Testosterone stimulates intracellular calcium release and mitogen-activated protein kinases via a G protein-coupled receptor in skeletal muscle cells

Involvement of intracellular Ca(2+) and ERK1/2 phosphorylation in the fast nongenomic effects of androgens in myotubes was investigated. Testosterone or nandrolone produced fast (<1 min) and transient increases in intracellular Ca(2+) with an oscillatory pattern. Calcium signals were slightly reduced in Ca(2+)-free medium, but lack of oscillations was evident. Signals were blocked by U-73122 and xestospongin B, inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway. Furthermore, IP(3) increased transiently 2- to 3-fold 45 sec after hormone addition. Cyproterone neither affected the fast Ca(2+) signal nor the increase in IP(3). Calcium increases could also be induced by the impermeant testosterone conjugated to BSA, and the effect of testosterone was abolished in cells incubated with guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin. Stimulation of myotubes with testosterone, nandrolone, or testosterone conjugated to BSA increased immunodetectable phosphorylation of ERK1/2 within 5 min, and this effect was not inhibited by cyproterone. Phosphorylation was blocked by the use of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester, U-73122, and xestospongin B as well as by dominant negative Ras, MAPK kinase (MEK), or the MEK inhibitor PD-98059. In addition, guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin blocked ERK1/2 phosphorylation. These results are consistent with a fast effect of testosterone, involving a G protein-linked receptor at the plasma membrane, IP(3)-mediated Ca(2+) signal, and the Ras/MEK/ERK pathway in muscle cells.     

Role of extra- and intracellular calcium and calmodulin in renin release from rat kidney

Renin release from the juxtaglomerular cell appears to be inversely related to calcium concentration. We studied the role of Ca+2 to confirm recent findings and to further explore the role of intracellular calcium as well as the calcium-calmodulin system in renin release. A rat renal cortical slice preparation was used. Isoproterenol (10(-6) M) caused significant stimulation of renin release, whereas angiotensin (AII; 5 X 10(-5) M) suppressed basal as well as isoproterenol-stimulated renin release. Removal of calcium from the buffer reversed AII suppression of renin release. Nifedipine (10(-5) M), a specific calcium channel blocker, induced a marked increase in basal renin release. TMB-8, an inhibitor of intracellular calcium release, also caused a dose-related increase in basal renin release. The calmodulin antagonists trifluoperazine and calmidazolium both caused significant dose-related increases; however, calmidazolium was a more potent stimulator. Low extracellular calcium or nifedipine concentrations did not alter isoproterenol-induced renin release. Isoproterenol further stimulated renin release in the presence of trifluoperazine and calmidazolium. These results suggest that acute beta-adrenergic stimulation of renin in independent of changes in levels of extracellular and intracellular calcium and calmodulin. These studies provide further evidence that unlike most other secretory systems, the reduction of intracellular calcium or inhibition of the calcium-calmodulin system in the juxtaglomerular cells of the kidney acts as a secretogogue.     

The recombinant rat glucagon-like peptide-1 receptor, expressed in an alpha-cell line, is coupled to adenylyl cyclase activation and intracellular calcium release.

The glucagon-like peptide-1 (GLP-1) receptor is expressed on alpha-cells, though its functional significance is unknown. The endogenous beta-cell GLP-1 receptor is coupled to adenylyl cyclase, cell depolarization, activation of voltage-dependent Ca2+ channels (VDCC) and extracellular Ca2+ influx (Lu et al., 1993 b). In contrast, the signaling pathways of the GLP-1 receptor in alpha-cells are poorly understood. To determine the signaling mechanisms of the alpha-cell GLP-1 receptor, we established a stable pancreatic islet alpha-cell line expressing the recombinant rat GLP-1 receptor (INR1-SF2), using INRl-G9 cells. These INRl-G9 cells do not express endogenous GLP-1 receptor. In INR1-SF2 cells, GLP-1 bound to the recombinant receptor (Kd = 0.9 nM) and increased cAMP (ED50 = 0.6 nM). GLP-1 increased the free cytosolic Ca2+ ([Ca2+]i) (ED50 = 50 nM) by release from intracellular stores, but did not affect INR1-SF2 cell phosphoinositol turnover. Despite expressing VDCC, the INR1-SF2 cells were not depolarized by GLP-1, even in the presence of glucose. This contrasts with the depolarizing action of GLP-1 in beta-cells in the presence of glucose (Lu et al., 1993 b). This study establishes that a single GLP-1 receptor species can mediate the effects of GLP-1 through multiple signaling pathways, including the adenylyl cyclase system and intracellular Ca2+ release, in an alpha-cell type. Furthermore, since GLP-1 is unable to cause cellular depolarization or activate VDCC in INR1-SF2 cells, these data suggest that glucose-induced membrane depolarization may be crucial for GLP-1 to further activate VDCC and potentiate glucose-stimulated insulin release in beta-cells. Finally this study describes a cell line that can be used as a model system for evaluation of GLP-1 signaling in alpha-cells.     

Adrenomedullin stimulates nitric oxide release from SK-N-SH human neuroblastoma cells by modulating intracellular calcium mobilization

We used SK-N-SH human neuroblastoma cells to test the hypothesis that adrenomedullin (ADM), a multifunctional neuropeptide, stimulates nitric oxide (NO) release by modulating intracellular free calcium concentration ([Ca2+]i) in neuron-like cells. We used a nitrite assay to demonstrate that ADM (10 pM to 100 nM) stimulated NO release from the cells, with a maximal response observed with 1 nM at 30 min. This response was blocked by 1 nM ADM(22-52), an ADM receptor antagonist or 2 microM vinyl-L-NIO, a neuronal NO synthase inhibitor. In addition, 5 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular calcium chelator, eliminated the ADM-induced NO release. Similar results were observed when the cells were incubated in calcium-free medium or when L-type calcium channels were inhibited with 5 microM nifedipine or 10 microM nitrendipine. Depletion of calcium stores in the endoplasmic reticulum (ER) with 1 microM cyclopiazonic acid or 150 nM thapsigargin, or inhibition of ryanodine-sensitive receptors in the ER with 10 microM ryanodine attenuated the ADM-induced NO release. NO responses to ADM were mimicked by 1 mM dibutyryl cAMP, a cAMP analog, and were abrogated by 5 microM H-89, a protein kinase A inhibitor. Furthermore, Fluo-4 fluorescence-activated cell sorter analysis showed that ADM (1 nM) significantly increased [Ca2+]i at 30 min. This response was blocked by nifedipine (5 microM) or H-89 (5 microM) and was reduced by ryanodine (10 microM). These results suggest that ADM stimulates calcium influx through L-type calcium channels and ryanodine-sensitive calcium release from the ER, probably via cAMP-protein kinase A-dependent mechanisms. These elevations in [Ca2+)]i cause activation of neuronal NO synthase and NO release.     

Adenylyl cyclase isoform v mediates renin release from juxtaglomerular cells

We have shown previously that decreasing intracellular calcium in the juxtaglomerular cells increases both cAMP formation and renin release. We hypothesized that this is because of an interaction between intracellular calcium and the calcium-inhibitable isoform of adenylyl cyclase, type-V. We used primary cultures of juxtaglomerular cells isolated from C-57/B6 mice at 70% to 80% confluence. Western blots were performed on isolated juxtaglomerular cells using antibodies against either of the 2 calcium inhibitable isoforms of adenylyl cyclase, types-V and -VI. Only the antibody against adenylyl cyclase-V gave us a strong band at 120 kDa as expected. Immunolabeling in juxtaglomerular cells with confocal microscopy found immunofluorescence for the adenylyl cyclase-V-specific antibody compared with either negative controls or cells stained with the adenylyl cyclase-VI antibody. Reducing isolated juxtaglomerular intracellular calcium with 100 micromol/L of the cytosolic calcium chelator BAPTA-AM stimulated both cAMP (3.49+/-0.70 to 10.09+/-0.81 pmol/mL per milligram of protein; P<0.002) and renin release (1001.8+/-81.5 to 1648.0+/-139.1 ng of angiotensin I per milliliter per hour per milligram of protein; P<0.01). The selective adenylyl cyclase-V inhibitor NKY80 completely blocked both BAPTA-AM-stimulated cAMP formation and renin release. We conclude that lowering intracellular calcium is permissive, allowing an increased activity of the calcium-inhibitable isoform adenylyl cyclase-V (but not adenylyl cyclase-VI) in the juxtaglomerular cell, producing cAMP, which stimulates renin secretion.     

Modification of intracellular calcium and plasma renin by dietary calcium in men

A double blind, placebo-controlled, parallel study was conducted on the effect of a high daily oral calcium supplementation of 1 g elemental calcium, given twice daily for 16 weeks in normal male subjects, on plasma renin, aldosterone, kallikrein, cGMP, cAMP, and calciotropic hormones, intracellular calcium concentrations, and plasma total and ionized calcium. After a 1-month run-in period on a limited use of dairy products, the subjects (n = 32) were allocated to a placebo or a calcium group. Placebo or 1 g elemental calcium was administered twice daily in the morning and evening for 16 weeks. All subjects were investigated at baseline and after 1, 2, 4, 8, and 16 weeks of placebo or calcium administration.

A decreased intraerythrocyte and intraplatelet Ca²⁺ concentration was observed in the calcium-treated subjects. Compared with the placebo group, an increase in the plasma renin activity (PRA) in the calcium group was observed after 4, 8, and 16 weeks of oral calcium administration. However, plasma aldosterone and urinary excretion of aldosterone, kallikrein, cGMP, and cAMP were not changed during calcium administration. Oral calcium supplementation in these men was also accompanied by a reduction in the plasma concentration of intact parathyroid hormone and 1,25-dihydroxyvitamin D₃, and an increase in 24-h urinary calcium excretion, but no change in the plasma total Ca²⁺ concentration, serum ionized Ca²⁺ level, and plasma phosphate or 25-hydroxyvitamin D₃.Our data show that the increase in PRA observed in men during oral calcium supplementation is accompanied by a reduction in the intracellular free and total Ca²⁺ concentration in platelets and erythrocytes and by a decrease in the plasma concentration of intact parathormone and 1,25-dihydroxyvitamin D₃.     

Decreased type VI adenylyl cyclase mRNA concentration and Mg(2+)-dependent adenylyl cyclase activities and unchanged type V adenylyl cyclase mRNA concentration and Mn(2+)-dependent adenylyl cyclase activities in the left ventricle of rats with myocardial infarction and longstanding heart failure.

OBJECTIVE: To address the effect of longstanding left ventricular (LV) hypertrophy and failure on LV adenylyl cyclase (AC) gene expression, mRNA concentrations of the main cardiac AC isoforms were measured in the non-infarcted area of LV from rats with myocardial infarction (MI), without (H) or with (F) LV failure, and in control (C) rats. Basal, GTP- and forskolin-stimulated Mg(2+)- and Mn(2+)-dependent AC activities were also measured in F and C rats. METHODS: Two- and six months after MI, steady-state AC mRNA concentrations were assessed by Northern blot analysis and RNase protection assay with isoform-specific cDNA and cRNA probes, respectively. AC activities were assessed on LV microsomal fractions using standard procedures. RESULTS: Types V and VI, and types IV and VII were the major and minor AC mRNA isoforms in both the LVs of F and C rats. Two months after MI, no difference in LV type V or VI mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratios was observed in rats with H or F compared to C. Six months after MI, no difference in LV type V mRNA concentration was observed between the three rat groups, whether this level was normalized to GAPDH, poly-(A+) or 18S RNAs. In contrast, a 35% decrease in the type VI mRNA to poly-(A+) RNA ratio and a 29% decrease in the type VI mRNA to 18S RNA ratio was observed only in rats with F compared to C (p < 0.05 vs. C for the two comparisons). Two- and six months after MI, basal and forskolin-stimulated Mg(2+)-dependent AC activities were decreased by 30-35% in F rats compared to C (p < 0.05), whereas Mn(2+)-dependent activities were unchanged. CONCLUSION: Longstanding LV hypertrophy and failure resulting from MI in rats is not associated with altered expression of the most abundant, type V, AC mRNA isoform, whereas that of type VI is decreased. The lack of change in Mn(2+)-dependent AC activities in the LV of F rats suggests that this decrease has no functional consequence on overall AC activity and that decreased Mg(2+)-dependent activities are related to alterations occurring upstream.     

beta-Endorphin stimulates plasma renin and aldosterone release in normal human subjects

To determine the effect of beta-endorphin on the renin-angiotensin-aldosterone system, human synthetic beta-endorphin (0.3, 1.0, and 3.0 micrograms/kg X min) was infused iv in normal subjects. Each dose was administered for 30 min, and a control infusion of 5% dextrose and water was given on another day. Ten subjects were studied recumbent and in balance while ingesting a 10-meq Na+ diet. Plasma renin activity (PRA), plasma aldosterone (PA), and plasma cortisol (F) were measured basally and every 30 min for 210 min. The increments in PRA and PA above basal significantly (P less than 0.05) increased (3.1 +/- 1.2 ng/ml X h and 12.2 +/- 5.3 ng/dl, respectively; P less than 0.05) at the end of the beta-endorphin infusion. beta-Endorphin also significantly (P less than 0.01) suppressed F levels. Since in the low salt study, beta-endorphin suppressed F release while stimulating renin secretion, an additional five subjects were pretreated with dexamethasone (0.5 mg every 6 h) and were studied in balance while ingesting a 200-meq Na+ diet to suppress the renin-angiotensin system. Significant (P less than 0.025) increments in PRA (2.1 +/- 0.7 ng/ml X h) and PA (4.1 +/- 1.7 ng/dl) levels above basal were again found during the sequential dose infusion of beta-endorphin (0.3, 1.0, and 3.0 micrograms/kg X min). However, PA elevations were sustained for at least 120 min after the beta-endorphin infusion was stopped despite a drop in PRA 90 min earlier. In additional studies, an attempt was made to define the minimal effective dose of beta-endorphin by 60-min infusions (0.03, 0.1, and 0.3 micrograms/kg X min) in subjects on a 200-meq Na+ diet who were dexamethasone pretreated. The PRA and PA levels rose significantly (P less than 0.05) above basal at the 0.3 micrograms/kg X min dose, but not at the 0.03 or 0.1 micrograms/kg X min dosage levels. There were no changes in blood pressure or potassium during either the 10 or 200-meq Na+ studies. Thus, beta-endorphin stimulates aldosterone release in vivo. However, the underlying mechanisms are complex, since renin levels also increased. The data suggest that the early aldosterone rise may be secondary to an increase in renin release, but renin cannot account for the sustained postinfusion elevations of aldosterone.     

The role of calcium in the control of renin release

The effects of removing external calcium and inhibiting entry of calcium into the cell by treatment with D-600 on renin release from renal cortical slices of male Sprague-Dawley rats were examined. Baseline renin release, angiotensin II (AII)-induced inhibition, and isoproterenol-induced stimulation of renin release were studied. Removal of external calcium by chelation with 5 mM EGTA inhibited basal renin release while treatment with 1 mM EGTA stimulated basal renin release slightly. Incubation of slices with zero calcium medium containing 1 mM EGTA had no effect on isoproterenol-induced stimulation of renin release. In contrast, similar treatment reduced the inhibitory effect of AII from 58.7% of baseline to 85.3% (p less than 0.001). Similarly, blockage of calcium entry into cells with D-600 had no effect on isoproterenol-induced stimulation of renin release but abolished AII-induced inhibition. Replacement of sodium in the bathing medium with choline had no effect on baseline renin release or on AII-induced inhibition of renin release, ruling out the possibility that D-600 altered renin release via an effect on sodium influx. Taken together, the data strongly suggest that AII-induced inhibition of renin release is partially dependent on the presence of external calcium but that isoproterenol-induced stimulation of renin release is not.     

Immunodetection of adenylyl cyclase protein in tissues

Immunodetection of the adenylyl cyclase isoforms has been difficult in tissues because of its low quantity of protein expression. We have developed a one-step cellular protein purification method that enables a simple immunodetection of the adenylyl cyclase isoforms. The type I isoform was detected exclusively in the brain. The type III isoform was detected in the brain and lungs. Further, the protein expression of type III adenylyl cyclase in lungs changed ontogenically and was the lowest in neonates. Thus, the comparison of the amount of certain adenylyl cyclase isoforms protein in each tissue is now feasible using our method.     

The calcium paradoxon of renin release: calcium suppresses renin exocytosis by inhibition of calcium-dependent adenylate cyclases AC5 and AC6

An increase in the free intracellular calcium concentration promotes exocytosis in most secretory cells. In contrast, renin release from juxtaglomerular (JG) cells is suppressed by calcium. The further downstream signaling cascades of this so called "calcium paradoxon" of renin secretion have been incompletely defined. Because cAMP is the main intracellular stimulator of renin release, we hypothesized that calcium might exert its suppressive effects on renin secretion via the inhibition of the calcium-regulated adenylate cyclases AC5 and AC6. In primary cultures of JG cells, calcium-dependent inhibitors of renin release (angiotensin II, endothelin-1, thapsigargin) suppressed renin secretion, which was paralleled by decreases in intracellular cAMP levels [cAMP]. When [cAMP] was clamped by membrane permeable cAMP derivates, renin release was not suppressed by any of the calcium liberators. Additionally, both endothelin and thapsigargin suppressed cAMP levels and renin release in isoproterenol or forskolin-pretreated As4.1 cells, a renin-producing cell line that expresses AC5 and AC6. The calcium-dependent inhibition of intracellular cAMP levels and renin release was prevented by small interfering RNA-mediated knockdown of AC5 and/or AC6 expression, underlining the functional significance of these AC isoforms in renin-producing cells. Finally, in isolated perfused mouse kidneys, angiotensin II completely inhibited the stimulation of renin secretion induced by adenylate cyclase activation (isoproterenol) but not by membrane permeable cAMP analogs, supporting the conclusion that the suppressive effect of calcium liberators on renin release is mediated by inhibition of adenylate cyclase activity.     

Aggregation of human platelets stimulates calcium ion movement and release reaction

We studied the effect of inhibition of platelet aggregation (obtained by omitting sample stirring and by the addition of ASA and GRGDS peptide) on ATP secretion and Ca(2+) movements. When collagen was the agonist, platelet aggregation stimulated release reaction and Ca(2+) movements in both the presence and absence of extracellular Ca(2+). When platelets were stimulated by thrombin, ATP release and Ca(2+) movements were largely independent of aggregation. Our data suggest that platelet aggregation stimulates Ca(2+) movement, and that this phenomenon of feedback amplification of platelet activation plays an important role in platelet function when collagen is the agonist, while it has little or no role when thrombin is the stimulus.     

Norepinephrine inhibits glucose-stimulated, Ca2+-independent insulin release independently from its action on adenylyl cyclase

Inhibition of insulin release by norepinephrine has been attributed to activation of ATP-sensitive K+ channels, inactivation of voltage-dependent Ca2+ channels, and inhibition of adenylyl cyclase. However, direct inhibitory action of norepinephrine at a distal site of stimulus-secretion coupling has also been suggested. To obtain more direct evidence for norepinephrine inhibition of insulin release at a distal site, we performed experiments in intact, non-permeabilized beta cells. In rat pancreatic islets, a combination of glucose, phorbol ester and forskolin under stringent Ca2+-free conditions was used as a trigger of insulin exocytosis at a distal site. Norepinephrine inhibited this Ca2+-independent insulin release in a concentration-dependent manner, with an IC50 of 50 nM. The inhibition was complete, reversible, and pertussis toxin-sensitive, and not associated with any reduction of cAMP content in the islet cells. In conclusion, norepinephrine strongly, yet reversibly, inhibits insulin release in intact beta cells at a late step of exocytosis, through pertussis toxin-sensitive, G protein-mediated mechanism(s).     

A membrane estrogen receptor mediates intracellular calcium release in astrocytes

Appreciating the physiology of astrocytes and their role in brain functions requires an understanding of molecules that activate these cells. Estradiol may influence astrocyte functions. We now report that estrogen altered intracellular calcium concentration ([Ca(2+)](i)) in neonatal astrocytes that expressed estrogen receptor (ER) mRNA in vitro. Western blotting revealed both ERalpha and ERbeta proteins in both the nuclear fractions and plasma-membrane fractions. Application of 17beta-estradiol (20 nm) to fura 2-loaded astrocytes in vitro stimulated [Ca(2+)](i) in 75% of astrocytes with an EC(50) of 12.7 +/- 3.1 nm. This rapid action of estradiol was blocked by the ER antagonist, ICI 182,780. The membrane-impermeable estradiol-BSA induced a [Ca(2+)](i) flux that was statistically similar to estradiol. Removal of extracellular Ca(2+) did not alter the effect of estradiol, but phospholipase C inhibitor U73122 (10 microm) and 2-aminoethoxydiphenyl borate (5 microm), an inhibitor of the inositol-1,4,5,-trisphosphate-gated intracellular Ca(2+) channel, significantly decreased the estradiol-induced [Ca(2+)](i) flux. Estradiol was unable to induce [Ca(2+)](i) flux in thapsigargin-depleted cells. These results indicate that estradiol mediates [Ca(2+)](i) flux in astrocytes through a membrane-associated ER that activates the phospholipase C pathway.     

The cyanobacterial tandem GAF domains from the cyaB2 adenylyl cyclase signal via both cAMP-binding sites

The tandem GAF domains from the cyanobacterium Anabaena PCC7120 cyaB2 adenylyl cyclase form an antiparallel dimer with cAMP bound to all four binding sites. cAMP binding causes highly cooperative allosteric enzyme activation (>500-fold; EC(50) = 1 microM; Hill coefficient >2.0). The cyaB2 GAF domains, like those of the cyclic nucleotide phosphodiesterases (PDEs), contain conserved NKFDE motifs that when mutated in the PDEs abrogate cyclic nucleotide binding. We mutated the aspartic acids within this motif in cyaB2 to determine which domains were required for signaling. Constructs containing an Asp/Ala mutation in either GAF domain still showed positive cooperative cAMP stimulation but with reduced Hill coefficients. The cyaB2 GAF domain NKFDE motifs contain inserts of 14 (GAF-A) and 19 (GAF-B) amino acids not present in PDE2 or cyaB1. Constructs having these inserts deleted could still be activated by cAMP (23- to 100-fold) but lost all positive cooperative activation, suggesting that the inserts play an important role in domain interaction and/or stabilization of the cAMP-binding pockets. In the shortened constructs, even those with a single Asp/Ala mutation in the NKFDE motifs could still be activated by cAMP. However, in a double Asp/Ala mutant of the shortened construct, stimulation by cAMP was almost completely lost, and the EC(50) shifted far to the right. Overall, the data suggest that in GAF domains without these inserts, only the canonical lysine:aspartate salt bridge keeps the alpha4-helix and the alpha4-beta5 linker that close over the cyclic nucleotide properly oriented, thereby stabilizing the binding pocket. The cyaB2 GAF ensemble appears to be an evolutionary intermediate where both GAF domains still participate in allosteric activation by cAMP.     

The adenylyl cyclase gene from Schizosaccharomyces pombe.

We cloned the adenylyl cyclase gene from the fission yeast Schizosaccharomyces pombe using low-stringency hybridization to the Saccharomyces cerevisiae adenylyl cyclase gene. The Sc. pombe gene encodes a 1692-amino acid-residue protein. The identity of this gene was confirmed by studies of its expression in Sa. cerevisiae. Expression of the carboxyl-terminal region of the Sc. pombe adenylyl cyclase protein will suppress a temperature-sensitive mutation in the Sa. cerevisiae adenylyl cyclase gene. Furthermore, Sa. cerevisiae that lack their endogenous adenylyl cyclase gene and express the carboxyl-terminal region of the Sc. pombe adenylyl cyclase protein have measurable adenylyl cyclase activity. The carboxyl-terminal region of this protein has strong homology with the catalytic domain of the Sa. cerevisiae adenylyl cyclase. Also, Sc. pombe adenylyl cyclase, like Sa. cerevisiae adenylyl cyclase, contains a tandemly repeated motif rich in leucine. Neither yeast protein is particularly homologous to the recently cloned Gs-responsive mammalian adenylyl cyclase [Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R. & Gilman, A. G. (1989) Science 244, 1558-1564].