Adenylate Cyclase Pathway Research Articles

Signal transduction pathways involved in macrophage migration induced by peritoneal fluid chemotactic factors in stages I and II endometriosis

Objective: To explore the role of G-protein coupled signaling pathways in activation of macrophage migration in endometriosis stages I and II. Design: Case controlled study. Setting: University hospital. Patient(s): Fifteen patients undergoing laparoscopy for elective sterilization (n = 5) or for diagnosis of endometriosis stages I and II associated with infertility (n = 10). Intervention(s): Peritoneal fluid samples were collected during laparoscopy. Main Outcome Measure(s): Macrophage migration induced by peritoneal fluid from patients with endometriosis stages I and II (PF SI-II) and potential G-protein coupled receptors and second messengers involved in macrophage activation. Result(s): Potential G-protein coupled receptors and second messengers involved in macrophage activation were evaluated after incubation of U-937 cells differentiated into macrophages with inhibitors of phospholipase A and C, adenylate cyclase, and protein kinase A and C. Macrophage chemotactic activity induced by PF SI-II was inhibited in the presence of a phospholipase C and A 2 inhibitor (IC 50= 30 μM) and after treatment with myristoylated protein kinase C peptide inhibitor (50 nM). An increase in inositol phosphate (IP 3) was also observed in macrophages exposed to PF SI-II. Activation of multiple G-proteins in macrophages was examined after exposure of cells to PF SI-II in the presence and absence of Bordettela pertussis and cholera toxins. No effect on macrophage migration was observed. Conclusion(s): Macrophage chemotaxis induced by PF SI-II appears to involve activation of pertussis toxin-insensitive G-protein coupled receptors in macrophages. Our data suggest that these events lead to subsequent activation of phospholipases followed by generation of IP 3 and potential mobilization of intracellular Ca 2+. Subsequent phosphorylation of target proteins by protein kinase C may regulate the chemotactic responses. The adenylate cyclase pathway does not appear to play a role in this process.

Analogs of Substance P modified at the C-terminus which are both agonist and antagonist of the NK-1 receptor depending on the second messenger pathway.

The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK-1 receptor. Therefore, the photoreactive amino acid para-benzoyl-phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa0[(pBzl)Phex]SP analogs. Biotinyl sulfone-5-aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK-1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK-1 receptor-binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa0[(pBzl)Phe11]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa0[Pro9, (pBzl)Hcy(O2)11]SP is a partial agonist, whereas Bapa0[Hcy(ethylaminodansyl)11]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK-1 receptor.

A novel Ala(-3)Thr mutation in the signal peptide of human luteinizing hormone beta-subunit: potentiation of the inositol phosphate signalling pathway and attenuation of the adenylate cyclase pathway by recombinant variant hormone.

Upon screening for polymorphisms in the human luteinizing hormone beta-subunit (LH beta) gene, we discovered a novel mutation in the LH beta signal peptide with functional consequences for signal transduction in mouse Leydig tumour cells (mLTC-1). This G(52)A point mutation in exon 2 of the LH beta gene, detected in heterozygous form in several normal DNA samples, caused an Ala(-3)Thr amino acid substitution. Recombinant forms of wild-type (WT) and Ala(-3)Thr variant (V) LH were produced in human embryonic kidney (HEK) 293 cells and purified. The immunoreactivities of the recombinant LH were determined by immunofluorometric assays and in-vitro bioactivities in mLTC-1 cells were assessed by using cAMP, progesterone and inositol trisphosphate (IP(3)), and activation of mitogen-activated protein kinase (MAPK) as end-points. Whereas both LH forms stimulated progesterone production and MAPK in similar fashion, WT-LH was more potent in stimulating cAMP, and V-LH was more potent in stimulating IP(3) generation. Both LH forms bound to LH receptors with similar affinities. No evidence was found for influence of the signal peptide mutation on efficacy of alpha- and beta-subunit dimerization. Sequencing of the recombinant V-LH beta protein also revealed that the mutation did not interfere with signal peptide cleavage. In summary, the present findings indicate that the Ala(-3)Thr mutation in the LH beta-subunit signal peptide has functional consequences, in the form of dissociation of stimulatory potency for different signal transduction pathways in vitro.

Potential signalling pathways underlying corticotrophin-releasing hormone-mediated neuroprotection from excitotoxicity in rat hippocampus.

In several neurological disorders including cerebral ischaemia, glutamate has been implicated as a neurotoxic agent in the mechanisms leading to neuronal cell death. The role of corticotrophin-releasing hormone (CRH), the 41-amino acid peptide, which activates the HPA axis in response to stressful stimuli, remains controversial. In this study, we report that CRH in low physiological concentrations (2 pM), prevented glutamate-induced neurotoxicity via receptor-mediated mechanisms when administered to organotypic hippocampal cultures both during and after the glutamate-induced insult. Detailed investigations on the mechanisms mediating this neuroprotective effect showed that activation of the adenylate cyclase pathway and induction of MAP kinase phosphorylation mediate the CRH action. In addition we showed that CRH can inhibit the phosphorylation of JNK/SAPK by glutamate. Most importantly, we showed that CRH can afford neuroprotection against neurotoxicity up to 12 h following the insult, suggesting that CRH is acting at a late stage in the neuronal death cycle, and this might be important in the development of novel neuroprotective agents in order to improve neuronal survival following the insult.

Substance P in the descending cholinergic projection to REM sleep-induction regions of the rat pontine reticular formation: anatomical and electrophysiological analyses.

Release of acetylcholine within the pontine reticular formation (PRF) from the axon terminals of mesopontine cholinergic neurons has long been hypothesized to play an important role in rapid eye movement (REM) sleep generation. As some of these cholinergic neurons are known to contain substance P (SP), we used anatomical, electrophysiological and pharmacological techniques to characterize this projection in the rat. Double immunofluorescence demonstrated that 16% of all cholinergic neurons within the mesopontine tegmentum contained SP; this percentage increased to 27% in its caudal regions. When double immunofluorescence was combined with retrograde tracing techniques, it was observed that up to 11% of all SP-containing cholinergic neurons project to the PRF. Whole-cell patch-clamp recordings from in vitro brainstem slices revealed that SP administration depolarized or evoked an inward current in a dose-dependent manner in all PRF neurons examined, and that these effects were antagonized by a SP antagonist. The amplitude of the SP-induced inward current varied with changes in the Na+ concentration, did not reverse at the calculated K+ or Cl- equilibrium potentials, and was not attenuated in the presence of tetrodotoxin, low Ca2+ concentration or caesium ions. These data suggest that activation of a tetrodotoxin-insensitive cation channel(s) permeable to Na+ is responsible for a SP-induced inward current at resting membrane potentials. The depolarizing actions of SP appeared to be primarily due to activation of the adenylate cyclase pathway, and were additive with cholinergic receptor activation even at maximal concentrations. These data indicate that SP is colocalized in a subpopulation of mesopontine tegmental cholinergic neurons projecting to REM sleep-induction regions of the PRF, and that actions of these two neuroactive substances on PRF neurons are additive. If SP is coreleased with acetylcholine, the additive actions of the two neurotransmitters might heighten the excitability of postsynaptic PRF neurons and ensure the initiation and maintenance of REM sleep.

Role of c-fos in the regulation of type X collagen gene expression by PTH and PTHrP: localization of a PTH/PTHrP-responsive region in the human COL10A1 enhancer.

PTH and PTHrP have been shown to inhibit maturation of growth plate chondrocytes and the expression of type X collagen. In order to examine the regulatory mechanisms involved, fetal bovine growth plate chondrocytes were incubated for 24-48 h under serum-free conditions with PTH and PTHrP and various aminoterminal, midregional, and carboxyterminal fragments of these hormones. Analysis of type X collagen mRNA levels by Northern hybridization showed a significant suppression by PTH (1-84), PTH (1-34), and PTHrP (1-40), but not by PTH (28-48) or PTH (53-84). PTH fragment (3-34) did not reduce alpha1(X) mRNA levels, while bis-indolylmaleimide, an inhibitor of the protein-kinase C pathway, did not affect alpha1(X) mRNA suppression by PTH, supporting the notion that the inhibition of type X collagen expression by PTH involves predominantly the adenylate cyclase pathway of the PTH/PTHrP-receptor. Since PTH and PTHrP have been shown to induce c-fos in osteoblasts and chondrocytes, the possibility was tested that c-fos mediated the suppressive effect of PTH/PTHrP on collagen X expression. In fetal bovine hypertrophic chondrocytes PTH (1-34), but not PTH (3-34) nor the midregional or C-terminal PTH fragments induced c-fos expression. In order to identify cis- and trans-acting elements in the COL10A1 gene involved in c-fos-mediated inhibition of collagen X expression by PTH/PTHrP, reporter gene constructs carrying various fragments of the human COL10A1 promoter coupled to the luciferase gene were transfected into hypertrophic chondrocytes. A tissue-specific, strong enhancer region, which we had previously located in the promoter of the human type X collagen gene COL10A1, was further narrowed down to a 530-bp sequence, located between - 1,870- and - 2,407 bp upstream of the transcription start site. The transcriptional activity of this enhancer element in transfected hypertrophic chondrocytes was significantly reduced after incubation with PTH (1-34) or PTHrP (1-40). Transcription of these reporter genes was also inhibited when chondrocytes were cotransfected with a c-fos expression vector. These results indicate the presence of a PTH/PTHrP responsive element in the human COL10A1 enhancer, which may be represented by multiple putative AP-1 sites located in this region.

Differential in vitro secretion of gonadotropin-releasing hormone (GnRH) and [hydroxyproline]GnRH from the rat hypothalamus during postnatal development.

The differential secretion of gonadotropin-releasing hormone (GnRH) and [hydroxyproline9]GnRH (HypGnRH) has been recently reported from the adult rat hypothalamus. We report here in vitro cosecretion of HypGnRH and GnRH by the hypothalamus of 2-45 day-old-rats and provide evidence that they are differentially regulated throughout development. The secretion of both forms of GnRH was increased in a dependent manner during depolarization by high K+ solutions, and was stimulated by forskolin and 12-O-tetradecanoylphorbol-13-acetate (TPA), activators of adenylate cyclase and protein kinase C pathways, respectively. The proportion of HypGnRH in the release of GnRH-like peptides remained stable and high (33-40%) under basal and K+-induced conditions until days 13 and 21, respectively. By contrast, the proportion of HypGnRH in the total GnRH-like content of the developing hypothalamus continuously decreased (from 37% to 14%). Similarly, the proportion of HypGnRH: total GnRH-like material released remained stable in TPA- (30%) and forskolin- (50%) induced secretion until postnatal day 8. Evaluation of release over tissue store ratios revealed a 1.3-to 2.8-fold higher release of HypGnRH compared to GnRH according to the different secretions and postnatal periods examined. The preferential recruitment of HypGnRH was maintained under basal and K+ conditions during postnatal development, but it disappeared under TPA stimulation from day 13 onwards. After forskolin stimulation, the preferential mobilization of HypGnRH was markedly reduced from day 2 to day 13 but recovered its high perinatal level during puberty. Taken together, our results support the hypothesis that HypGnRH may play a specific role in development. In addition, a specific function of this peptide taking place during puberty through the activation of the adenylate cyclase pathway is suggested.

A role for muscarinic excitation: control of specific singing behavior by activation of the adenylate cyclase pathway in the brain of grasshoppers.

Muscarinic acetylcholine receptors exert slow and prolonged synaptic effects in both vertebrate and invertebrate nervous systems. Through activation of G proteins, they typically decrease intracellular cAMP levels by inhibition of adenylate cyclase or stimulate phospholipase C and the turnover of inositol phosphates. In insects, muscarinic receptors have been credited with two main functions: inhibition of transmitter release from sensory neuron terminals and regulation of the excitability of motoneurons and interneurons. Our pharmacological studies with intact and behaving grasshoppers revealed a functional role for muscarinic acetylcholine receptors as being the basis for specific arousal in defined areas of the brain, underlying the selection and control of acoustic communication behavior. Periodic injections of acetylcholine into distinct areas of the brain elicited songs of progressively increasing duration. Coinjections of the muscarinic receptor antagonist scopolamine and periodic stimulations with muscarine identified muscarinic receptor activation as being the basis for the underlying accumulation of excitation. In contrast to reports from other studies on functional circuits, muscarinic excitation was apparently mediated by activation of the adenylate cyclase pathway. Stimulation of adenylate cyclase with forskolin and of protein kinase A with 8-Br-cAMP mimicked the stimulatory effects of muscarine whereas inhibition of adenylate cyclase with SQ22536 and of protein kinase A with H-89 and Rp-cAMPs suppressed muscarine-stimulated singing behavior. Activation of adenylate cyclase by muscarinic receptors has previously been reported from studies on membrane preparations and heterologous expression systems, but a physiological significance of this pathway remained to be demonstrated in an in vivo preparation.

Different domains in the third intracellular loop of the GLP-1 receptor are responsible for Gα s and Gα i/Gα o activation

It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Gα s proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP production. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Gα s but also to the Gα i/Gα o type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.

Receptor-G-protein signalling in Alzheimer's disease.

Based on radioligand binding studies, it has long been assumed that the neurochemical pathology of Alzheimer's disease (AD) does not involve widespread changes in post-synaptic neurotransmitter function. However, more recent studies suggest that receptor function in AD may be compromised due to disrupted post-receptor signal transduction, in particular that mediated by the G-protein regulated phosphoinositide hydrolysis and adenylate cyclase (AC) pathways. The phosphoinositide hydrolysis pathway has been shown to be altered at a number of levels in AD post-mortem brains, including impaired agonist and G-protein regulation of phospholipase C, decreased protein kinase C (PKC) levels and activity, and a reduced number of receptor sites for the second messenger, Ins(1,4,5)P3. Of these, loss of Ins(1,4,5)P3 receptors and PKC in the entorhinal cortex and hippocampus correlates with AD-related neurofibrillary changes, as staged according to Braak's protocol. Disregulation of the phosphoinositide hydrolysis pathway may therefore have consequences for the progression of AD pathology. In contrast to the extensive pattern of disruption seen with the phosphoinositide hydrolysis pathway, changes to AC signalling in AD appear more circumscribed. Disruptions include a lesion at the level of Gs-protein stimulation of AC and, at least in the hippocampus, reduced enzyme activities in response to forskolin stimulation. Of these, the latter change has been shown to precede neurofibrillary changes. Apart from a loss of calcium/calmodulin sensitive AC isoforms, other components of this signalling pathway, including G-protein levels, Gi-protein mediated inhibition and protein kinase A levels and activity, remain relatively preserved in the disorder.

Differential alterations in cardiac adrenergic signaling in chronic hypoxia or norepinephrine infusion.

Norepinephrine (NE)-induced desensitization of the adrenergic receptor pathway may mimic the effects of hypoxia on cardiac adrenoceptors. The mechanisms involved in this desensitization were evaluated in male Wistar rats kept in a hypobaric chamber (380 Torr) and in rats infused with NE (0.3 mg. kg(-1). h(-1)) for 21 days. Because NE treatment resulted in left ventricular (LV) hypertrophy, whereas hypoxia resulted in right (RV) hypertrophy, the selective hypertrophic response of hypoxia and NE was also evaluated. In hypoxia, alpha(1)-adrenergic receptors (AR) density increased by 35%, only in the LV. In NE, alpha(1)-AR density decreased by 43% in the RV. Both hypoxia and NE decreased beta-AR density. No difference was found in receptor apparent affinity. Stimulated maximal activity of adenylate cyclase decreased in both ventricles with hypoxia (LV, 41%; RV, 36%) but only in LV with NE infusion (42%). The functional activities of G(i) and G(s) proteins in cardiac membranes were assessed by incubation with pertussis toxin (PT) and cholera toxin (CT). PT had an important effect in abolishing the decrease in isoproterenol-induced stimulation of adenylate cyclase in hypoxia; however, pretreatment of the NE ventricle cells with PT failed to restore this stimulation. Although CT attenuates the basal activity of adenylate cyclase in the RV and the isoproterenol-stimulated activity in the LV, pretreatment of NE or hypoxic cardiac membranes with CT has a less clear effect on the adenylate cyclase pathway. The present study has demonstrated that 1) NE does not mimic the effects of hypoxia at the cellular level, i.e., hypoxia has specific effects on cardiac adrenergic signaling, and 2) changes in alpha- and beta-adrenergic pathways are chamber specific and may depend on the type of stimulation (hypoxia or adrenergic).

Altered coronary dilation in deoxycorticosterone acetate-salt hypertension.

To compare coronary dilation in uninephrectomized hypertensive deoxycorticosterone acetate (DOCA)-salt rats (HTRs), treated for 2 or 4 weeks, with age-matched uninephrectomized normotensive rats (NTRs). DESIGN AND METHODS Coronary perfusion pressure was recorded in isolated hearts perfused at a constant flow rate to evaluate coronary resistance. A decreased vasoconstriction due to NG-nitro-Larginine (NNLA, 30 pmol/I) in hearts from HTRs suggested a reduced basal nitric oxide (NO) release. In contrast, coronary vasodilation due to the NO donor, sodium nitroprusside (3 pmol/I), remained unaffected in 2-week HTRs, and was enhanced in 4-week HTRs. Cumulative dose-response curves to bradykinin induced an important vasodilation in NTRs, with a maximal response that remained unaffected in the presence of either NNLA (30 pmol/I), indomethacin (10 pmol/l) or the two combined. In contrast, hearts from HTRs showed a diminished maximal relaxation to bradykinin, suggesting an altered endothelium-dependent relaxation. The presence of NNLA or indomethacin had no effect on the weak relaxation observed in HTRs. However, NNLA and indomethacin combined unmasked an important relaxation due to bradykinin in HTRs. The addition of clotrimazole (1 pmol/I) to NNLA and indomethacin blunted the relaxation due to bradykinin in both NTRs and HTRs. Perfusion with superoxide dismutase (120 IU/ml) restored most of the coronary relaxation due to bradykinin in hearts from HTRs. Bradykinin-induced prostaglandin 12 (PGI2) and E2 (PGE2) production was unaffected by hypertension. No increase in thromboxane A2 (TXA2) due to bradykinin was detected. Finally, reduced reactivity to papaverine and forskolin was observed in hearts from HTRs. DOCA-salt hypertension is associated with alterations in coronary reactivity. Basal NO formation appears to be reduced in HTRs, but the intact relaxation to exogenous NO suggests a preserved guanylate cyclase pathway. In addition, alteration in adenylate cyclase activity, and not in prostaglandin production, may explain the blunted cAMP-mediated responses in HTRs. The combined nitric-oxide synthase (NOS) and cyclo-oxygenase (COX) inhibition unmasked an endothelium-derived hyperpolarizing factor (EDHF) involvement in the coronary dilation due to bradykinin in hearts from HTRs, suggesting that endothelial NO and PGI2, although unable to induce coronary smooth-muscle relaxation, can inhibit EDHF production in HTRs. Impairment in the adenylate cyclase pathway and the suppression of NO by free radicals may explain the blunted vasodilation in DOCA-salt hypertension.

Involvement of Cyclooxygenase 2 in the Protective Effect of 17β-Estradiol on Hypercholesterolemic Rabbit Aorta

The involvement of cyclooxygenase (COX) in the effects of 17β-estradiol was investigated on hypercholesterolemic rabbits aorta. Acetylsalycilic acid, nimesulide, or SQ22536 was used as respective antagonist of COX-1, COX-2, or adenylate cyclase using aortic rings precontracted with phenylephrine and exposed to cumulative concentrations of acetylcholine (ACh). The relaxation effect of ACh was impaired by hypercholesterolemia and restored by an 8-week 17β-estradiol treatment. In the control group treated with estrogen, nimesulide, acetylsalycilic acid, or SQ22536 slightly reduced the response to ACh. In hypercholesterolemic rabbits treated with estrogen, nimesulide significantly reduced the maximal relaxation and shifted to the right the relaxation curve of ACh, whereas acetylsalycilic acid did not modify the maximal response to ACh but displaced slightly the concentration–response curve. SQ22536 reduced the relaxant effect of ACh down to the level obtained in the presence of nimesulide. These results suggest that the protective effect of 17β-estradiol against hypercholesterolemia involved COX-2/adenylate cyclase pathway.

Receptor – G-protein signalling in Alzheimer's disease

Based on radioligand binding studies, it has long been assumed that the neurochemical pathology of Alzheimer's disease (AD) does not involve widespread changes in post-synaptic neurotransmitter function. However, more recent studies suggest that receptor function in AD may be compromised due to disrupted post-receptor signal transduction, in particular that mediated by the G-protein regulated phosphoinositide hydrolysis and adenylate cyclase (AC) pathways. The phosphoinositide hydrolysis pathway has been shown to be altered at a number of levels in AD post-mortem brains, including impaired agonist and G-protein regulation of phospholipase C, decreased protein kinase C (PKC) levels and activity, and a reduced number of receptor sites for the second messenger, Ins(1,4,5)P3. Of these, loss of Ins(1,4,5)P3 receptors and PKC in the entorhinal cortex and hippocampus correlates with AD-related neurofibrillary changes, as staged according to Braak's protocol. Disregulation of the phosphoinositide hydrolysis pathway may therefore have consequences for the progression of AD pathology. In contrast to the extensive pattern of disruption seen with the phosphoinositide hydrolysis pathway, changes to AC signalling in AD appear more circumscribed. Disruptions include a lesion at the level of Gs-protein stimulation of AC and, at least in the hippocampus, reduced enzyme activities in response to forskolin stimulation. Of these, the latter change has been shown to precede neurofibrillary changes. Apart from a loss of calcium/calmodulin sensitive AC isoforms, other components of this signalling pathway, including G-protein levels, Gi-protein mediated inhibition and protein kinase A levels and activity, remain relatively preserved in the disorder.

Optimized binding of [35S]GTPgammaS to Gq-like proteins stimulated with dopamine D1-like receptor agonists.

Subtypes of dopamine D1-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [35S]GTPgammaS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [35S]GTPgammaS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [35S]GTPyS binding reaction include protein concentration at 40 microg/ml, cationic concentrations of 120 mM Na+, 1.8 mM K+, and 20 mM Mg(2+), a molar guanine nucleotide ratio of 100,000 GDP to [35S]GTPgammaS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30-120 min. Under the optimized conditions, the D1-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [35S]GTPgammaS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [35S]GTPgammaS-bound proteins with specific Galpha antibodies showed that at least 70% of SKF38393-stimulated [35S]GTPgammaS binding was to Galphaq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Galphas and the adenylate cyclase pathway or to Galphaq and the phospholipase C pathway.

Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus-oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 μg/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 μg/ml) resulted in high rates of maturation to the MII stage (92.6 ± 2.5 and 98.5 ± 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 ± 4.2 and 45.1 ± 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 ± 2.9%). Finally, the addition of 0.1 μg/ml iAC and 0.5 mM 3-isobutyl 1-methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 μg/ml iAC (31.9 ± 5.5 vs. 12.1 ± 1.6 and 45.5 ± 2.9 vs. 19.1 ± 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86–91,1999. © 1999 Wiley-Liss, Inc.

Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization.

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus-oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 microgram/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 microgram/ml) resulted in high rates of maturation to the MII stage (92.6 +/- 2.5 and 98.5 +/- 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 +/- 4.2 and 45.1 +/- 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 +/- 2.9%). Finally, the addition of 0.1 microgram/ml iAC and 0.5 mM 3-isobutyl 1-methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 microgram/ml iAC (31.9 +/- 5.5 vs. 12.1 +/- 1.6 and 45.5 +/- 2.9 vs. 19.1 +/- 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86-91,1999.

Existence of multiple novel Gs alpha splice variants in acute leukemia patients.

The alpha subunit of the stimulatory G protein, Gs alpha, is involved in stimulation of the adenylate cyclase pathway of signal transduction. In this study, we investigated the status of the Gs alpha gene in 29 acute leukemia patients and identified three novel splice variants (designated Gs alpha L-1, Gs alpha L-2, and Gs alpha L-3), possibly derived from aberrant splicing. All of the splice variants have in-frame deletions, removing the functional domain responsible for GTPase activity of Gs alpha, and would encode truncated proteins of 160(Gs alpha L-1), 90(Gs alpha L-2) and 70(Gs alpha L-3) amino acids, respectively. The data suggest that these novel products may be implicated in an as-yet-unidentified signal transduction pathway in hematopoietic cells.

Mechanism of action of cholera toxin on the opossum internal anal sphincter smooth muscle.

Cholera toxin (CTX), an activator of G(s) protein, is an important pharmacological tool in G protein research. The effect and the mechanism of action of CTX in the gastrointestinal smooth muscle, including the internal anal sphincter (IAS), are not known. The present investigation was carried out to examine the effects of CTX on the signal transduction associated with the adenylate cyclase (AC) pathway on the basal tone of the IAS smooth muscle. CTX caused a prompt and dose-dependent fall in the basal tone of the IAS that was not affected by the neurotoxins TTX and omega-conotoxin or the nitric oxide synthase inhibitor N(G)-nitro-L-arginine. The cyclooxygenase inhibitor indomethacin, cAMP-dependent protein kinase inhibitor Rp-8-bromoadenosine 3',5' cyclic monophosphorothioate inhibited CTX-induced IAS smooth muscle relaxation. Furthermore, CTX caused a concentration-dependent relaxation of the isolated smooth muscle cells (SMC) of the IAS, which was blocked by G(s)alpha antibody (G(s)alpha-Ab). The IAS smooth muscle relaxation was accompanied with an increase in the GTPase activity that was also specifically blocked by G(s)alpha-Ab. We conclude that a major part of the inhibitory action of CTX in the IAS is via the direct response of the SMC that is linked with G(s) protein to the AC pathway. A part of the inhibitory action of CTX on the smooth muscle occurs via the activation of cyclooxygenase pathway. The relative contribution of such actions of CTX in the smooth muscle in the gastrointestinal motility disturbances following cholera infection remains to be determined.

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose.

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.