The frequency of gonadotropin-releasing hormone secretion regulates expression of alpha and luteinizing hormone beta-subunit messenger ribonucleic acids in male rats

The influence of GnRH pulse frequency on LH subunit mRNA concentrations was examined in castrate, testosterone-replaced male rats. GnRH pulses (25 ng/pulse) or saline to controls, were given via a carotid cannula at intervals of 7.5-240 min for 48 h. alpha and LH beta mRNA concentrations were 109 +/- 23 and 30 +/- 5 pg cDNA bound/100 micrograms pituitary DNA, respectively, in saline controls. GnRH pulse intervals of 15, 30, and 60 min resulted in elevated alpha and LH beta mRNAs (P less than 0.01) and maximum responses (4-fold, alpha; 3-fold, LH beta) were seen after the 30-min pulses. Acute LH release to the last GnRH pulse was seen after the 15-, 30-, and 60-min pulse intervals. In contrast, LH subunit mRNAs were not increased and acute LH release was markedly impaired after the rapid (7.5 min) or slower (120 and 240 min) pulse intervals. Equalization of total GnRH dose/48 h using the 7.5- and 240-min intervals did not increase LH subunit mRNAs to levels produced by the optimal 30-min interval. These data indicate that the frequency of the pulsatile GnRH stimulus regulates expression of alpha and LH beta mRNAs in male rats. Further, GnRH pulse frequencies that increase subunit mRNA concentrations are associated with continuing LH responsiveness to GnRH.     

Effects of acute stress, (1-24)ACTH administration and changes in superfusion temperature and flow rate on the in vitro secretion of glucocorticosteroids and aldosterone from the Mongolian gerbil (Meriones unguiculatus) adrenal gland

The release of glucocorticosteroids and aldosterone rapidly decreased after start of superfusion and reached a steady base-line within 60-90 min of superfusion. While secretion markedly varied between experiments, it was very constant in the same experiment (coefficient of variation: 7.4-2.2% for glucocorticosteroids and 5.8-3.9% for aldosterone). After repeated exposure of adrenal tissue to 1 IU/ml (1-24)ACTH, glucocorticosteroid release progressively increased; under the same conditions aldosterone secretion was not changed. Glucocorticosteroid secretion from glands of animals stressed by 1-hr confinement or of animals injected with 6 IU (1-24)ACTH was significantly higher than that of controls over the 60-min superfusion period. Aldosterone secretion was not affected significantly by these pretreatments. After reduction of temperature from 35 to 1 degrees C, steroid release ceased. Elevation of temperature from 12 to 32 degrees C resulted in a linear increase of glucocorticosteroid and aldosterone secretion. A highly significant positive correlation was found between glucocorticosteroid and aldosterone amounts secreted from adrenals superfused at temperatures between 1 and 35 degrees C (r = 0.91, n = 116, P less than 0.0001). Changes of flow rate from 0.5 to 1.5 ml/min for 5 min induced a short term (1 min) stimulation of glucocorticosteroid and aldosterone release; reduction of flow rate to 0.5 ml/min for 5 min drastically diminished secretion of steroids below control levels for 1 min.     

Aldosterone-mediated regulation of Na+, K(+)-ATPase gene expression in adult and neonatal rat cardiocytes.

By altering the Na+/K+ electrochemical gradient, Na+,K(+)-ATPase activity profoundly influences cardiac cell excitability and contractility. The recent finding of mineralocorticoid hormone receptors in the heart implies that Na+,K(+)-ATPase gene expression, and hence cardiac function, is regulated by aldosterone, a corticosteroid hormone associated with certain forms of hypertension and classically involved in regulating Na+,K(+)-ATPase gene expression and transepithelial Na+ transport in tissues such as the kidney. The regulation by aldosterone of the major cardiac Na+,K(+)-ATPase isoform genes, alpha-1 and beta-1, were studied in adult and neonatal rat ventricular cardiocytes grown in defined serum-free media. In both cell types, aldosterone-induced a rapid and sustained 3-fold induction in alpha-1 mRNA accumulation within 6 h. beta-1 mRNA was similarly induced. alpha-1 mRNA induction occurred over the physiological range with an EC50 of 1-2 nM, consistent with binding of aldosterone to the high affinity mineralocorticoid hormone receptor. In adult cardiocytes, this was associated with a 36% increase in alpha subunit protein accumulation and an increase in Na(+)-K(+)-ATPase transport activity. Aldosterone did not alter the 3-h half-life of alpha-1 mRNA, indicating an induction of alpha-1 mRNA synthesis. Aldosterone-dependent alpha-1 mRNA accumulation was not blocked by the protein synthesis inhibitor cycloheximide, whereas amiloride inhibited both an aldosterone-dependent increase in intracellular Na+ [Na+]i) and alpha-1 mRNA accumulation. This demonstrates that aldosterone directly stimulates Na+,K(+)-ATPase alpha-1 subunit mRNA synthesis and protein accumulation in cardiac cells throughout development and suggests that the heart is a mineralocorticoid-responsive organ. An early increase in [Na+]i may be a proximal event in the mediation of the hormone effect.     

Differential regulation of hCG alpha and beta subunit mRNAs in JEG-3 choriocarcinoma cells by 8-bromo-cAMP

The coordinate regulation of human chorionic gonadotropin (hCG) subunit synthesis by JEG-3 choriocarcinoma cells was studied at the pretranslational level. The responses of the hCG alpha and beta mRNAs were measured during stimulation with the potent cAMP analog 8-bromo-cAMP (8-Br-cAMP) using 32P-labeled hCG alpha and beta cDNA probes. The hCG alpha mRNA (850 bases) and beta mRNA (1050 bases) from JEG-3 cells were identical in size to that of their respective mRNAs from placenta, by Northern blot analysis. After 48 h of stimulation with 2 mM 8-Br-cAMP, production of immunoreactive alpha and beta subunits increased 25- and 52-fold, respectively; corresponding levels of the alpha and beta mRNAs increased 36- and 43-fold, respectively, in a dot blot hybridization assay. Total cellular protein, DNA content, and messenger RNA pools were not altered by treatment with 8-Br-cAMP. The temporal coordination of the expression of the hCG alpha- and beta-subunit genes was examined by comparing the time course of stimulation of the respective mRNAs and the production of immunoreactive subunits. The kinetic responses of the alpha and beta mRNAs differed: the increase in hCG alpha mRNA preceded the increase in hCG beta mRNA, while levels of free alpha subunit and intact hCG increased in parallel with the increase in beta mRNA. hCG alpha mRNA levels increased rapidly between 8 and 24 h after the addition of 8-Br-cAMP, and approached a plateau by 48 h. The levels of hCG beta mRNA increased steadily throughout the 8-48 h period. These results demonstrate that the cAMP analog 8-Br-cAMP differentially regulates hCG subunit biosynthesis in JEG-3 cells at a pretranslational level, and that the stimulation by 8-Br-cAMP in this system appears to be relatively selective for hCG subunits.     

Influence of gonadotropin-releasing hormone pulse amplitude, frequency, and treatment duration on the regulation of luteinizing hormone (LH) subunit messenger ribonucleic acids and LH secretion

The effects of GnRH pulse amplitude, frequency, and treatment duration on pituitary alpha and LH beta subunit mRNA concentrations were examined in castrate-testosterone replaced male rats. Experimental groups received iv GnRH pulses (5, 25, or 125 ng) at 7.5-, 30-, or 120-min intervals for 8, 24, or 48 h. Saline pulses were given to control rats. Acute LH secretion was measured in blood drawn before and 20 min after the last GnRH pulse. In saline controls, alpha and LH beta mRNAs (150 +/- 14, 23 +/- 2 pg cDNA bound/100 micrograms pituitary DNA) fell to 129 +/- 14 and 18 +/- 2, respectively, after 48 h. In animals receiving GnRH pulses (7.5-min intervals), the 125-ng dose stimulated a slight increase (P less than 0.01) in alpha mRNA levels after 8 and 24 h and both LH subunit mRNAs were increased by the 25- and 125-ng doses after 48 h. The 30-min pulse interval injections (25- and 125-ng doses) increased LH beta mRNA levels after 8 h, but alpha mRNAs were not elevated until after 24 h. Maximum (3-fold) increases in alpha and LH beta mRNAs were seen in rats receiving 25-ng pulses every 30 min for 48 h. Using 120-min pulses, LH subunit mRNAs were not increased by any GnRH dose through 48 h. Acute LH release was not seen in rats receiving 5 ng GnRH pulses at any pulse interval.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the endothelins on aldosterone secretion by rat zona glomerulosa cells in vitro.

Endothelins are thought to be involved in the local regulation of blood flow and tissue function. These experiments were carried out to investigate the possible role of endothelins in the control of aldosterone secretion by the rat adrenal. Suspensions of zona glomerulosa cells were prepared by collagenase digestion of capsular tissue, and incubated in the presence of increasing concentrations of endothelin. Aldosterone was measured by RIA. All three peptides caused a dose-dependent increase in the secretion rate of aldosterone by zona glomerulosa cells. The minimum concentration of peptide required to give a significant response was 10(-14) mol/l for endothelins 2 and 3 and 10(-13) mol/l for endothelin 1. At a concentration of 10(-7) mol/l endothelin 2 elicited a 20-fold increase over basal aldosterone secretion, while both endothelins 1 and 3 elicited a 30-fold increase (P less than 0.001 in all cases). These results show that the endothelins are potent stimulators of aldosterone secretion, and suggest that these peptides may have a role in the control of zona glomerulosa function.     

Characterization of a gain of function mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa).

Integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) is a prototype of integrins involved in cellular adhesive functions. As part of a structure-function analysis of this molecule, we constructed a mutant, designated alpha IIb beta 3 (beta 1-2), by replacing 6 amino acids within a putative ligand binding domain of the beta 3 subunit with sequences derived from beta 1. The alteration did not affect the capacity of beta 3(beta 1-2) to combine with transfected alpha IIb, nor did it cause it to combine with endogenous alpha 5. Integrin alpha IIb beta 3(beta 1-2) was in a "resting" state on Chinese hamster ovary cells as judged by minimal binding of an activation-specific anti-alpha IIb beta 3, PAC1. Nevertheless, cells expressing alpha IIb beta 3(beta 1-2) spontaneously bound fibrinogen with low affinity (Ka = (4.85 +/- 0.84) x 10(6) M-1). Activation with an anti-beta 3 antibody (monoclonal antibody 62) resulted in a 10-fold increase in fibrinogen binding affinity (Ka = (4.55 +/- 0.77) x 10(7) M-1), which was 3-fold greater than fibrinogen binding to activated wild type alpha IIb beta 3 (Ka = (1.66 +/- 0.33) x 10(7) M-1, F = 7.46, p = 0.008). The mutant receptor also bound fibrinogen mimetic peptide ligands with enhanced affinity as measured by the conformation-specific antibody, anti-LIBS1. This indicates that the increased affinity for fibrinogen was caused by enhanced interaction of alpha IIb beta 3(beta 1-2) with known recognition sequences in fibrinogen. Thus, this gain of function mutant augments ligand binding function, supporting a role for this region of the beta subunit in ligand binding to integrins.     

Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump.

In order to understand the molecular mechanism of ouabain resistance in the toad Bufo marinus, Na,K-ATPase alpha and beta subunits have been cloned and their functional properties tested in the Xenopus laevis oocyte expression system. According to sequence comparison between species, alpha 1, beta 1, and beta 3 isoforms were identified in a clonal toad urinary bladder cell line (TBM 18-23). The sequence of the alpha 1 isoform is characterized by two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop and no charged amino acid at the C terminus, a pattern distinct from the ouabain-resistant rat alpha 1 isoform. The coexpression of alpha 1 beta 1 or alpha 1 beta 3 TBM subunits in the Xenopus oocyte resulted in the expression of identical maximum Na,K-pump currents with identical inhibition constant for ouabain (Ki) (alpha 1 beta 1: 53 +/- 3 microM; n = 7 vs. alpha 1 beta 3: 57 +/- 3.0 microM; n = 8) but distinct potassium half activation constant (K1/2) (alpha 1 beta 1: 0.87 +/- 0.08 mM, n = 16; alpha 1 beta 3: 1.29 +/- 0.07 mM, n = 17; p less than 0.005). We conclude that (i) the TBM alpha 1 isoform is necessary and sufficient to confer the ouabain resistant phenotype; (ii) the beta 3 or beta 1 subunit can associate with the alpha 1 equally well without affecting the ouabain-resistant phenotype; (iii) some specific sequence of the beta subunit can modulate the activation of the Na,K-pump by extracellular potassium ions.     

Age-related specifics of aldosterone reception in distal segments of the rat nephrons and of Na(+), K(+)-ATPase expression induction

The reason of unresponsiveness of young 10-day rats kidney to aldosterone was explored. The aldosterone binding in distal segments of renal nephrons and the influence of hormonal induction on the mRNA of the alpha and beta subunits of the Na+, K(+)-ATPase in 10-day and 2-month old rats were investigated. There was no age related difference in the aldosterone specific binding in presence of RU-38486 (10(-7) M; Russel Uclaf) in the cortical collecting tubules: 0.26 +/- 0.04 (n = 9) and 0.22 +/- 0.03 (n = 8) mMol/mm of tubule lengths in 10 day and adult rats, respectively. By Nozern blot analysis and RT-PCR more then three and two fold increase of the mRNA abundance of both subunits was found in young and adult renal cortex compare to the adrenalectomized control after aldosterone induction (5 micrograms/100 g. v. b. w. 4 times i/p injections in 3 hour interval between injections) (p < 0.01). By RT-PCR no expression of the alpha 2, alpha 3 and beta 2 isoforms has been observed in all experimental conditions. The age difference was discovered when aldosterone was injected together with spironolactone (5 micrograms and 12 mg per 100 g. b. w. respectively). It was shown, that spironolactone inhibits the effect of aldosterone in adults whereas the latter was unaffected in young rats. The scheme of the age-related differences in the aldosterone regulation of the sodium pump induction in the target cell of the distal part of the rat nephrons is presented.     

Aldosterone induces a vascular inflammatory phenotype in the rat heart

Vascular inflammation was examined as a potential mechanism of aldosterone-mediated myocardial injury in uninephrectomized rats receiving 1% NaCl-0.3% KCl to drink for 1, 2, or 4 wk and 1) vehicle, 2) aldosterone infusion (0.75 microg/h), or 3) aldosterone infusion (0.75 microg/h) plus the selective aldosterone blocker eplerenone (100 mg. kg(-1). day(-1)). Aldosterone induced severe hypertension at 4 wk [systolic blood pressure (SBP), 210 +/- 3 mmHg vs. vehicle, 131 +/- 2 mmHg, P < 0.001], which was partially attenuated by eplerenone (SBP, 180 +/- 7 mmHg; P < 0.001 vs. aldosterone alone and vehicle). No significant increases in myocardial interstitial collagen fraction or hydroxyproline concentration were detected throughout the study. However, histopathological analysis of the heart revealed severe coronary inflammatory lesions, which were characterized by monocyte/macrophage infiltration and resulted in focal ischemic and necrotic changes. The histological evidence of coronary lesions was preceded by and associated with the elevation of cyclooxygenase-2 (up to approximately 4-fold), macrophage chemoattractant protein-1 (up to approximately 4-fold), and osteopontin (up to approximately 13-fold) mRNA expression. Eplerenone attenuated proinflammatory molecule expression in the rat heart and subsequent vascular and myocardial damage. Thus aldosterone and salt treatment in uninephrectomized rats led to severe hypertension and the development of a vascular inflammatory phenotype in the heart, which may represent one mechanism by which aldosterone contributes to myocardial disease.     

Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex

GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.