa/fa than in Fa/fa pups (Kact. increased by 30 and 20% respectively). At the high-affinity site (mainly beta 1) these two agonists were more potent in fa/fa than in Fa/fa rats (Kact. decreased by 40 and 80% respectively). In good agreement with the latter result, the beta 1-adrenergic receptor (beta 1-AR)-selective antagonist CGP20712A had more effect on the Iso-stimulated AC activity in pre-obese than in lean pups (2-fold decreased in IC50). Binding experiments with [3H]CGP12177 show that in BAT of suckling rats, beta 3-ARs represent 80% of the total beta-ARs. Bmax values for the two sites were not affected by the genotype, although the beta 3-AR mRNA concentration in BAT (quantitative reverse-transcriptase PCR) was 3-fold lower in fa/fa rats than in Fa/fa pups. In conclusion, these results provide evidence for alterations in beta 1- and beta 3-AR signalling in BAT of 14-day-old suckling pre-obese Zucker rats with a decreased activity of alpha Gs. The impaired AC responsiveness to catecholamines might be a primary contributor to the development of this genetic obesity.

Effects of tandospirone on second messenger systems and neurotransmitter release in the rat brain

1. 1. We studied the effects of tandospirone, a novel serotonin (5-HT) 1A receptor-related anxiolytic, on the intracellular second messenger systems and neurotransmitter release. 2. 2. Tandospirone inhibited forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes by activation of 5-HT 1A receptors and had high efficacy comparable to 5-HT 1A receptor agonists such as 5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). 3. 3. Tandospirone suppressed carbachol-stimulated phosphatidyl-inositol metabolism (PI response), which was shown to be a 5-HT 1A receptor-mediated event. 4. 4. Tandospirone did not affect the release of 5-HT, norepinephrine (NE), dopamine (DA) and acetylcholine (ACh) from rat brain slice preparations. 5. 5. These findings suggested that tandospirone shows high agonistic efficacy on the postsynaptic 5-HT 1A receptors but does not affect the presynaptic autoreceptors located on nerve endings. The modulation of the second messenger system via postsynaptic 5-HT 1A receptors might be involved in the anxiolytic efficacy of tandospirone.

Identification and characterization of guanine nucleotide-sensitive melatonin receptors in chicken brain

Specific melatonin receptors present in crude membrane preparations of chicken forebrain were identified by specific binding activity of 2-[ 125I]iodomelatonin and melatonin-dependent inhibition of forskolin-stimulated adenylate cyclase activity by a pertussis toxin-sensitive mechanism. The membrane-bound melatonin receptors were successfully solubilized with digitonin, and the ligand binding activity was shown to be preserved in solubilized membranes when compared with that of crude membranes. In both fractions, melatonin receptors consisted of two binding components with picomolar and nanomolar affinity. The ligand binding activity of solubilized receptors was decreased after treatment with guanine nucleotides, and guanosine 5 - ́ (γ-thio)triphosphate (GTPγS) induced a transformation of the high affinity receptors to low affinity state. Moreover, [ 35S]GTPγS was indicated to bind to solubilized membranes in a melatonin-dependent fashion. These results demonstrate the existence of functional melatonin receptors-G proteins coupling in digitonin-solubilized membranes of chicken forebrain membranes.

Effects of lactation on the signal transduction systems regulating lipolysis in sheep subcutaneous and omental adipose tissue

The effect of lactation on the regulation of lipolysis by beta- and alpha 2-adrenergic agents and by adenosine has been investigated. When changes in adipocyte mean cell volume (which decreases with lactation) are allowed for, lactation increased the maximum response both to beta-adrenergic agents and to the adenosine analogue N6-phenylisopropyladenosine, but had no apparent effect on the responsiveness of the alpha 2-adrenergic system in both subcutaneous and omental adipocytes. For subcutaneous adipocytes, there was no significant change in the number of beta-adrenergic or alpha 2-adrenergic receptors, but the amount of Gs and the maximum (forskolin-stimulated) adenylate cyclase activity were increased by lactation. In contrast, in omental adipocytes, the number of beta- (but not alpha 2-) adrenergic receptors and the amount of Gs were increased, whereas forskolin-stimulated adenylate cyclase activity was unchanged by lactation. In both types of adipocyte, cyclic AMP phosphodiesterase and total protein kinase A activities were unchanged. Lactation had no effect on the number of adenosine receptors but increased the amounts of the Gi isoforms expressed in both types of adipocyte. These various adaptations differ markedly in a number of respects from those described previously in the rat. Lactation, then, while having a similar overall effect on the response to beta-adrenergic agonists of adipocytes, achieves this by depot-specific mechanisms. In contrast, changes in response to adenosine appear to involve the same mechanism in the two depots investigated.

Pancreatic changes in somatostatin content and receptor/effector system after intrapancreatic injection of 5,7-dihydroxytryptamine

To date, it is unknown whether intrapancreatic serotonergic nerves can influence pancreatic somatostatin (SS) content and the SS receptor/effector system in the exocrine pancreas. In this study, the intrapancreatic serotonergic nerves were chemically ablated by injecting a specific serotonin (5-HT) neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), into the substance of the gland. Three days after the injection, the 5-HT-like immunoreactive levels in the pancreas were reduced by more than 85% whereas somatostatin-like immunoreactive levels had increased (86%). The number of SS receptors in the pancreatic acinar cell membranes of the 5,7-DHT-treated rats was also increased (72%). No significant differences were seen in basal or forskolin-stimulated adenylate cyclase (AC) enzyme activities in the control and the 5,7-DHT-treated groups. In spite of the increase in the number of SS receptors in the pancreatic acinar cell membranes of 5,7-DHT-treated rats, SS caused a significantly lower inhibition of AC activity in these membranes. This finding is related to the observed decrease of a 41 kD pertussis toxin-sensitive substrate, presumably the alpha i subunit of the guanine nucleotide inhibitory protein, in pancreatic acinar cell membranes 3 days after intrapancreatic 5,7-DHT administration when compared with the corresponding controls. The functions of pancreatic serotonergic nerves seem to be associated with enteropancreatic communication. These data together with the present results suggest that pancreatic SS content and the SS receptor/effector system in the exocrine pancreas may be regulated by enteropancreatic serotonergic nerve fibers and may participate in enteropancreatic reflexes.

Decreased Adenylate Cyclase Activity and Expression of G sα in Human Myocardium After Orthotopic Cardiac Transplantation

We studied several aspects of guanine nucleotide-stimulated adenylate cyclase function in patients after orthotopic cardiac transplantation. In 28 patients, adenylate cyclase activity was measured in endomyocardial biopsy samples obtained just before and at monthly intervals after cardiac transplantation. In biopsies obtained > or = 6 months after transplantation, basal adenylate cyclase activity was decreased by 67% (n = 12; P < .05), GTP gamma S-stimulated adenylate cyclase activity was decreased by 78% (n = 12; P < .05), Mn+2+forskolin-stimulated adenylate cyclase activity was decreased by 80% (n = 8; P < .05), and Mn+2-stimulated adenylate cyclase activity (a measure of activity of the catalytic subunit of adenylate cyclase) was decreased by 83% (n = 8, P < .05). Western blot analysis demonstrated that 6 months after cardiac transplantation, the level of Gs alpha protein was decreased by 61 +/- 12% (n = 8; P < .001). There was no change in the level of Gi alpha as assessed by pertussis toxin-catalyzed ADP-ribosylation (n = 4; P = NS). With the use of the quantitative polymerase chain reaction, a 50 +/- 10% (n = 6; P < .001) reduction in the steady-state level of Gs alpha mRNA was observed. There was no change in the level of mRNA for Gi-3 alpha. Thus, after orthotopic cardiac transplantation in humans, guanine nucleotide-stimulated adenylate cyclase activity is decreased in parallel with decreased levels of Gs alpha protein and mRNA.

Tannin inhibits adenylate cyclase in airway epithelial cells

Tannin, isolated from cotton bracts extract and implicated in the pathogenesis of byssinosis, inhibits adenosine 3',5'-cyclic monophosphate (cAMP) production and Cl- secretion in bovine airway epithelial cells in part by inhibiting adrenergic receptor binding. The purpose of this study was to determine whether tannin affected other parts of the adrenergic-cAMP signal transduction pathway by examining the effect of tannin on guanosine 5'-triphosphate (GTP)-regulatory pathways (G proteins) and on adenylate cyclase activity. cAMP production in confluent airway epithelial cells was measured in the presence of cholera toxin (100 micrograms/ml), an activator of GS proteins, and forskolin (0.1-1,000 microM), a direct activator of adenylate cyclase. Cholera toxin stimulated cAMP production; this response, however, was inhibited in cells pretreated with 50 micrograms/ml tannin. Forskolin (100 microM) stimulated cAMP production 13-fold above baseline values. Tannin pretreatment inhibited the stimulatory effect of forskolin on cAMP release in a dose-dependent manner with a tannin concentration causing 50% inhibition of 7.5 micrograms/ml. The stimulatory effect of forskolin on cAMP release was completely inhibited in cells pretreated with 50 micrograms/ml tannin. The inhibition was reversible 3 h after removal of tannin from the solution. Tannin also inhibited forskolin-stimulated adenylate cyclase activity in a dose-dependent, noncompetitive manner. We conclude that forskolin and cholera toxin stimulate cAMP production in airway epithelial cells and that tannin inhibits the production of cAMP in airway epithelial cells by a direct effect on adenylate cyclase activity.