Adenosine Monophosphate Formation Research Articles

Dopamine D1-like Receptors and Reward-related Incentive Learning

BENINGER, R. J., R. MILLER. Dopamine D1-like receptors and reward-related incentive learning. NEUROSCI BIOBEHAV REV 22(2) 335–345, 1998.—There now is general agreement that dopaminergic neurons projecting from ventral mesencephalic nuclei to forebrain targets play a critical role in reward-related incentive learning. Many recent experiments evaluate the role of dopamine (DA) receptor subtypes in various paradigms involving this type of learning. The first part of this paper reviews evidence from these studies that use antagonists or agonists relatively specific for D1- or D2-like receptors in operant paradigms with food, brain stimulation, self-administered stimulant or conditioned rewards or place conditioning. The focus is on studies that directly compare agents acting at the two DA receptor families, especially those studies where the agents produce differential actions. Results support the conclusion that D1-like receptors play a more critical role in reward-related learning than D2-like receptors. D1-like receptors initiate a cascade of intracellular events including cyclic adenosine monophosphate (cAMP) formation and activation of cAMP-dependent protein kinase (PKA). The final section of this paper reviews evidence from a wide range of neuroscience experiments that implicates the cAMP/PKA pathway in learning in general and in reward-related incentive learning in particular. We conclude that the molecular mechanism underlying DA-mediated incentive learning may involve DA release in association with reward, stimulation of D1-like receptors, activation of the cAMP/PKA cascade and additional intracellular events leading to modification of cortico-striatal glutamatergic synapses activated by stimuli encountered in close temporal contiguity with reward. Thus, when reward-related incentive learning takes place, it may be the action of DA acting at D1-like receptors that leads to plastic changes in the striatum that form the substrate of that learning.

A Naturally Occurring Basically Charged Human Follicle-Stimulating Hormone (FSH) Variant Inhibits FSH-Induced Androgen Aromatization and Tissue-Type Plasminogen Activator Enzyme Activity in vitro

It is well known that deglycosylation of gonadotropins by enzymatic or chemical procedures or by deletion of sites for N-linked glycosylation produces antagonistic analogs which are able to interact strongly with the receptor and to inhibit binding of the wild-type hormone. In the present study, we analyzed the antagonistic properties of a naturally occurring basic follicle-stimulating hormone (FSH) charge isoform obtained after high-resolution chromatofocusing of human anterior pituitary glycoprotein extracts. Coincubation of increasing amounts of this isoform with a highly purified human pituitary FSH preparation or with recombinant human FSH at doses equivalent to their corresponding ED₅₀ for estradiol and tissue-type plasminogen activator (tPA) production, inhibited FSH-induced estrogen production and tPA enzyme activity by cultured rat granulosa cells in a dose-dependent manner. These inhibitory effects were apparently exerted at steps following 3′,5′-cyclic adenosine monophosphate (cAMP) formation and did not involve activation of the protein kinase C pathway since: (a) at low doses, this basic FSH isoform moderately increased FSH-induced cAMP production by cultured rat granulosa cells; (b) coincubation of the antagonist isoform with dibutyryl cAMP completely inhibited the effects of this cAMP analog on estrogen and tPA production; (c) the isoform was able to stimulate production of cAMP in a human fetal cell line expressing the recombinant human FSH receptor, and (d) the inhibitory effects of the isoform were not affected by staurosporine, a protein kinase C inhibitor. The effects of this isoform upon dibutyryl cAMP-induced estrogen and tPA production were blocked by the addition of a highly specific antibody directed against human FSH, further demonstrating that the antagonistic effects observed were due to FSH-like molecules. In contrast to the inhibitory effects exhibited by this basic FSH isoform, a more acidic FSH charge variant consistently acted as an agonist of pituitary and recombinant FSH on both estrogen production and induction of tPA enzyme activity. These results indicate that the anterior pituitary gland normally produces FSH isoforms which act as either agonists or antagonists of FSH at the target cell level.

Desialylated and deglycosylated human chorionic gonadotropin are superagonists of native human chorionic gonadotropin in human thyroid follicles.

Highly purified human chorionic gonadotropin (hCG) interacts with the thyrotropin (TSH) receptor and stimulates triiodothyronine (T3) secretion, iodide uptake and organification, and cyclic adenosine monophosphate (cAMP) formation in human thyroid follicles. Because of interest in the role of the carbohydrate component in the structure-function relationships of hCG we undertook to deplete hCG of its sialic acid or carbohydrate residues and assess the thyrotropic activity of the carbohydrate-modified forms. For this purpose, we used our assay system consisting of human thyroid follicles cultured and suspended in collagen gel in serum-free medium. Under these conditions, the cells are organized as follicular three-dimensional structures with normal polarity, enabling enhanced responsiveness to hormonal stimulation, and T3 secretion can be measured as a response parameter. Desialylated (ds)-hCG and deglycosylated (dg)-hCG dose-dependently stimulated T3 secretion, iodide uptake and organification, and in each case did so with about twice the intrinsic activity of native hCG. Indeed, removal of the sialic acid or carbohydrate residues from native hCG transformed it into a thyroid stimulator that elicited a maximal response in terms of iodide uptake, organification and T3 secretion by human thyroid follicles as high as TSH and almost twice as high as native hCG. Not only were ds-hCG and dg-hCG more intrinsically active than hCG, they were more than five times as potent. As with hCG, both ds-hCG and dg-hCG managed to elicit such responses in human thyrocytes while evoking minimal amounts of cAMP, illustrating the concept of cAMP superfluity and highlighting the potential pitfalls of using cAMP as a measure of hormonal bioactivity. hCG, and to a greater extent ds-hCG and dg-hCG, inhibited, as did TSH, gamma-interferon-induced human leukocyte antigen-DR (HLA-DR) expression in human thyrocytes, again reflecting the intrinsic thyrotropic activity of native hCG and its variants depleted of sialic acid or carbohydrate residues. In conclusion, this is the first report on the thyrotropic activity of ds-hCG and dg-hCG using the physiologically relevant hormonal end-point response, thyroid hormone secretion. The study was conducted in a serum-free culture system of human thyroid follicles and shows that removal of the sialic acid or carbohydrate residues from native hCG transform hCG variants into thyroid stimulating superagonists. The hCG variants inhibited, as did TSH, gamma-interferon-induced HLA-DR expression.

Regulation of transforming growth factor beta 1 messenger ribonucleic acid expression in porcine thyroid follicles in vitro by growth factors, iodine, or delta-iodolactone.

Transforming growth factor beta 1 (TGF beta 1) is an autocrine growth factor for thyrocytes and is supposed to be the mediator of iodine-induced growth inhibition of thyroid epithelial cells, but this is still controversial. We further investigated this hypothesis using intact porcine thyroid follicles ex vivo in a three-dimensional culture system. In this culture system it has been shown previously that both iodide as well as delta-iodolactone, the putative iodocompound mediating thyroid cell proliferation, inhibit growth of these follicles. We measured the amount of TGF beta 1 mRNA expression in these follicles after treatment either with thyrotropin (TSH), epidermal growth factor (EGF), or transforming growth factor alpha (TGF alpha) for growth stimulation or with inorganic iodine or delta-iodolactone in concentrations known to inhibit growth. TGF beta 1-mRNA was detected by Northern blot analysis. The known major transcript of 2.5 kb was detected in a steady state level up to 48 hours in untreated thyroid follicles. EGF and TGF alpha (5 ng/mL each) enhanced TGF beta 1 mRNA about threefold within 4 and 8 hours. This increase of TGF beta 1 mRNA was slightly decreased by simultaneous incubation with delta-iodolactone (1 microM) or iodide (40 microM KI). In contrast, both TSH (1 mU/mL) and forskolin (16 microM) decreased TGF beta 1 mRNA expression to about 70%, and this effect was abolished when follicles were pretreated with iodide (40 microM KI) in a concentration known to inhibit TSH action on cyclic adenosine monophosphate (cAMP) formation and proliferation. Iodide or delta-iodolactone alone had no significant effect on basal TGF beta 1 mRNA expression. We conclude that the growth inhibitory effect of iodide as well as of delta-iodolactone is not mediated through TGF beta 1 in intact porcine thyroid follicles ex vivo. The stimulatory effect of EGF and TGF alpha on TGF beta 1 expression might be related to extracellular matrix modulation during proliferation.

Oxidation and Nitration of Catecholamines by Nitrogen Oxides Derived from Nitric Oxide

The reactivity of catecholamines with nitrogen oxides formed from NO in aerated solutions, nitrite, and peroxynitrite was evaluated. Dopamine and norepinephrine in aerobic buffer (pH 7.4) were almost completely converted to their 6-nitro-derivatives by nitric oxide (NO) at room temperature, while epinephrine was nitrated and above all oxidized. The products obtained from each catecholamine treated with sodium nitrite at pH 4–7 were compared to those produced by NO at pH 7.4. Peroxynitrite, which can nitrate tyrosinyl residues, did not produce nitro-derivatives, only oxidized ones. The physiological relevance, particularly for the vascular and nervous system, is discussed. Catecholamine oxidation reactions could be relevant to physiological conditions and also explain neurotoxicity in Parkinson's disease and aging. Nitration reactions, requiring such high NO concentrations, do not seem possible to occur directly under normal physiological conditions, but could take place in acidic vesicules where nitrite, catecholamines, and their nitrated products could accumulate. Finally, the ability of dopamine to increase 2′,5′-cyclic adenosine monophosphate (cAMP) formation in cultured striatal neurons was blocked by its nitration by NO or its nitrogen oxide derivatives.

Mutants in the ADP-ribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity.

Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties. We have constructed two CT-A subunit mutants, e.g., serine-->phenylalanine at position 61 (S61F), and glutamic acid-->lysine at 112 (E112K) by site-directed mutagenesis. Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops. Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses. Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs. OVA-specific CD4+ T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response. These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.

Effector coupling of somatostatin receptor subtypes on human endocrine tumors

Effector coupling of somatostatin receptor subtypes sst 1 and sst 2 was examined in a reconstituted system. Forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation was inhibited 66% by somatostatin (SRIF-14) in CHO cells expressing somatostatin receptor 1 (sst 1) (CHO-SR1), but not sst 2, in a dose-dependent manner with an ED 50 of 1 × 10 −9 mol/L SRIF-14. The inhibition was blocked by pertussis toxin (PTX), indicating that sst 1 is coupled to adenylyl cyclase via PTX-sensitive G i protein. In CHO cells, G iα2 and G iα3 mRNAs were detected. In adenylyl cyclase assays, 1 μmol/L SRIF-14 caused a 16% inhibition of forskolin-stimulated adenyly cyclase activity. Preincubation with G iα3, but not G i α1 G i α2 , antiserum blocked this inhibition. By contrast, sst 2 is coupled to adenylyl cyclase via G iα1. In cells expressing sst 2 with G iα1 (CHO-SR2G1), SRIF-14 significantly inhibited forskolin-stimulated cAMP formation by 53% and with an ED 50 at 4 × 10 −9 mmol/L SRIF-14, which was completely blocked by PTX; ED 50 values for sst 1 and sst 2 agree with the IC 50 values in binding assays. In CHO-SR1, the rank of potency of agonists affecting adenyl cyclase was SRIF-14 = SRIF-28 > RC 160 > SMS 201-995. In CHO-SR2G1, the rank was RC-160 > SRIF-14 = SRIF-28 > SMS201-995.

Platelet receptor desensitization induced by elevated prostacyclin levels causes platelet-endothelial cell adhesion

The purpose of this study was to investigate the role of platelet prostacyclin receptor desensitization in platelet-endothelial cell adhesion. Platelet-endothelial cell adhesion is regulated by endothelial cell-derived mediators, such as prostacyclin and endothelium-derived relaxing factor. Prostacyclin activates platelet adenylate cyclase and augments cyclic adenosine monophosphate formation by way of specific membrane receptors. Platelet exposure to prostacyclin or chemically stable analogs results in a time- and dose-dependent prostacyclin receptor desensitization as it occurs during infusion therapy with prostacyclin analogs or in pathophysiologic situations such as acute myocardial infarction. Adhesion of washed and radiolabeled human platelets stimulated with thrombin to cultured umbilical vein endothelial cells was measured under control conditions and under conditions of platelet prostacyclin receptor desensitization induced by incubation with the prostacyclin analog iloprost (10 to 100 nmol/liter) for 3 h. Thrombin (0.08 to 0.2 U/ml) increased platelet adhesion in a dose-dependent manner from 2.7 +/- 0.3% to 6.4 +/- 0.6% (mean value +/- SEM). Preincubation of platelets resulted in a dose-dependent down-regulation of 3H-iloprost binding up to 58.8 +/- 6.7% of control platelets with 100 nmol/liter of iloprost. Co-incubation of prostacyclin receptor-desensitized platelets with endothelial cells resulted in a marked augmentation of thrombin-induced adhesion up to 28.6 +/- 4.5%. Approximately the same increase in platelet adhesion was seen after complete abrogation of endothelial cell prostacyclin synthesis by pretreatment with aspirin. Comparison of iloprost-induced receptor desensitization and increased platelet-endothelial cell adhesion indicated a positive correlation. Platelet prostacyclin receptor desensitization was observed in humans in vivo during acute myocardial infarction or during therapeutic administration of prostacyclin analogs. In vitro platelet prostacyclin receptor desensitization caused a marked augmentation of platelet-endothelial cell adhesion. This increase in adhesion might result in an enhanced tendency toward thrombus formation in humans.

Stimulation of cyclic adenosine monophosphate formation by the novel vasorelaxant peptide adrenomedullin in cultured rat mesangial cells

The present study was designed to examine the effect of synthetic adrenomedullin (AM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on intracellular cyclic adenosine monophosphate (cAMP) formation in cultured rat mesangial cells. The effect of AM on cAMP formation in rat mesangial cells was compared with its effect in cultured rat vascular smooth muscle cells, cAMP levels were determined by radioimmunoassay after stimulation for 30 minutes with different concentrations (10 −10 to 10 −7 mol/L) of rat and human AM. Rat and human AM concentration-dependently (10 −9 to 10 −7 mol/L) stimulated cAMP formation in cultured mesangial cells. This stimulatory effect of rat AM was significantly greater than human AM. The stimulatory effect of rat AM in mesangial cells was significantly weaker than its potency in vascular smooth muscle cells. These preliminary data suggest that mesangial cells, as well as vascular smooth muscle cells, possess AM receptors functionally coupled to adenylate cyclase.

Characterisation of a new human neuroblastoma cell line, NAL-GT

This paper describes the establishment of a new polyclonal human neuroblastoma cell line NAL-GT. Pharmacological characterisation of a cell line comprising > 70% neurones using radioligand binding, Ins(1,4,5)trisphosphate (Ins(1,4,5)P 3) mass formation, intracellular Ca 2+ ([Ca 2+] i) determinations and cyclic adenosine monophosphate (cAMP) formation has been performed. Carbachol (1 mM) and noradrenaline (l0, μM) increased Ins(1,4,5)P 3 formation 2-3-fold basal. Noradrenaline (l0 μM) and morphine (10 μM) reduced forskolin stimulated cAMP formation by 19.7 and 30.5%, respectively. Carbachol (1 mM) and K+ (50 mM) increased [Ca 2+] i. These data indicate that polyclonal (heterogeneous) NAL-GT cells express muscarinic, alphas and alpha 2 adrenoceptors, opioid receptors and voltage-sensitive Ca 2+ channels and may prove useful in the study of the cellular basis of drug action.

Bursal antisteroidogenic peptide alters the activity of steroidogenic enzymes in chicken granulosa cells

We have previously reported that a peptide from chicken bursa of Fabricius, bursal antisteroidogenic peptide (BASP), inhibits luteinizing hormone-stimulated progesterone biosynthesis by chicken ovarian granulosa cells. The objective of this study was to determine the site(s) of BASP inhibition within the steroidogenic pathway of chicken granulosa cells. The effects of BASP on key steroidogenic enzymes, including adenylyl cyclase (AC), phosphodiesterase, the cholesterol side-chain cleavage enzyme complex and 3ß-hydroxysteroid dehydrogenase were determined. Luteinizing hormone (10 ng/tube) stimulated a fivefold increase in granulosa cell progesterone production that was inhibited by BASP (0.06, 0.12 or 0.25 bursal equivalents) in a dose-dependent manner. Luteinizing hormone stimulated a sixfold increase in cyclic 3′,5′-adenosine monophosphate (cAMP) formation, and this increase was potentiated by BASP in a dose-dependent manner. In addition, BASP stimulated cAMP formation in the absence of luteinizing hormone without affecting progesterone production. The AC activator forskolin (0.1 mM) stimulated a 4.5-fold increase in progesterone synthesis, which was inhibited by BASP. In the presence of forskolin. BASP increased cAMP formation in a dose-dependent manner. A fivefold increase in progesterone synthesis induced by the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (1.0 mM) was inhibited by BASP. In the presence of 3-isobutyl-l-methylxanthine, BASP increased cAMP formation in a dose-dependent manner. Finally, 22(R)-hydroxycholesterol (250, 500, 1,000, or 2,500 ng/tube) or pregnenolone (50, 100, 200, or 500 ng/tube) resulted in up to 15- or 10-fold increases in progesterone production, respectively. Increasing concentrations of BASP caused a dose-dependent suppression of the conversion of 22(R)-hydroxycholesterol, but not pregnenolone, to progesterone. The inhibition of steroidogenesis by BASP is not associated with reduced cAMP levels, and BASP appears to strongly stimulate AC activity. In addition, these findings suggest that BASP may limit the availability of progesterone precursors by inhibiting the activity of the cholesterol side-chain cleavage enzyme complex.

Changes in G Protein Pattern and in G Protein-Dependent Signaling During Erythropoietin- and Dimethylsulfoxide-Induced Differentiation of Murine Erythroleukemia Cells

We have studied the expression of G protein subtypes and the role of G protein-dependent signaling in two subclones of RED-1 cells, an erythropoetin(Epo)-sensitive, murine erythroleukemia cell line. Clone 6C8 showed terminal erythroid differentiation in response to a combined treatment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation. We measured G protein alpha subunit levels by toxin-catalyzed adenosine diphosphate (ADP)-ribosylation with [32P]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera. Native RED-1 cells expressed G alpha i2, alpha i3, alpha s, and alpha q/11, but not alpha i1 and alpha o. Terminal differentiation was associated with a selective loss (approximately 80%) of G alpha i3 and an increase in a truncated cytosolic form of G alpha i2, while the membrane levels of alpha i2, alpha q/11, and alpha s did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for alpha s, G3 cells contained significantly higher levels of the different G protein alpha subunits tested. Stimulation of G protein-coupled receptors by thrombin and ADP caused a pertussis toxin (PTX)-inhibitable transient increase in intracellular Ca2+ that was markedly reduced in differentiated cells. In G3 cells, but not in 6C8 cells, thrombin also caused a PTX-sensitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine monophosphate (cAMP) formation. Our results show that specific alterations in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal relationship. The loss of G alpha i3 may affect cellular responses that are mediated via P2T purine or thrombin receptors.

Lack of vasoactive intestinal peptide-releasing property in prolactin cells from ovariectomized rats: contribution of post-transductional impairments.

We have demonstrated recently that in menopausal women and in ovariectomized rats the deficiency of circulating oestrogens impairs vasoactive intestinal peptide (VIP) efficacy in stimulating prolactin (PRL) release. The present study was designed to investigate whether the lack of VIP-induced PRL release after ovariectomy is a consequence of a defect at the receptor-transductional or post-transductional level. For this purpose we evaluated the VIP receptor function, by measuring VIP-stimulated cyclic adenosine monophosphate (AMP) formation, and the efficacy of the cyclic AMP-dependent PRL release in pituitary cells from control and ovariectomized animals. We observed that VIP induced a significantly higher stimulation of adenylate cyclase in pituitary homogenates from ovariectomized rats than in those from control animals. This effect appeared to be linked to an increased efficiency of the Gs coupling protein, because superimposable results were obtained by using the non-hydrolysable guanosine triphosphate (GTP) analogue, 5-guanylylimidodiphosphate. On the contrary, the cyclic AMP analogue, 8-Br-cAMP, that potently stimulated PRL release from control pituitary cells was completely ineffective in cells from ovariectomized rats. The present data indicate that in PRL-secreting cells from ovariectomized rats a defect in the post-transductional mechanism that couples the VIP receptor to PRL secretion, rather than a reduction of receptor function, possibly accounts for the lack of VIP efficacy.

Expression of Muscarinic M2 Receptors in Cultured Human Airway Smooth Muscle Cells

[3H]-inositol phosphate formation and the inhibition of isoproterenol-induced [3H]-cyclic adenosine monophosphate (AMP) formation in response to the muscarinic agonist carbachol were studied in cultured human airway smooth muscle cells. Stimulation with carbachol produced concentration-dependent inhibition of isoproterenol (1 microM)-induced cyclic AMP formation (EC50, 0.15 microM; maximal inhibition, 60%). This response was itself reversed by pertussis toxin (100 ng/ml) and was competitively inhibited by the muscarinic antagonists pirenzepine (pA2, 6.5), methoctramine (pA2, 8.0), 4-diphenylacetoxy-N-methyl-piperidine (pA2, 8.0), and parafluorohexahydrosiladifenidol (pA2, 6.5), indicating that the M2 receptor subtype was mediating this response. In addition, carbachol also induced [3H]-inositol phosphate formation in these cells (EC50, 11 microM; 2.1-fold stimulation over basal), although the response observed was markedly down-regulated compared with the response seen in noncultured airway smooth muscle preparations. Growth arrest of cells failed to increase the magnitude of the inositol phosphate response to carbachol. These results demonstrate that cultured human airway smooth muscle cells express functionally coupled M2 receptors and probably also low levels of coupled M3 receptors.

Formation of neutralizing antibodies after treatment with human calcitonin

purpose: Calcitonin is used for the treatment of Paget's disease of bone, hypercalcemia, and postmenopausal osteoporosis. The formation of antibodies against heterologous calcitonins, such as salmon calcitonin(sCT), has been described frequently. Neutralizing effects of these antibodies have been demonstrated in many cases. As far as antibody formation against human calcitonin (hCT) is concerned, only a single case has been reported in the literature; however, investigations concerning the biologic activity of the antibodies were not performed. We have now assessed the sera of 33 patients treated with hCT for postmenopausal osteoporosis for a period of at least 12 months to evaluate the occurrence of hCT-binding and hCT-neutralizing antibodies. patients and methods: Binding antibodies were detected by incubation of patient sera with 1251-labeled hCT; neutralizing activity was assessed in an in vitro bioassay that measured the impairment of the hCT-induced cyclic adenosine monophosphate (cAMP) formation in the human breast cancer cell line T47D. results: Prior to hCT treatment, none of the patients showed evidence of the presence of either binding or neutralizing antibodies. During the course of treatment, binding antibodies occurred in a single patient. These antibodies had a neutralizing activity characterized by 15% impairment of cAMP formation after 6 months and 27% impairment after 12 months of treatment compared with pretreatment control values. The neutralizing effect observed in this particular patient was comparatively mild compared with the effects seen after the formation of neutralizing antibodies against sCT, so major clinical sequelae were not expected in this patient. This may be due to the lower antigenicity of hCT as compared with sCT. conclusion: Although antibody formation against hCT is a rare phenomenon, we nonetheless recommend monitoring of postmenopausal osteoporosis patients treated with sCT or hCT for neutralizing antibody formation in order to evaluate the therapeutic effect of treatment.

Severe endocrine and nonendocrine manifestations of the McCune-Albright syndrome associated with activating mutations of stimulatory G protein G s

McCune-Albright syndrome (MCAS) is a sporadic disease classically including polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and other hyperfunctional endocrinopathies. An activating missense mutation in the gene for the alpha subunit of GS, the G protein that stimulates cyclic adenosine monophosphate formation, has been reported to be present in these patients. The mutation is found in variable abundance in different affected endocrine and nonendocrine tissues, consistent with the mosaic distribution of abnormal cells generated by a somatic cell mutation early in embryogenesis. We describe three patients with MCAS who had profound endocrine and nonendocrine disease and who died in childhood. Two of the patients were severely ill neonates whose complex symptoms did not immediately suggest MCAS. A mutation of residue Arg201 of GS alpha was found in affected tissues from all three children. A review of the literature and unpublished case histories emphasizes the existence of other patients with severe and unusual clinical manifestations. We conclude that the manifestations of MCAS are more extensive than is generally appreciated, and may include hepatobiliary disease, cardiac disease, other nonendocrine abnormalities, and sudden or premature death.

Intraventricular injection of agents that enhance cyclic adenosine monophosphate formation leads to inhibition of proestrous luteinizing hormone surge in rats.

The effect of increasing hypothalamic levels of 3',5'-cyclic adenosine monophosphate (cAMP) on the preovulatory surge of luteinizing hormone (LH) and ovulation was studied in cycling rats. Animals hearing chronically implanted guiding cannulae into the third ventricle were injected with agents known to enhance the cellular levels of cAMP. Hourly blood samples from the unanesthetized, unrestrained rats were obtained between 11.00 and 17.00 h through a plastic cannula inserted into the jugular vein. Intraventricular injections of serotonin (7.5 mg/ml; 2 microliters) in the morning of proestrous blocked the preovulatory surge of LH and ovulation. This effect was assigned to an increased neuronal level of cAMP because it was prevented by a serum anti-cAMP. Third-ventricle injections of 2 microliters of forskolin (0.5 mmol/l), guanosine 5'-O-(3-thiotriphosphate)(2 mmol/l) or dibutyryl-cAMP (1 mmol/l) at 11.00 h on the day of proestrus mimicked the inhibitory effect of serotonin on the proestrous release of LH. It is suggested that serotonin inhibits LH surge by acting directly on LH-releasing hormone neurons and/or on neurons that provide inputs to these neurons involving cAMP as a second messenger. Neurons releasing gamma-aminobutyric acid (GABA) may serve as interneurons sensitive to serotonin, as well as to cAMP, inasmuch as the inhibitory effect of forskolin on the release of LH was partially blocked by the GABA antagonists, picrotoxin and bicuculline.

Parathyroid hormone is able to enhance cyclic adenosine monophosphate formation without causing an increase in cytoplasmic Ca2+ in osteoblasts.

There are several reports indicating that parathyroid hormone (PTH), besides inducing the formation of cyclic adenosine monophosphate (cAMP), also causes an increase in cytoplasmic free Ca2+ ([Ca2+]i) in osteoblasts, and it has been speculated that both of these second messengers are necessary to mediate PTH-induced bone resorption. In the osteoblastic cell line MC3T3-E1, bovine PTH 1-34 (10 nmol/l-1 mumol/l) stimulated cAMP formation but did not cause an increase in [Ca2+]i in adherent single cells (basal [Ca2+]i = 151 +/- 5 nmol/l, mean +/- SEM; N = 98). In contrast, subsequent addition of bradykinin (1 mumol/l) resulted in a transient increase in [Ca2+]i from a basal level of 155 +/- 11 nmol/l to a peak value of 351 +/- 60 nmol/l (N = 14). When the PTH challenge was followed by the addition of thrombin (10 U/ml), the latter induced a transient rise in [Ca2+]i from a basal level of 173 +/- 12 nmol/l to a peak at 341 +/- 33 nmol/l (N = 20). Primary cultures of human osteoblasts were obtained from trabecular bone. These cells were also PTH-responsive in terms of cAMP formation. On the other hand, human PTH 1-34 (100 nmol/l) did not affect [Ca2+]i in the isolated human osteoblasts, while bradykinin (1 mumol/l) caused a transient increase in [Ca2+]i (from a basal value of [Ca2+]i at 154 +/- 10 nmol/l to a peak value of 757 +/- 147 nmol/l within 30 s; N = 16).(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C activation and positive and negative agonist regulation of 3',5'-cyclic adenosine monophosphate levels in cultured rat Sertoli cells.

The present study examines the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on agonist-regulated 3',5'-cyclic adenosine monophosphate (cAMP) formation and cAMP-mediated effects in cultured Sertoli cells from immature rats. Concentration-dependent stimulation of cAMP levels by follicle-stimulating hormone (FSH) was inhibited dramatically by the coaddition of 100 nmol/l TPA, which exerted a similar inhibition of glucagon- and isoproterenol-stimulated cAMP production. These results show that protein kinase C (PKC) activation by TPA attenuates Gs-protein-mediated agonist activation of cAMP production. (-)-N6(R)-Phenylisopropyladenosine (L-PIA), an A1-adenosine receptor agonist, inhibited cAMP stimulation by FSH in a concentration-dependent manner. When L-PIA was added in increasing concentrations simultaneously with 100 nmol/l TPA, the L-PIA still inhibited FSH-stimulated cAMP production in a concentration-dependent manner. In the presence of TPA, the half-inhibitory concentration (IC50) for L-PIA inhibition of cAMP formation was reduced by more than one order of magnitude, indicating that PKC activation by TPA increases the sensitivity of Sertoli cells to Gi-protein-mediated agonist inhibition of cAMP production. The inhibitory effects of TPA on FSH-stimulated cAMP production were still observed when cAMP phosphodiesterase activity was inhibited by 1 mmol/l methylisobutylxanthine or when the activity of G alpha i-protein was eliminated by pretreatment with 100 micrograms/l pertussis toxin. Taken together, the results indicate that PKC activation inhibits agonist-dependent stimulation of cAMP production by phosphorylation of components common to all the activating agonists used, and not via stimulation of G(i)-protein activity or degradation of cAMP by cAMP phosphodiesterase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone increases basal 3',5'-cyclic adenosine monophosphate formation and down-regulates the agonist-induced inositol phosphates generation in human term placenta.

Whether the placenta is a target tissue for estrogens and progesterone, and their putative mechanism of action, is still a controversial question in the literature. The effect of progesterone and estradiol on 3',5'-cyclic adenosine monophosphate (cAMP) and inositol phosphates generation in human term placenta was investigated. Placental explants were incubated in vitro for up to 48 h in the absence and in the presence of estradiol, progesterone or both steroids (0.1 mumol/l final concentration in all cases), and were stimulated with terbutaline, a beta-adrenergic agonist, (0.1 mmol/l) or angiotensin II (1 mumol/l). The cAMP content was measured by a competitive protein binding assay, and the generation of labelled inositol phosphates formation in explants prelabelled with 3H-myo-inositol was measured by anion exchange chromatography. Progesterone increased significantly basal cAMP concentrations in comparison with control or estradiol-treated tissues (169 +/- 13, 72 +/- 8, and 69 +/- 2 pmol/g wet wt tissue, mean +/- SEM, respectively). However, following terbutaline stimulation cAMP levels (mean +/- SEM) increased to similar values under all conditions (182 +/- 33, 197 +/- 36, and 237 +/- 17 pmol/g wet wt tissue for control, estradiol-, and progesterone-treated tissues, respectively). Angiotensin II stimulated inositol phosphates generation in placental explants by an average of fivefold, but this increase was significantly reduced in the presence of progesterone (5.2 +/- 0.7, 3.7 +/- 0.4, and 2.2 +/- 0.3 fold increase vs non-angiotensin-stimulated tissues, for control, estradiol-, and progesterone-treated placenta, mean- +/- SEM, respectively). These data suggest that progesterone modulates the formation of second messengers in human placenta at term.