Comparison of the glucose-lowering properties of vanadyl sulfate and bis(maltolato)oxovanadium(IV) following acute and chronic administration

The possibility that progesterone or estradiol may regulate expression of G protein in the rat myometrium during the course of pregnancy has been investigated using 1) immunoblot analysis of Gi2 alpha, Gi3 alpha, and Gq alpha subunits and 2) hybridization blot analysis of subunit mRNA. Eighteen hours after administration, estradiol had significantly increased the levels of both Gi2 alpha subunit and Gi2 alpha mRNA (by 40% and 32%, respectively). In control pregnant rats, we observed similar changes at the end of pregnancy, when myometrial concentrations of estradiol had increased, i.e., a 41% increase in immunoreactive Gi2 alpha subunit that correlated with a parallel 45% increase in mRNA levels. In contrast, levels of immunoreactive Gi3 alpha subunit and mRNA, which decreased with advancing gestation, were not influenced by estradiol or progesterone administration. Progesterone administration resulted 30 h later in a significantly decreased level of Gq alpha immunoreactivity (32%) and Gq alpha mRNA (30%). In control rats, Gq alpha protein and mRNA were also significantly lower at midpregnancy under progesterone dominance vs. term. At this stage, a twofold increase in Gq alpha subunit correlated with a 40% increase in mRNA levels. These results demonstrate that myometrial Gi2 alpha and Gq alpha subunits are physiological targets for estradiol and progesterone, respectively, in vivo. Alterations of these G protein levels are discussed in relation to their mediating effects on adenylyl cyclase activity or the phospholipase C pathway during the course of pregnancy.     

Guanine nucleotide binding proteins mediate the chemotactic signal of transforming growth factor-beta 1 in rat IL-2 activated natural killer cells

Transforming growth factor (TGF)-beta 1 induced rat IL-2-activated natural killer (IANK) cell chemotaxis. Various doses of cholera toxin (CT) or pertussis toxin (PT) inhibited the activity of TGF-beta 1 suggesting a role for guanine nucleotide binding (G) proteins. ADP-ribosylation assay showed that rat IANK cell membranes possess a 39 kDa PT substrate and two, 41 and 42 kDa, CT substrates. ADP-ribosylation also showed that incubating IANK cell membranes with TGF-beta 1 in the presence of guanosine 5'-O-(3-thiotriphosphate) resulted in the disappearance of the PT substrate. Immunoblot analysis showed that rat IANK cell membranes possess one Gi (39 kDa), one G0 (39 kDa) and three Gs (40, 41, and 42 kDa) proteins. Pretreatment of IANK cell membranes with TGF-beta 1 in the presence of guanosine-5'-O-(3-thiotriphosphate) reduced the intensity of the 39 kDa G(0) and the 40 kDa Gs but not the 39 kDa Gi or the 41 kDa or 42 kDa Gs. Furthermore, TGF-beta 1 stimulated GTP binding and increased GTPase activity in IANK cell membranes. Both activities were inhibited by PT or CT. This inhibition was associated with the modification of G proteins by the toxins suggesting that bacterial toxin substrates are linked to TGF-beta 1 receptors. Our results suggest that G0 and Gs are involved in mediating the chemotactic signal of TGF-beta 1 in rat IANK cells.     

Agonist-induced release of splice variants of the alpha subunit of the stimulatory G-protein from rat cardiac membranes

It was the aim of the present study to evaluate whether, in physiological cardiac tissue, long and short splice variants of G(s)alpha (the alpha subunit of the stimulatory G-protein) differ in their susceptibility to guanine nucleotide-mediated activation. As a measure of G(s)alpha activation, we determined the proportion of G(s)alpha subunits which were released from the plasma membrane upon stimulation. Membrane preparations from heart ventricles of Wistar rats were incubated with increasing concentrations of the non-hydrolyzable GTP analogue guanylyl-imidodiphosphate (GppNHp, 0-100 micromol/L) in the absence or presence of the beta-adrenoceptor agonist isoprenaline (1 micromol/L). The 45 and 52 kDa forms of G(s)alpha (G(s)alpha-S and -L, respectively) were measured in the supernatant of the incubation mixture by immunoblotting and densitometry. The increase in cyclic AMP induced by GppNHp was measured in the same supernatant. In the absence of isoprenaline, GppNHp increased cyclic AMP formation and the concentration-dependent release of G(s)alpha-L (Friedman test, P < 0.05), whereas the amount of soluble G(s)alpha-S was not affected. After addition of isoprenaline, the redistribution of G(s)alpha-S into the soluble fraction could be stimulated by GppNHp in a concentration-dependent manner (P < 0.05). Kinetic experiments revealed that activation of G(s)alpha-S by GppNHp was rather slow, but could be markedly enhanced by isoprenaline. Thus, it is likely that the different susceptibilities of G(s)alpha-S and -L towards GppNHp reflects differences in the rate of spontaneous GDP release.     

Activation of protein kinase C by oxytocin inhibits the biological activity of the human myometrial corticotropin-releasing hormone receptor at term

The role of placental CRH in human pregnancy is currently unknown. The myometrium expresses CRH receptors that during pregnancy become coupled to adenylate cyclase. Oxytocin (OT) is one of the main regulators of uterine activity, acting via activation of the inositol triphosphate pathway. In view of the possible cross-talk between the CRH and OT signal transduction pathways we have sought to examine in more detail the second messenger mechanisms involved. CRH receptor binding affinity for CRH and activation of adenylate cyclase were reduced in the presence of OT in pregnant (at term, but not preterm) human myometrium. OT action was mediated via pertussis toxin-sensitive G proteins, which directly inhibit adenylate cyclase and, via activation of protein kinase C, phosphorylate the CRH receptor, leading to desensitization. Activation of protein kinase C by OT could be partially inhibited in human pregnant myometrial cells by OT antagonists (F327 and CAP476; 1 microM) or phospholipase C inhibitors (U73122; 10 microM). These results suggest that in term myometrium, CRH receptor function is modulated by OT, leading to reduced biological activity, lower cAMP levels, and a subsequent shift in favor of contractility rather than relaxation.     

The human and dog 5-HT1D receptors can both activate and inhibit adenylate cyclase in transfected cells

The cloned human serotonin 1D (5-HT1D) receptor has been shown to inhibit adenylate cyclase while the corresponding cloned dog receptor has been characterized by its enhancement of cAMP accumulation. To resolve this apparent discrepancy, the human 5-HT1D receptor has been cloned and expressed in Chinese hamster ovary (CHO) cells and the corresponding dog receptor expressed in mutant Y1 adrenal (Y1 Kin-8) cells. It is shown that both receptors when activated by sumatriptan depress forskolin induced adenosine 3'5'-cyclic monophosphate (cAMP) accumulation by a pertussis toxin sensitive mechanism, presumably involving Gi (the adenylate cyclase inhibitory GTP transducing protein). In the absence of forskolin, the dog receptor enhances cAMP accumulation, thus activating Gs (the adenylate cyclase stimulatory GTP transducing protein). When its overriding action on Gi is blocked by pertussis toxin pretreatment, the human receptor also enhances cAMP accumulation. Thus both 5-HT1D receptors activate markedly Gi and to a lesser extent Gs and can exert opposite effects on the same effector system, adenylate cyclase.     

Cell cycle phase specificity of putative cyclin-dependent kinase variants in synchronized alfalfa cells

OBJECTIVE: Impaired beta-adrenergic signal transduction has been proposed as a mechanism contributing to myocardial depression after cardiac surgery. This study determined the changes in the beta-adrenergic system in a model of postoperative myocardial dysfunction induced by myocardial ischaemia and reperfusion under cardiopulmonary bypass (CPB). Those changes were then related to contractility and responsiveness to beta-adrenergic stimulation. METHODS: Four groups of dog hearts were studied: 7 hearts harvested immediately after anaesthesia induction (control group representing the preoperative cardiac condition); 6 hearts harvested after three hours of chest opening by sternotomy (open chest group serving as control for the effects of anaesthesia and surgery); 7 hearts harvested during CPB after 30 minutes of global ischaemia (ischaemia group); and 10 hearts from dogs submitted to one hour of CPB involving 30 minutes of global cardiac ischaemia, harvested 30 minutes after CPB (ischaemia-reperfusion group). Myocardial membranes were prepared to assess: (1) beta-adrenergic receptor density using the radioligand [125I]iodocyanopindolol; (2) GTP-sensitive adenylate cyclase activity and its regulation by isoprenaline and forskolin; (3) G protein levels, using an immunoblotting technique. Ventricular trabeculae or papillary muscles served to assess contractility and responsiveness to isoprenaline. RESULTS: The control and open chest groups had comparable beta-adrenergic receptor density, adenylate cyclase activity and cardiac contractility. In the ischaemia group, the left ventricular membranes had a 55% decrease in receptor density as compared to the controls (P < 0.005), similar GTP-sensitive adenylate cyclase activity and significantly lower adenylate cyclase responses to stimulation with isoprenaline and forskolin. In the ischaemia-reperfusion group, a 144% increase in the left ventricular receptor density was found as compared to the controls (P < 0.005), with a 70% increase in GTP-sensitive adenylate cyclase activity (P < 0.05), a similar adenylate cyclase response to isoprenaline and a 61% increase in response to forskolin (P < 0.005). As compared to the controls, the ischaemia and ischaemia-reperfusion groups had comparable Gs alpha levels, but markedly decreased Gi alpha-2 and Gi alpha-3 levels. The baseline tension of the isolated muscles in the ischaemia and ischaemia-reperfusion groups was comparable, but was 61% and 47% lower than the controls, respectively (P < 0.05). The maximal isoprenaline stimulated tension in the ischaemia and ischaemia-reperfusion groups was 66% and 36% lower than the controls, respectively (P < 0.05 between all groups). CONCLUSIONS: The beta-adrenergic system is severely depressed during global cardiac ischaemia under CPB, but recovers to supranormal values after CPB. However the increased cAMP generation by myocardial membranes after CPB is associated with decreased tension generation by corresponding cardiac muscles. Thus decreased contractility after CPB may be better explained by cellular alterations distal to cAMP generation rather than by changes in the beta-adrenergic system.     

Structure of adenylate cyclase and the coupling with the receptor-G protein system

Adenylate cyclase is a key enzyme that couples with both the stimulatory and inhibitory G proteins (Gs and Gi). The cyclase has been purified and shown to be a glycoprotein of molecular weight 115,000-180,000. Cloning of cDNAs for adenylate cyclase showed that the cyclase is a member of a large family consisting of a variety of subtypes of the enzyme. These subtypes show different responses to calmodulin and G protein beta gamma subunits, and their distributions in tissues and organs are also different. This suggests that each subtype is involved in a particular physiological function. The general structure of adenylate cyclase is composed of two cytoplasmic domains and two membrane-spanning domains, each of which contains 6 transmembrane spans (12 spans in a molecule). The amino acid sequence of each cytoplasmic domain, which is thought to contain a nucleotide (ATP) binding site, is well-conserved among the various subtypes. This review also focuses on the regulation of adenylate cyclase activity by G protein subunits, particularly on several models for adenylate cyclase inhibition by Gi. As one of these mechanisms, direct inhibition of adenylate cyclase by the beta gamma subunits recently demonstrated by us will be discussed.     

Urinary cyclic adenosine 3',5'-monophosphate response in McCune-Albright syndrome: clinical evidence for altered renal adenylate cyclase activity

The recent finding of an activating mutation in the Gs alpha protein, the protein that couples receptors to stimulation of adenylate cyclase, from endocrine and nonendocrine tissues of patients with McCune-Albright syndrome (MAS) suggests that alterations in adenylate cyclase activity may account for the clinical abnormalities in these patients. Many patients with MAS have hypophosphatemia. This may result from the presence of the activating Gs alpha mutation in proximal renal tubules or the elaboration of a phosphaturic factor from fibrous dysplasia. We, therefore, sought to characterize renal cAMP generation and phosphate handling in MAS patients. Intravenous infusion of PTH is a classic clinical test used to evaluate hormonal responsiveness of renal proximal tubule adenylate cyclase and examine PTH-dependent phosphate clearance. We performed PTH infusion in 6 MAS patients, 10 normal subjects, and 6 patients with pseudohypoparathyroidism (PHP). The basal urinary cAMP (UcAMP) level in the MAS group [5.5 +/- 2.6 nmol/dL glomerular filtration (GF)] was elevated (P < 0.05) compared to those in both normal subjects (3.2 +/- 1.2 nmol/dL GF) and patients with PHP (1.9 +/- 0.6 nmol/dL GF). However, PTH-stimulated peak UcAMP (15.0 +/- 7.0 nmol/dL GF) and the peak/basal UcAMP ratio (3.1 +/- 1.7) in MAS were significantly lower than the respective values in normal subjects (30.8 +/- 16.9 nmol/dL GF and 9.3 +/- 2.9; P < 0.05 for both) and were statistically similar to the blunted levels in PHP (respectively, 3.1 +/- 1.5 nmol/dL GF and 2.0 +/- 1.7). By contrast, the PTH-induced phosphaturic response in MAS patients was similar to that in the normal subjects. Our study provides clinical evidence that MAS patients have altered renal adenylate cyclase activity, manifested by an elevated basal UcAMP, but a blunted UcAMP response to PTH stimulation. These observations are presumably due to a mutation in the Gs alpha protein in the renal tubules. Despite the blunted UcAMP excretion, the phosphaturic response to PTH in MAS patients is intact.     

The urinary ratio of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid in surgical patients with chronic alcohol misuse

Interactions among growth factors are important in a variety of physiological and pathological processes. The regulation of IGF-I mRNA expression by bFGF was investigated in cultured rat Müller cells and the mechanism of regulation studied. Müller cells from 1- to 3-day-old Sprague-Dawley rats were isolated and cultured with Eagle MEM+10% FCS. Cultured cells were identified by immunocytochemistry using antibodies against vimentin, carbonic anhydrase C, and glutamine synthetase. Cells of passage 1-4 were treated with bFGF, the PKC inhibitor H-7, calphostin C, the PKC activator PMA or the PKA inhibitor H-89, as well as the adenylate cyclase activator forskolin, or adenylate cyclase inhibitor SQ22536. IGF-I and bFGF expression levels were assessed by Northern blot analysis. The addition of bFGF to culture medium down-regulated IGF-I expression in a dose- and time-dependent manner. Decrease of IGF-I expression started at a bFGF concentration of 1 ng ml-1. IGF-I mRNA level declined to 44% of baseline level at 10 ng ml-1 of bFGF, and reached a trough of 40% at 50 ng ml-1. At 10 ng ml-1 of bFGF, down-regulation of IGF-I expression was observed as early as 4 hr (60%) after treatment, and reached a trough of 42% by 8 hr. The temporal and concentration dependence of IGF-I expression by addition of the PKC activator PMA, to culture medium was similar to that due to the addition of bFGF. The down-regulation of IGF-I expression by bFGF (10 ng ml-1) and PMA (0.1 microM) was blocked by the PKC inhibitors H-7 (30 microM) and calphostin C (1 microM). Forskolin (5 microM), an adenylate cyclase activator, had activator, had no effect on IGF-I expression. SQ22536 (100 microM), an adenylate cyclase inhibitor, and H-89, a PKA inhibitor, had no inhibitory effect on bFGF-induced down-regulation of IGF-I expression. These results indicate that bFGF down-regulates IGF-I expression in cultured rat M uller cells through PKC activation.     

Catabolism of adipose tissue by a tumour-produced lipid-mobilising factor

Induction of lipolysis in murine white adipocytes by a tumour lipid-mobilising factor (LMF) was associated with stimulation of adenylate cyclase in adipocyte plasma membrane preparations. Induction of lipolysis was attenuated by the adenylate cyclase inhibitor MDL12330A and the protein kinase A inhibitor H8, suggesting that cAMP was the intracellular mediator of induction. The effect of LMF on adenylate cyclase was responsive to GTP, with low concentrations (0.1 microM) causing stimulation and high concentrations (10 microM) causing inhibition, suggesting the involvement of both stimulatory (Gs) and inhibitory (Gi) guanine nucleotide-binding proteins. At a concentration of 10 microM, propranolol noncompetitively reduced the induction of lipolysis by LMF. Thus, lipolysis in white adipose tissue during the process of cancer cachexia is mediated by a tumour factor which stimulates cAMP production, possibly through a beta-adrenergic receptor.     

Signal transduction through trimeric G proteins in megakaryoblastic cell lines

The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.     

Changes in steroid hormones in plasma and myometrium and uterine activity in ewes during late pregnancy and parturition

In 7 ewes during late pregnancy, peripheral plasma concentrations of oestrogens were correlated with uterine activity (P less than 0-01; r=+0-47). In myometrial tissue, the concentration of progesterone was similar to that in plasma; it rose to a plateau from Days 115 to 130 of pregnancy and then fell, but was still detectable during parturition. Myometrial oestrogen concentration was much higher than in plasma; from a peak at Days 100-115 it decreased, but rose sharply just before parturition.     

Stable cholinergic-muscarinic and alpha-adrenergic inhibition of rat parotid adenylate cyclase

(-) Isoproterenol-stimulated adenylate cyclase activity of washed at parotid membrane preparations from gland slices previously treated with the alpha-adrenergic agonist, methoxamine, was inhibited approximately 30%. The action of methoxamine required exogenous Ca2+ and was blocked by the alpha-adrenergic blocking agent, phentolamine. Incubation of gland slices with carbamylcholine resulted in a dose-dependent inhibition (50%) of (-) isoproterenol-stimulated adenylate cyclase activity in washed membranes. The action of carbamylcholine was independent of exogenous Ca2+ and was blocked by preincubation with atropine. Cholinergic inhibition of parotid adenylate cyclase was compared to the cholinergic inhibition of adenylate cyclase in dog heart homogenates. While carbamylcholine caused a limited (20-30%) inhibition of basal and (-) isoproterenol-stimulated activities when added to dog heart homogenates, it failed to produce any effect in parotid homogenates prepared in an identical manner. Cholinergic inhibition of parotid adenylate cyclase activity was stable and persisted in washed particulate fractions while the inhibition in dog heart homogenates was reversible by washing. Cholinergic inhibition of adenylate cyclase was markedly dependent on the presence of GTP and was abolished when Mn2+ was subsituted for Mg2+ in both systems. Guanyl-5'-yl imidodiphosphate had little effect on the inhibition in parotid preparations but abolished the inhibition in heart homogenates. It is concluded that, in contrast to the cholinergic action in dog heart homogenates, the exposure of parotid slices to carbamylcholine results in a stable lesion in the adenylate cyclase activity.     

Multiple coupling of human D5 dopamine receptors to guanine nucleotide binding proteins Gs and Gz

We have demonstrated previously that D1 dopamine receptors are coupled to both Gs alpha and Go alpha. We examine here the coupling between human D5 dopamine receptors and G proteins in transfected rat pituitary GH4C1 cells. Similar to D1 receptors, cholera toxin treatment of cells reduced, but did not abolish, D5 agonist high-affinity binding sites, indicating D5 receptors couple to both Gs alpha and cholera toxin-insensitive G proteins. The interaction between D5 receptors and Gs alpha was confirmed by immunoprecipitation studies and by the ability of D5 receptors to stimulate adenylyl cyclase. Unlike D1 receptors, D5 receptors did not display any pertussis toxin-sensitive G-protein coupling to Go alpha or Gi alpha. D5 receptors were also not coupled to Gq alpha and were unable to mediate phosphatidylinositol metabolism. Instead, D5 sites appeared to be coupled to an AIF(-)4-sensitive, N-ethylmaleimide-resistant G protein. Anti-Gz alpha caused immunoprecipitation of 24.2 +/- 5.2% of G protein-associated D5 receptors, indicating coupling between D5 and Gz alpha. The coupling to Gz alpha was specific for D5 receptors, because similar associations were not detected between D1 receptors and Gz alpha.     

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE1 receptors and stimulatory G proteins

Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 mu M; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 mu M) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE1 receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE1 receptor number (Bmax) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPgammaS (100 mu M) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of GSalpha and S49 cyc- reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE1 receptors and GS in situ by application of a C-terminal anti-GSalpha antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE1 receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available GSalpha within the PGE1 receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 mu M)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE1 receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.     

Mechanism underlying histamine-induced desensitization of amylase secretion in rat parotid glands

1. Histamine acted on H2 receptors in rat parotid tissues and induced the amylase secretion. Immunoblot analysis by using anti-H2 receptor protein antiserum demonstrated that histamine induced the increase and decrease in the amounts of H2 receptor proteins in basolateral and intracellular membranes, respectively. 2. Short-term treatment with histamine resulted in decreases in amylase secretion, the density of H2 receptors and their affinity for the agonists during further incubation with histamine, but showed an unaltered secretory response to isoproterenol, indicating that the histamine-induced desensitization was confined to H2 receptors. 3. This treatment triggered a 20% decrease in the histamine-stimulated adenylate cyclase activity and a 40% decrease in the phosphorylation level of Gi2alpha protein in the tissues, resulting in an increase in pertussis toxin (IAP)-catalyzed ADP-ribosylation of the protein. An enhancement of cholera toxin-catalyzed ADP-ribosylation of Gs protein was observed only during the first incubation with histamine. 4. This treatment triggered a 30% decrease and a 60% increase in the histamine-stimulated activities of protein kinase A and protein phosphatase 2A in the tissues, respectively. 5. Pretreatment with okadaic acid completely blocked the histamine-induced decrease in amylase secretion and increase in IAP-catalyzed ADP-ribosylation of Gi protein. The levels of Gi2alpha and Gs alpha proteins in the tissues were not modified by histamine treatment and the level of Gi2alpha protein was not affected by pretreatment with okadaic acid, as assessed by immunoblot analyses with anti-Gi2alpha and anti-Gs alpha protein antiserum. 6. The regulation of Gi2alpha protein phosphorylation in parotid tissues plays an important role in the histamine-induced desensitization of amylase secretion.     

Immunohistochemical localization of estradiol and progesterone receptors in human uterus throughout pregnancy: expression in endometrial blood vessels

Although progesterone and estrogens are essential to maintain human pregnancy after implantation, the localization of their specific receptors in different uterine cell types during pregnancy has not been investigated. We studied uteri (n = 40) obtained during the first 3 months of pregnancy (n = 21) and in late pregnancy (n = 9) as well as from women 5-14 weeks pregnant (n = 10) who had received the antiprogestagen RU 38486 (Roussel-UCLAF) to induce cervical dilation. Frozen tissues were processed for indirect immunocytochemical staining with specific monoclonal antibodies against estrogen receptors (ER; Abbott Laboratories) and progesterone receptors (PR; Li 417). Specific staining for steroid receptors was only detected in the nucleus. In the endometrium, PR staining remained fairly constant throughout pregnancy, whereas ER staining was initially weak and then undetectable. PR was widely expressed in stromal cells and in spiral arterial wall cells, whereas ER was expressed in scattered stromal cells and arterial cells. Both PR and ER were absent from glandular epithelium, contrasting with the secretory activity during the first trimester. Spiral arteries of the endometrium and myometrial smooth muscle cells showed intense PR and moderate ER staining in early pregnancy. The progesterone antagonist RU 38486 (mifepristone), given in early pregnancy at a dose of 200 mg, caused a marked increase in ER staining and a smaller increase in PR staining in stromal cells, whereas the glandular epithelium remained negative for both ER and PR (except for one and two specimens, respectively). We conclude the following. 1) Stromal cells retain PR despite the high progesterone levels during pregnancy, in keeping with the role of progesterone in stromal decidualization. The absence of PR from the secretory glandular epithelium suggests a paracrine link between decidualized stromal cells and epithelial cells. 2) Significant PR down-regulation by progesterone during pregnancy occurs only in epithelial cells of the endometrium. 3) In contrast, the absence or low level of ER staining in the various cell types of the endometrium during gestation concurs with the known effect (down-regulation) of steroid hormones on ER mRNA or protein levels. The increase in ER in human decidua after RU 38486 treatment indicates that the main cause of the low ER levels is progesterone secretion. 4) The intense PR staining in smooth muscle cells of spiral arteries during early pregnancy suggests that progesterone is essential for modulating blood flow during pregnancy.     

Influence of aluminum on the regulation of PTH- and 1,25(OH)2D3-dependent pathways in the rat osteosarcoma cell line ROS 17/2.8

The role of hormonal status in the development of aluminum (Al)-dependent renal osteodystrophy, which is characterized by reduced bone matrix deposition, still remains largely unknown. To address this question, we used the osteoblast-like osteosarcoma cell line ROS 17/2.8 to evaluate the role of Al on parathyroid hormone (PTH)- and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent activities in these cells. Al (1 microM) caused an inhibition of basal and 1,25(OH)2D3-induced alkaline phosphatase, but only at low doses (< 1 nM) of the steroid. Al partly inhibited basal osteocalcin (OC) secretion in ROS cells (p < 0.001), and the dose-dependent increase in 1,25(OH)2D3-induced OC release by these cells was also reduced by 1 microM Al at low concentrations of the steroid (< or = 1 nM), whereas high doses of 1,25(OH)2D3 (> or = 5 nM) totally prevented the inhibiting effects of Al. Al also had strong inhibitory actions on PTH-dependent cAMP production by ROS cells over the concentration range tested (0.5-50 nM). This inhibitory action of Al was also observed for PTH-related peptide- (PTHrp, 50 nM) but not for Isoproterenol-dependent (100 nM) cAMP formation. To evaluate more fully the mechanism of this inhibition of cAMP formation, we investigated the effect of Al on toxin-modulated, G protein-dependent regulation of cAMP formation and on the activation of adenylate cyclase by Forskolin. Cholera toxin (CT, 10 micrograms/ml), applied to cells for 4 h prior to PTH challenge, enhanced cAMP production about 2-fold above PTH alone (p < 0.001), a process that was further stimulated by Al. Pertussis toxin (PT, 1 microgram/ml, 4 h) did not modify basal PTH-dependent cAMP formation by ROS cells. However, PT treatment prevented the inhibitory effect of Al on cAMP formation by these cells (p < 0.025). The stimulation of adenylate cyclase by Forskolin (0.1 and 1 microM), which bypasses G protein regulation, was not modified by Al, indicating that Al does not affect adenylate cyclase directly. Northern blot analysis of PTH receptor mRNA levels showed that Al did not modify PTH receptor message in ROS cells. Likewise, Western blot analyses of G protein subunits showed that Al did not significantly alter Gs alpha subunit levels, in accordance with the results obtained for cAMP-dependent formation in response to CT. In contrast, Gi alpha-1 and Gi alpha-2 subunits were decreased by Al treatment, consistent with PT-restricted increases in cAMP formation in Al-treated ROS cells. Taken together, these results suggest that Al has multiple actions in osteoblast-like ROS cells. The effects of Al are modulated by hormonal control of the pathways investigated. Al affects 1,25(OH)2D3-regulated functions only when this steroid is low. Al has large inhibitory effects on PTH- and PTHrp-dependent cAMP formation. This last feature is related to the ability of Al to alter the G protein transducing pathway for PTH/PTHrp-dependent formation of cAMP since it does not affect adenylate cyclase activity directly and does not affect the PTH receptor message level. Thus, Al has stronger deleterious effects in osteoblast-like cells with an already compromised 1,25(OH)2D3 status and can modulate specifically PTH/PTHrp-mediated cAMP formation at the postreceptor level.