Basal Accumulation Research Articles

GTP Binding and Signaling by Gh/Transglutaminase II Involves Distinct Residues in a Unique GTP-binding Pocket

G(h) is a dual function protein. It has receptor signaling activity that requires GTP binding and Ca(2+)-activated transglutaminase (TGase) activity that is inhibited by GTP binding. G(h) shows no homology with other GTP-binding proteins, and its GTP-binding site has not been defined. Based on sequence analysis of [alpha-(32)P]GTP-photolabeled and proteolytically released internal peptide fragments, we report localization of GTP binding to a 15-residue segment ((159)YVLTQQGFIYQGSVK(173)) of the G(h) core domain. This was confirmed by site-directed mutagenesis; a G(h)/fXIIIA chimera (in which residues 162-179 of G(h) were substituted with the equivalent but nonhomologous region of the non-GTP-binding TGase factor XIIIA) and a G(h) point mutant, S171E, retained TGase activity but failed to bind and hydrolyze GTP and did not support alpha(1B)-adrenergic receptor signaling. Slight impairment of GTP binding (1.5-fold) and hydrolysis (10-fold) in the absence of altered TGase activity did not affect signaling by the mutant K173N. However, greater impairment of GTP binding (6-fold) and hydrolysis (50-fold) abolished signaling by the mutant K173L. Mutant S171C exhibited enhanced GTP binding and signaling. Thus, residues Ser(171) and Lys(173) are critical for both GTP binding and signaling but not TGase activity. Mutagenesis of residues N-terminal to Gly(170) impaired both GTP binding and TGase activity. From computer modeling of G(h), it is evident that the GTP-binding region identified here is distinct from, but interacts with, the TGase active site. Together with structural considerations of G(h) versus other GTP-binding proteins, these findings indicate that G(h) has a unique GTP-binding pocket and provide for the first time a mechanism for GTP-mediated regulation of the TGase activity of G(h).

Cellular Release of and Response to ATP as Key Determinants of the Set-Point of Signal Transduction Pathways

The determinants of "basal" activity of signaling pathways regulating cellular responses are poorly defined. One possibility is that cells release factors to establish the set-point of such pathways. Here we show that treatment of Madin-Darby canine kidney cells with the nucleotidase apyrase decreases basal arachidonic acid release and cAMP production 30-40% and that inhibitors of P2Y receptor action also affect basal and forskolin-stimulated cAMP accumulation. Changing medium prominently increases extracellular levels of ATP in Madin-Darby canine kidney, COS-7, and HEK-293 cells. Mechanical stimulation of ATP release likely occurs in virtually every experimental protocol with cultured cells, implicating such release and P2Y receptor activation as critical in establishing the set-point for signal transduction pathways.

17β-Estradiol acts separately on the LDL particle and artery wall to reduce LDL accumulation

Estrogen replacement therapy has been shown to attenuate atherogenesis, although the mechanisms for this effect are incompletely defined. Previously, we showed that 17-β estradiol (estradiol) attenuated oxidant stress-induced increases in vascular low density lipoprotein (LDL) accumulation. It was unclear whether estradiol's effect was imparted on the lipoprotein particle or the artery wall. To examine this, we chronically treated rats with the following sex hormones: low estradiol, high estradiol, progesterone, low estradiol + progesterone, placebo, or control. Carotid arteries (n = 8/group) were isolated and perfused with fluorescently labeled LDL. Rates of LDL accumulation were measured before and after treatment with 10 ng/ml tumor necrosis factor-α (TNF) using quantitative fluorescence microscopy. We observed a 50% decrease in basal LDL accumulation rates (P < 0.01) and a 25% decrease in endothelial layer permeability (P < 0.01) in arteries from estradiol-treated animals. There was no effect of hormone replacement on rate of TNF-induced LDL accumulation (P = 0.451), while incubation of LDL with 65 pg/ml estradiol attenuated the TNF effect (P < 0.01). These experiments suggest two independent mechanisms of anti-atherogenic protection by estradiol: 1) decreased endothelial layer permeability; and 2) incorporation of estradiol into the LDL particle and prevention of LDL binding to the artery wall. — Walsh, B. A., A. E. Mullick, C. E. Banka, and J. C. Rutledge. 17β-Estradiol acts separately on the LDL particle and artery wall to reduce LDL accumulation.

Susceptibility to β-Amyloid-Induced Toxicity Is Decreased in Goto-Kakizaki Diabetic Rats: Involvement of Oxidative Stress

The response of synaptosomes isolated from Wistar non-diabetic rats and Goto–Kakizaki (GK) diabetic rats to the β-amyloid fragment Aβ25–35 was compared. The synaptosomal redox activity, evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, was shown to be decreased in GK rats (72.8±7.45% of MTT reduction). However, the reduction of MTT was decreased in synaptosomes of Wistar rats upon Aβ25–35 treatment (84.47±3.73%), while in GK rats it was not affected. Aβ25–35 induced lipid peroxidation in synaptosomes of Wistar rats, but not in that of GK rats, leading to an 1.5-fold increase in thiobarbituric acid reactive substances (TBARS) levels. In the absence of Aβ25–35, basal TBARS levels were 1.6-fold higher in GK rats. In the former preparations, the content in vitamin E was also higher (2-fold). A decrease in ATP levels, of about 2-fold, was observed in synaptosomes of Wistar rats treated with Aβ25–35, while no significant changes were observed in synaptosomes of GK rats. No significant differences between the two groups were detected in the basal ATP levels. The extrasynaptosomal accumulation of aspartate and glutamate increased upon Aβ25–35 treatment, only in synaptosomes of Wistar rats (aspartate and glutamate accumulation increased by 1.1-fold and 1.5-fold, respectively), while the accumulation of glycine increased in both Wistar (by 1.8-fold) and GK (by 2.2-fold) rats. No statistical differences in the basal accumulation of aminoacids were observed. These results show that synaptosomes of GK diabetic rats have a lower redox activity, but are less susceptible to the Aβ25–35-induced toxicity. Data also suggest that oxidative stress occurs in this hyperglycemia animal model and that an increase in the antioxidant defense systems may exert protection against toxic insults. This mechanism, occuring in the early phases of diabetes, may correspond to an adaptive response.

Downregulation of the cAMP signal transduction cascade in the prothoracic glands is responsible for the fenoxycarb-mediated induction of permanent 5th instar larvae in Bombyx mori

Fenoxycarb application at 48 h (day 2) of the 5th instar of Bombyx mori induced permanent larvae with prothoracic glands (PGs) exhibiting weak ecdysteroidogenic activity. Although glands from control and fenoxycarb-treated larvae exhibited similar responses to dibutyl cAMP and forskolin on day 2, forskolin could not stimulate ecdysteroid secretion from PGs of fenoxycarb-treated larvae on day 3. Glands from control larvae incubated with cholera toxin (CTX) on day 3 had increased cAMP content and enhanced ecdysteroid secretion. Cholera toxin did not stimulate ecdysteroid secretion and marginally increased cAMP content in day 3 PGs of fenoxycarb-treated larvae. After application of fenoxycarb on day 2, crude brain extracts (cBRAIN) could not increase the glandular cAMP content throughout the rest of the 5th instar of the treated larvae. Fenoxycarb did not affect the basal or cBRAIN-stimulated cAMP accumulation in control PGs on day 2 and day 3 in vitro. Application of fenoxycarb on day 2 did not affect the recombinant PTTH (rPTTH)-stimulated ecdysteroid secretion on day 3, but reduced the cBRAIN-stimulated ecdysteroid secretion on day 3 to levels similar to that of rPTTH. The combined results suggest that the cAMP signalling cascade in the PGs of B. mori becomes nonfunctional after fenoxycarb application on day 2 of the 5th instar.

Involvement of the carboxyl terminus of the third intracellular loop of the cannabinoid CB1 receptor in constitutive activation of Gs.

The human cannabinoid receptor CB1 functionally couples primarily to Gi-, but also to Gs-mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl-terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist-independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs- and not a Gi-mediated pathway. Treatment with the CB1-specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP-55,940 to the L341A/A342L receptor was not markedly different from that for the wild-type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.

Beta-adrenergic receptor-dependent and -independent stimulation of adenylate cyclase is impaired during severe sepsis in humans.

a) To investigate the functional consequences of sepsis on the beta-adrenergic signal transduction in human circulating lymphocytes; b) to appreciate sepsis-associated catecholamine and cytokine release. Experimental, comparative study. Research laboratory in a university hospital. Healthy controls (n = 10); critically ill patients who were not septic (n = 7); septic patients with severe sepsis or septic shock (n = 11). Experiments were carried out using freshly isolated peripheral blood mononuclear cells (PBMC). We measured beta-adrenergic receptor (betaAR) number and affinity, and intracellular cAMP content at baseline and after the pharmacological stimulation of each component of the beta-adrenergic complex: betaAR with isoproterenol, Gs-protein with sodium fluoride (NaF), adenylate cyclase with forskolin. Catecholamine (adrenaline, noradrenaline) and cytokine (TNFalpha, IL-1alpha, IL-1beta, IL-6) serum levels were measured. In both septic and non-septic patients we observed a similar 40 % down-regulation of betaARs compared to controls, and a reduced basal and isoproterenol-stimulated cAMP accumulation (p < 0.05). The cAMP production elicited by NaF or forskolin was lower in septic patients than in the controls (p < 0.01). Forskolin-stimulated cAMP accumulation was significantly lower in septic patients than it was in non-septic ones (p < 0.001). Catecholamine serum concentrations were increased in the two patient groups without any significant difference. Elevated cytokine serum levels were detected in 45% of the septic patients (versus 14% of non-septic patients p < 0.05). Patients presenting with severe sepsis or septic shock have extended postreceptor defects of the beta-adrenergic signal transduction. This finding suggests a heterologous desensitization of adenylate cyclase stimulation.

Ca2+-Induced Inhibition of Adenylyl Cyclase in Turkey Erythrocyte Membranes

We investigated the effects of calcium ions (Ca²⁺) on the adenylyl cyclase activity in purified turkey erythrocyte membranes. Results showed the following: (i) Ca²⁺ inhibits cAMP accumulation stimulated by isoproterenol (1 µmol/l), NaF + AlCl₃ (10 mmol/l + 20 µmol/l) or forskolin (10 µmol/l) in EGTA-washed turkey erythrocyte membranes. IC₅₀ of free [Ca²⁺] is approximately 0.1 mmol/l in the presence of Mg²⁺ (2.5 mmol/l) and isobutylmethylxanthine (1 mmol/l). (ii) The potency of Ca²⁺ to inhibit cAMP accumulation is independent of the type of stimulus used to activate the adenylyl cyclase. We also evaluated the calcium sensitivity of the basal cAMP accumulation in the presence of GTP (10 µmol/l) and Mg²⁺ (2.5 mmol/l) which was also inhibited by Ca²⁺ with the same potency. (iii) The inhibition pattern of cAMP accumulation is not affected by the presence of added calmodulin (100 nmol/l). (iv) Ca²⁺ is ineffective on the binding of isoproterenol to the β-adrenoceptors. (v) Increasing the concentration of Ca²⁺ does not induce an observable activation of cyclic nucleotide phosphodiesterase in the present experimental conditions. Thus, we concluded that the inhibition of cAMP accumulation is due to an inhibition of the adenylyl cyclase rather than the activation of phosphodiesterase(s). The presence of a yet unidentified isoform of adenylyl cyclase that can be directly inhibited by Ca²⁺ or a G_i protein that can be activated by Ca²⁺ seems to explain these results. In either case, these results provide an additional mode of cross-talk that can take place between the Ca²⁺- and cAMP-signaling systems.

Hybrid tumors or salivary gland tumors sharing common differentiation pathways?: Reexamining adenoid cystic and epithelial-myoepithelial carcinomas

Three adenoid cystic carcinomas and two epithelial-myoepithelial carcinomas, which focally shared common histologic features, were studied to examine the common differentiation pathways manifested by these tumors and to discuss criteria for hybrid salivary gland tumors. Regions of the adenoid cystic carcinomas had cellular features ranging from simple clear cell change of basal/myoepithelial cells to combined clear cells and prominent ductal structures mimicking epithelial-myoepithelial carcinoma. Conversely, two epithelial-myoepithelial carcinomas had adenoid cystic carcinoma-like regions caused by the formation of “pseudocysts”; this resulted in a focal cribriform pattern. Electron microscopy of two additional but typical epithelial-myoepithelial carcinomas revealed both excess basal lamina at the margins of cellular nests and widened intercellular spaces containing reduplicated basal lamina and accumulations of glycosaminoglycans; these ultrastructural features were identical to those seen in adenoid cystic carcinomas. The five current cases are not examples of hybrid tumors, but they demonstrate the effects of gene expression and the resulting differentiation of synthetic products and tumor cells that are generally restricted to one or the other of these two tumor types by as-yet-unknown means. To avoid misdiagnosis and its prognostic implications, adenoid cystic carcinoma-like regions in epithelial-myoepithelial carcinoma and epithelial-myoepithelial-like regions in adenoid cystic carcinoma should be recognized simply as anomalous differentiation.

Heat shock protein 72 restores cyclic AMP accumulation after heat shock in N18TG2 cells

Although there are several reports on the alteration of intracellular signal transduction during heat shock in somatic cells, the long term effects of heat shock on neuronal cells remain unknown. In this report, we investigated cyclic AMP (cAMP) accumulation and the expression of heat shock proteins following heat shock in mouse neuroblastoma N18TG2 cells. Basal cAMP accumulation, or that stimulated by serotonin (10 μM), cholera toxin (1 μg/ml), and forskolin (1 μM) was suppressed at 0, 3, and 6 h following heat shock (45°C for 30 min). The cAMP levels were restored at 15 and 24 h after heat shock, corresponding with the expression of stress-induced heat shock protein 72 (HSP72). Quercetin, an inhibitor of HSP expression, decreased the expression of HSP72 and inhibited the recovery of cAMP levels 24 h after heat shock. Quercetin also decreased the basal expression of the constitutive heat shock cognate protein 70 (HSC70) and suppressed cAMP accumulation in non-heat shocked cells. These results suggest that stress-induced HSP72 restores cAMP accumulation to control levels following heat shock and that constitutive HSC70 is related to cAMP levels in non-stress conditions.

Reversible induction of mucous cell hyperplasia and parietal cell loss in rats dosed with DMP-777

Parietal cell loss and mucous cell hyperplasia are important histological components of a spectrum of gastric mucosal diseases including Menetrier's disease and fundic H. pylori gastritis. Recent investigations using transgenic mouse strains have highlighted the importance of intrinsic mucosal growth factors such as TGFct in the differentiation of specific cell lineages in the gastric mucosa. Unfortunately, experiments in transgenic mice have been hampered by the irreversibility of atrophic-gastritis phenotypes. In three-month toxicity studies in rats, oral dosing with DMP-777 (50-200 mg/kg/day), a neutrophil elastase inhibitor, resulted in an unexpected phenotype of gastric mucous cell hyperplasia and parietal cell loss associated with hypergastrinemia. Routine histological staining (diastase-resistant PAS) and immunocytochemistry for BrdU, H/K-ATPase, spasmolytic peptide, gastrin, somatostatin and chromogranin A were utilized to determine alterations in mucosal lineages. Rats did not demonstrate any significant inflammatory infiltrate. Fundic mucosa demonstrated a prominent hyperplasia of surface mucous cells, 4 to 8-fold increases in isthmus zone BrdU-labeling index and a profound decrease in the numbers of parietal ceils, chief cells, somatostatin ceils and ECL ceils after 9 days of dosing with 200 mg/kg/day. The numbers of mucous neck cells were unaffected by drug treatment. Hyperplasia of gastrin cells was seen in the gastric antrum, but there was no hyperplasia of antral mucous cells. These changes were completely reversible after a threemonth drug-free recovery period. To examine the marked hypergastrinemia observed in DMP 777-treated rats, the effects of the drug on acid secretion were studied in the rat pylorus-ligation model, where DMP-777 was shown to be a potent inhibitor of gastric acid secretion when dosed orally. Additional in vitro tests showed that DMP-777 did not block histamine (H2), acetylcholine (M3) or gastrin receptors (novel and CCK-B receptors). In isolated rabbit parietal cells, 3 pM DMP-777 completely inhibited basal, histaminestimulated and forskolin-stimulated aminopyrine accumulation (ECso---0.6 pM). While the drug had no direct effect on the activity of the H/K-ATPase, 1-3 laM DMP-777 could dissipate completely an established stimulated proton gradient in isolated tubulovesicles loaded with acridine orange. These latter results suggest that DMP-777 is a protonophore. Since other regions of the gastrointestinal mucosa are not affected by the drug, unexpected effects of DMP-777 on the gastric mucosa may be due to a specific interaction with parietal cell, perhaps involving a specific partitioning of the drug into the parietal cell secretory canalicolus. Short term DMP-777 dosing of rats provides the first readily reversible model of parietal cell loss and mucous cell hyperplasia. This work was funded by DuPont-Merck Pharmaceuticals.

Deletions in the Third Intracellular Loop of the Thyrotropin Receptor: A NEW MECHANISM FOR CONSTITUTIVE ACTIVATION

Gain-of-function mutations of the thyrotropin receptor (TSHR) gene have been invoked as one of the major causes of toxic thyroid adenomas. In a toxic thyroid nodule, we recently identified a 9-amino acid deletion (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This finding exemplifies a new mechanism of TSHR activation and raises new questions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein interaction in many receptors, we systematically reexamined the role of the TSHR's i3 loop for G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We identified an optimal deletion length responsible for constitutive activity. If the number of deleted amino acids was reduced, elevated basal cAMP accumulation was found to be concomitantly diminished. Expansion of the deletion dramatically impaired cell surface expression of the receptor. Shifting the deletion toward the N terminus of the i3 loop resulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significantly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the first time that amino acid deletions within the i3 loop of a G protein-coupled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G protein coupling can be deleted without rendering the mutant receptor unresponsive to thyrotropin. These findings support a novel model explaining the molecular events accompanying receptor activation by agonist.

Adenosine A1 receptor activation inhibits basal accumulation of inositol phosphates in rat hippocampus.

The ability of the adenosine A1 receptor selective agonist, N6-cyclopentyladenosine, to modify basal accumulation of inositol phosphates in rat hippocampal slices, was investigated. Cyclopentyladenosine (10-300 nM) inhibited the basal accumulation of total [3H]inositol phosphates, with an EC50 of 10 nM and an Emax of 24%. This effect of cyclopentyladenosine was prevented by the adenosine A1 receptor selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (30 nM). Cyclopentyladenosine (100 nM) also inhibited histamine (300 nM)-stimulated accumulation of [3H]inositol phosphates, this effect being quantitatively similar to that observed on basal [3H]inositol phosphates accumulation. The results suggest that adenosine A1 receptor activation is able, per se, to inhibit the formation of phosphatidylinositol-derived second messengers in hippocampus.

Ocular hypotensive and vasodilative effects of two β-adrenergic blockers with intrinsic sympathomimetic activity

Purpose. The ocular hypotensive and vasodilative effects of vaninolol and eugenolol, two β-adrenergic blockers with intrinsic sympathomimetic activity, were tested in rabbits and their pharmacologic mechanisms were also studied in vitro.Methods. Intraocular pressure was measured in ocular hyper-tonic rabbits which were induced by infusing 20% NaCl or 5% glucose solution. The rabbit's ocular blood flow was determined using the colored microsphere technique. The concentrations of cAMP were evaluated in porcine ciliary body and cultured A7r5 smooth muscle cells by radioimmunoassay. Ca+2 concentration was measured in A7r5 cells by spectrofluorometry after loading cells with Fura-2-AM.Results. It was found that 0.5% eugenolol and vaninolol could suppress the intraocular pressure in glucose-induced ocular hypertensive rabbits and delay the intraocular pressure recovery in NaCl-induced ocular hypertensive rabbits. In addition, both agents improved the ocular blood flow in the iris, ciliary body, retina and choroid. Vaninolol and eugenolol of 10 μM inhibited the basal cAMP accumulation from 23.9 ± 2.0 of control to 8.7 ± 0.4 and 2.4 ± 0.1 respectively and inhibited the isoproterenol-induced cAMP accumulation from 154.3 ± 13.6 to 120.6 ± 8.3 and 74.2 ± 6.1 respectively in the porcine ciliary body. The cellular cAMP concentration was significantly increased from 10 ± 1 of control to 96 ± 5 (vaninolol) and 38 ± 3 (eugenolol) in cultured A7r5 smooth muscle cells. Both agents also increased the intracellular calcium concentration in A7r5 cells.Conclusions. These results indicate that the lowering of intraocular pressure by vaninolol and eugenolol may be due to cAMP suppression in the ciliary body by β-antagonist and/or α2-agonist activities. Both agents cause vasodilation via β2-agonist action that increase the smooth muscle cellular cAMP level more than vasoconstriction via α-agonist activity by increasing an influx of extracellular Ca+2. Curr. Eye Res. 17: 700–707, 1998.

Ocular hypotensive and vasodilative effects of two β-adrenergic blockers with intrinsic sympathomimetic activity

Purpose. The ocular hypotensive and vasodilative effects of vaninolol and eugenolol, two β-adrenergic blockers with intrinsic sympathomimetic activity, were tested in rabbits and their pharmacologic mechanisms were also studied in vitro.Methods. Intraocular pressure was measured in ocular hyper-tonic rabbits which were induced by infusing 20% NaCl or 5% glucose solution. The rabbit's ocular blood flow was determined using the colored microsphere technique. The concentrations of cAMP were evaluated in porcine ciliary body and cultured A7r5 smooth muscle cells by radioimmunoassay. Ca+2 concentration was measured in A7r5 cells by spectrofluorometry after loading cells with Fura-2-AM.Results. It was found that 0.5% eugenolol and vaninolol could suppress the intraocular pressure in glucose-induced ocular hypertensive rabbits and delay the intraocular pressure recovery in NaCl-induced ocular hypertensive rabbits. In addition, both agents improved the ocular blood flow in the iris, ciliary body, retina and choroid. Vaninolol and eugenolol of 10 μM inhibited the basal cAMP accumulation from 23.9 ± 2.0 of control to 8.7 ± 0.4 and 2.4 ± 0.1 respectively and inhibited the isoproterenol-induced cAMP accumulation from 154.3 ± 13.6 to 120.6 ± 8.3 and 74.2 ± 6.1 respectively in the porcine ciliary body. The cellular cAMP concentration was significantly increased from 10 ± 1 of control to 96 ± 5 (vaninolol) and 38 ± 3 (eugenolol) in cultured A7r5 smooth muscle cells. Both agents also increased the intracellular calcium concentration in A7r5 cells.Conclusions. These results indicate that the lowering of intraocular pressure by vaninolol and eugenolol may be due to cAMP suppression in the ciliary body by β-antagonist and/or α2-agonist activities. Both agents cause vasodilation via β2-agonist action that increase the smooth muscle cellular cAMP level more than vasoconstriction via α-agonist activity by increasing an influx of extracellular Ca+2. Curr. Eye Res. 17: 700–707, 1998.

Constitutive activity of a chimeric D2/D1 dopamine receptor.

Chimeric D1/D2 receptors were constructed to identify structural determinants of drug affinity and efficacy. We previously reported that chimeras that had D1 receptor transmembrane domain VII together with amino-terminal sequence from the D2 receptor were nonfunctional. D2/D1 chimeras were constructed that contained D2 receptor sequence at the amino- and carboxyl-terminal ends and D1 receptor sequence in the intervening region. Chimeric receptors with D2 sequence from transmembrane domain 7 to the carboxyl terminus together with D2 receptor sequence from the amino terminus through transmembrane helix 4 (D2[1-4,7]) and 5 (D2[1-5,7]) bound [3H]spiperone with high affinity, consistent with the hypothesis that D2 receptor transmembrane domain I or II is incompatible with D1 receptor transmembrane domain VII. D2[1-4,7] and D2[1-5,7] had affinities similar to D1 and D2 receptors for most nonselective dopamine antagonists and had affinities for most of the selective antagonists that were intermediate between those of the parent receptors. D2[1-4,7] and D2[1-5,7] mediated dopamine receptor agonist-induced stimulation and inhibition, respectively, of cAMP accumulation. The more efficient coupling of D2[1-5,7] to inhibition of cAMP accumulation, compared with the coupling of D2[5-7] and D2[3-7], supports the view that multiple D2 receptor cytoplasmic domains acting in concert are necessary for receptor activation of Gi. In contrast, D2[1-4,7], which contains only one cytoplasmic loop (the third) from the D1 receptor, is capable of activating Gs. D2[1-4,7] exhibited several characteristics of a constitutively active receptor, including enhanced basal (unliganded) stimulation of cAMP accumulation, high affinity for agonists even in the presence of GTP, and blunted agonist-stimulated cAMP accumulation. A number of dopamine receptor antagonists were inverse agonists at D2[1-4,7], inhibiting basal cAMP accumulation. Some of these drugs were also inverse agonists at the D1 receptor. Interestingly, several antagonists also potentiated forskolin-stimulated cAMP accumulation via D2[1-5,7] and via the D2 receptor, which could reflect inverse agonist inhibition of native constitutive activity of this receptor.

Insulin stimulation of lactate accumulation in isolated human granulosa-luteal cells: a comparison between normal and polycystic ovaries.

Polycystic ovary syndrome (PCOS) is often associated with hyperinsulinaemia and peripheral insulin resistance. Whether the ovary is resistant to insulin is a matter of controversy. The aim was therefore to study the effect of insulin on lactate accumulation, an indicator of glucose metabolism, in granulosa-luteal cells from women with PCOS and from women with normal ovarian function. The cells were obtained from women undergoing clinical in-vitro fertilization-embryo transfer, either from patients with normal ovarian function and tubal or male infertility, or from women with PCOS, with or without tubal factor. The patients were down-regulated with buserelin and stimulated with urofollitrophin and human chorionic gonadotrophin (HCG). Follicle aspiration was performed under ultrasound guidance. Following oocyte recovery the granulosa-luteal cells were isolated, washed and cultured (2-3 x 10(4) viable cells/well) in serum-free Eagle's minimal essential medium for 48 h. After washing, the cells were then cultured in medium containing HCG (0.1-10 IU/ml) or insulin (0.05-0.5 microg/ml) for 24-48 h. Lactate accumulation in the media and cellular protein were analysed. Basal lactate accumulation did not differ in granulosa-luteal cells obtained from normal or PCOS ovaries, and averaged 46 and 49 nmol/g protein/24 h, respectively. A significant stimulation (40-60%) was obtained by HCG in both groups. Insulin caused a dose-dependent increase in lactate in granulosa-luteal cells obtained from normal ovaries (control: 45.5 +/- 6.3; insulin 0.5 microg/ml: 77 +/- 10 nmol/microg protein). Lactate accumulation in granulosa-luteal cells from PCOS ovaries was not altered in the presence of insulin. These results suggest that granulosa-luteal cell glucose metabolism is resistant to insulin in PCOS.

Interleukin-1 β inhibits progesterone accumulation in rat corpora luteal cell cultures in a mechanism dissociated from its effects on nitric oxide and prostaglandin E accumulation

The objective of this study was to examine the effect of interleukin-1 β (IL-1) on progesterone (P) biosynthesis and the potential intermediary involvement of prostaglandin (PG) E and nitric oxide (NO) in P accumulation in PMSG/hCG-primed rat corpora luteal (CL) cell cultures. Exposure of primed CL cells to IL-1 (10 ng/ml) for 48 h resulted in a 65–86% reduction ( P<0.01) in P accumulation concurrent with a 2–3.4-fold increase in PGE content, a 70% increase in PGF 2 α content and a 1.9–3.3-fold increase in nitrite generation. These effects were abolished by the IL-1 receptor antagonist, suggesting specific IL-1 receptor-mediated effects. Indomethacin, a cyclooxygenase inhibitor, abolished PGE and PGF 2 α production and attenuated the basal (but not IL-1-stimulated) accumulation of P. N G-Nitro- l-arginine (NNLA), a competitive inhibitor of nitrite synthesis, slightly reduced basal P accumulation but had no effect on IL-1-induced suppression of P accumulation. NNLA reduced basal PGE accumulation and IL-1-stimulated PGE accumulation (55 and 61%, respectively). Transforming growth factor β1 (TGF- β1; 10 ng/ml) significantly attenuated the IL-1-stimulated PGE and NO production (61 and 42%, respectively), but did not affect the ability of IL-1 to suppress P accumulation. Thus, NO, PGF 2 α and PGE are not obligatory intermediaries of IL-1-mediated suppression of P accumulation in rat CL, but are involved in basal P biosynthesis and NO seems to have a regulatory role in the biosynthesis of PGE. The present observations suggest a pleiotropic response of PMSG/hCG-primed CL cells to IL-1, characterized by an independent suppression of P accumulation and a concomitant increase in NO, PGF 2 α and PGE generation. Since IL-1 attenuates P accumulation, these findings may imply a direct autocrine/paracrine function for IL-1 in the maintenance or the demise of rat CL.

Sodium Saccharin Inhibits Adenylyl Cyclase Activity in Non-Taste Cells

We have studied the in vitro effect of sodium saccharin (NaSacch) on the rat adipocyte adenylyl cyclase complex. NaSacch (2.5–50 mM) inhibited significantly in a dose-dependent manner basal and isoproterenol-stimulated cAMP accumulation on isolated rat adipocytes. Similarly, NaSacch (2.5–50 mM) inhibited forskolin-stimulated adenylyl cyclase activity measured in the presence of Mg 2+-ATP on adipocyte, astrocyte and thyrocyte membrane fractions. In contrast, NaSacch did not inhibit but slightly increased the forskolin-stimulated adenylyl cyclase activity measured in the presence of Mn 2+-ATP and GDPβS, a stable GDP analogue. The effect of NaSacch was not mediated through either the A 1-adenosine receptor (A 1R) or the α 2-adrenergic receptor (α 2AR). The inhibitory effect of NaSacch was additive to that of A 1R agonist and was not blocked by the addition of the α 2AR antagonist RX 821002. Pretreatment of adipocytes with pertussis toxin slightly attenuated but did not abolish the inhibitory effect of NaSacch on forskolin-stimulated adenylyl cyclase activity on membrane fractions. These data suggest that the inhibitory effect of NaSacch on forskolin stimulated-adenylyl cyclase in adipocytes does not imply only Gi protein but also other direct or indirect inhibitory pathway(s) which remain to be determined.

Diversity and prevalence of somatic mutations in the thyrotropin receptor and Gs alpha genes as a cause of toxic thyroid adenomas.

A total of 33 different autonomous hot nodules from 31 patients, originating mainly from Belgium, were investigated for the presence of somatic mutations in the TSH receptor and Gs alpha genes. This constitutes an extension of our previous study, including the first 11 nodules of the series. The complete coding sequence of the TSH receptor gene and the segments of Gs alpha known to harbor mutations impairing guanosinetriphosphotase activity were studied by direct sequencing of genomic DNA extracted from the nodules. DNA from the juxtanodular tissue or peripheral white blood cells was analyzed in all patients to confirm that the mutations identified were somatic. Twenty-seven mutations (82%) were found in the TSH receptor gene, affecting a total of 12 different residues or locations. All these mutations but 2 (see below) have been identified previously as activating mutations. Only 2 mutations were found in Gs alpha (6%). In 4 nodules, no mutation was detected. Five residues (Ser281, Ile486, Ile568, Phe631, and Asp633) were found mutated in 3 or 4 different nodules, making them hot spots for activating mutations. Phe631 and Asp633 belong to a cluster of 5 consecutive residues (629-633) in the N-terminal half of transmembrane segment VI; which harbor together 44% of the mutations identified in this cohort. Two novel mutations were identified: a point mutation causing substitution of Phe for Leu at position 629 (L629F); and a deletion of 12 bases removing residues 658-661 at the C-terminal portion of exoloop 3 (del658-661). When tested by transfection in COS-7 cells, both mutant receptors display increase in constitutive stimulation of basal cAMP accumulation. Although it is still capable of binding TSH, the del658-661 mutant has completely lost the ability to respond to the stimulation by the hormone. Our results demonstrate that, in a cohort of patients from a moderately iodine deficient area, somatic mutations increasing the constitutive activity of the TSH receptor are the major cause of autonomous hot nodules.