Regulation of hormone biosynthesis in guinea pig adrenal glands by potassium ions as affected by dihydropyridines. 1. Analysis of changes in steroidogenesis evoked by dihydropyridines]

Influence of Ca2+ channel modulators BAY K 8644, nitrendipine and newly synthesized derivative of 1,4-dihydropyridine: 4-(3', 4'-dimethoxyphenyl)-2,6-dimethyl-3,5- diethoxy-carbonyl-1,4-dihydropyridine-(DHP-51) on aldosterone production by adrenocortical slices depends on K+ content in the incubation medium. The modulators only slightly influence the hormone output at low K+ level in the medium. Intensive synthesis of aldosterone at high level of potassium in the medium was prevented in the presence of DHP-51 and low concentration of BAY K 8644. DHP-51 inhibited [3H]-cholesterol incorporation into all main corticosteroids in the high-potassium medium.     

The role of phosphoinositides, protein kinase C and protein kinase A in the K+ regulatory signal transduction in human adrenocortical cells

The messenger mechanisms mediating K+ regulatory signals in human adrenocorticocytes were studied. It was shown that potassium ions initiated decay of polyphosphoinositides to inositolphosphates and obviously diacylglycerol. The latter compounds activate protein kinase C as affected by different agonists. Using western blotting method we showed translocation of PKCalpha from cytosol to membranes after adrenal tissue preincubation in the medium with increased K+ content (8.5 mM). Translocation means activation of the enzyme. Activity of PKC increased in the microsomal fraction and did not change in cytosol. Increased concentration of K+ in the incubation medium also activates protein kinase A, although to a lesser extent compared to PKC. Unlike PKC activity of PKA was changed in cytosol as well. The possibility of involvement of several messenger systems in K+ signal transduction in human adrenocortical cells as well as the hypothesis on cross-talk between messenger mechanisms for main physiological agonists controlling aldosterone biosynthesis in the adrenals are discussed.     

Aldosterone biosynthesis induced by ACTH and angiotensin II in newborn rat adrenocortical cells transfected by c-EJ-Ha-ras oncogene.

Adrenocortical cells were obtained by fractionated trypsination of newborn rat adrenal glands and transfected with a plasmid containing the EJ/T24-Ha-ras oncogene. Isolation of adhesive cells led to a proliferative cell line with an overexpression of 21 kDa ras protein. These cells incubated with corticosterone or deoxycorticosterone as the precursor produced a high level of 18-hydroxycorticosterone and aldosterone as identified by gas chromatography- mass spectrometry. ACTH and angiotensin II increased the basal production of aldosterone nineteen-fold and six-fold respectively. Under ACTH stimulation the ratio between aldosterone and 18-hydroxycorticosterone production was 1:3. The transformation of corticosterone under angiotensin II stimulation yielded up to 41% of 18-hydroxycorticosterone (4.7 micrograms/mg of cell protein per 24h) and 4.4% of aldosterone (0.5 microgram/mg of cell protein per 24h) in a low potassium concentration medium (6 mmol/l). To our knowledge this is the first report of continuous proliferative adrenocortical cells producing aldosterone.     

Enhanced secretion of oxytocin from bovine granulosa cells treated with adrenal steroids

Bovine granulosa cells were exposed in vitro to various adrenal steroids (cortisol, cortisone, corticosterone, aldosterone; 1 mumol/l), in the presence and absence of stimulation by ascorbic acid (0.5 mmol/l), to determine the possible effects of these hormones on ovarian oxytocin and progesterone secretion. Only cortisol produced a consistent stimulation of the cells; the response was dose-related over the range 0.01 to 1.0 mumol/l and was greatly enhanced in the presence of ascorbate. The secretion of oxytocin was stimulated to a greater extent and with more consistency than was that of progesterone. Although the secretion of oxytocin could be stimulated by cortisol on the day of treatment, the cells also showed a delayed and persistent response to exposure earlier in the culture. It is concluded that cortisol may directly stimulate the secretion of ovarian oxytocin in the cow and that granulosa cells may respond in such a way as to smooth out the effects of short-term fluctuations in cortisol concentration.     

In vitro metabolism of adrenocortical hormones by mammary glands of lactating rats. A comparative study

A comparative study was made of the metabolism of tritium-labeled corticosterone, cortisol and aldosterone on incubation with minced mammary glands of lactating rats. The yield of total nonpolar (acylated) radiometabolites was highest for [3H]corticosterone, lowest for [3H]cortisol and intermediate for [3H]aldosterone. Unlike [3H]corticosterone, [3H]aldosterone yielded two 21-acyl derivatives (Metabolites I and II) in comparable amounts. Metabolite I (39%) was identified as [3H]aldosterone 21-oleate by isotope dilution analysis. Metabolite II (54%) could not be identified: it is intermediate in polarity between corticosterone 21-oleate and the less polar, corticosterone 21-stearate, and is distinctly less polar than the 21-palmityl, linoleoyl (and presumably also less polar than the arachidonyl) derivatives of aldosterone. The [3H]cortisol metabolites were not further investigated.     

Steroid biosynthesis by zona glomerulosa-fasciculata cells in primary culture of guinea-pig adrenals

Steroidogenesis was studied in guinea-pig glomerulosa-fasciculata cells maintained in primary culture for up to 7 days. The basal secretion which remained stable for the first 2 days in culture rapidly rose to reach a plateau on day 4 at levels 6-7-fold higher than those observed during the first 2 days of culture while the maximal response to ACTH in terms of cortisol and androstenedione secretion was fairly stable throughout the 7-day period. Exposure of glomerulosa-fasciculata cells to ACTH caused a stimulation of pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, corticosterone, 11-deoxy-corticosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione, 11 beta-hydroxyandrostenedione and aldosterone while, after 48 h of incubation, a marked accumulation of end-products, namely cortisol and 11 beta-hydroxyandrostenedione, was observed. The half-maximal steroidogenic response to ACTH occurred at concentrations varying between 1.7 x 10(-11) and 1.1 x 10(-10) mol/l for the 12 steroids examined. Addition of 8-bromoadenosine 3', 5'-cyclic monophosphate stimulated steroid secretion in a dose-dependent manner. Maximal response to 8-bromoadenosine 3', 5'-cyclic monophosphate was obtained at 1 mmol/l, and no further rise of steroid secretion was observed after addition of ACTH. Incubation of glomerulosa-fasciculata cells with labeled corticosterone, cortisol and androstenedione indicates that only androstenedione can be converted into 11 beta-hydroxyandrostenedione, thus suggesting that this end-product is a good parameter of the C-19 steroid production by guinea-pig glomerulosa-fasciculata cells in primary culture. The present data confirm that guinea-pig glomerulosa-fasciculata cells in primary culture provide an interesting model for the study of the regulation of C-19 steroid formation by the adrenals.     

Incorporation and metabolism of cortisol in oocytes of tilapia (Oreochromis mossambicus)

The entry and metabolism of 3H-cortisol in oocytes were investigated using isolated follicles of the tilapia (Oreochromis mossambicus) in order to examine the mechanisms of incorporation of maternal hormones into oocytes. The composition of 3H-labeled steroids in the oocyte was analyzed by high-performance liquid chromatography. A significant amount of cortisol was converted to cortisone and an unidentified molecule by the follicular layer. The contents of 3H-cortisol and 3H-cortisone in the oocyte reached an equilibrium level within 12 hr, whereas the content of the unidentified metabolite continued to increase for 36 hr. The total content of the incorporated cortisol and its metabolites was proportional to cortisol in the medium over the concentration range of 5 ng/ml to 5 microg/ml. The amounts of cortisone and the unidentified molecule increased proportionally when the concentration of cortisol in the medium was lower than 500 ng/ml, whereas they reached a plateau when the concentration of cortisol exceeded 500 ng/ml. Cortisol entry was reversible, because 90% of cortisol and cortisone in the oocyte was lost within 18 hr when the medium was changed to that without 3H-cortisol. On the other hand, 50% of the unidentified molecule was preserved at the end of the incubation. In conclusion, the entry of cortisol into the oocyte was considered to be nonspecific and due probably to simple diffusion. However, a considerable amount of cortisol (50-70%) was specifically converted to cortisone and another unidentified molecule during passage through the follicular layer.     

Hippocampal slice (K+)e: depth profiles and changes induced by stimulation or anoxia

In an attempt to develop a technique which would allow early assessment of the functional state of explanted brain tissue, (K+)e was measured in the CA1 region of rat hippocampal slices using K+-selective microelectrodes. In slices (450 micron) maintained at the boundary between the incubation medium and 95% O2/5% CO2 atmosphere, (K+)e was highest (up to 25-30 mmol/l) immediately below the exposed surface and gradually decreased with depth to (K+) of the bathing fluid (5 mmol/l). (K+)e below the exposed surface remained high throughout the 2 h of incubation. In submersed slices, (K+)e was the highest in the center of the slice (200 micron, 10 mmol/l) and decreased towards both surfaces. During 2 h incubation, (K+)e decreased in the center of the slice to 6 mmol/l in viable preparations remaining high in the deteriorating ones. Electrical stimulation of Schaffer's collaterals (15 V; 0.2 ms; 10 Hz) increased (K+)e of viable slices 200 micron below the surface by 2-3 mmol/l. Similar but slower (K+)e changes were elicited by brief (3 min) anoxic episodes (perfusion with incubation medium equilibrated with 95% N2/5% CO2). It is concluded that submersed slices have a more uniform (K+)e profile as compared to the exposed ones and that low (K+)e in the early phase of incubation is a good predictor of slice viability.     

The regulation of purine ribonucleotide biosynthesis by glucocorticoid hormones

The influence of the glucocorticoid hormones (cortisone, cortisol, corticosterone) on the biosynthesis of purine nucleotides (inosinic acid, guanylic acid and adenylic acid) in different organs was investigated in vivo, by following the incorporation of formate-14C into the acid-soluble nucleotides, after administration of the hormones to adrenalectomized rats. Cortisone and corticosterone show a remarkable and comparable increase of the incorporation of formate-14C only in the purine bases of the liver: cortisol is much more effective, increasing the incorporation of formate-14C into the purine bases even ten times over the basal values. No specific effect is evident either in the kidney or in the heart after glucocorticoid administration. Results are interpreted considering that the action of an individual hormone is specifically restricted to the purine nucleotide synthesis in the liver, and that cortisol seems to be the most efficient from this point of view.     

Significance of adenylic acid catabolites for mouse thymus regeneration]

The distribution of [8-14C]adenylic acid catabolites in mouse thymocytes in cortisone-resistant and total thymocyte population has been studied. The accumulation of labelled catabolites in a form of hypoxanthine was found preferentially in cortisone resistant thymocytes but not in total population. This accumulation considerably grows after incubation of cortisone resistant thymocytes with non-peptide mitogenic factor. The excretion of labelled hypoxanthine into medium was also observed. In order to investigate a significance of hypoxanthine accumulation cortisone resistant thymocytes were incubated in the presence of hypoxanthine range concentration and [14C]thymidine incorporation into thymocyte DNA was determined. It was found that [14C]thymidine incorporation into thymocyte DNA increases after incubation in presence of 0.5-5.0 micrograms of hypoxanthine or 0.0005-5.0 micrograms of uric acid. It has been concluded that stimulation of [14C]thymidine incorporation and thymocyte proliferation by non-peptide mitogenic factor is caused by hypoxanthine accumulation.     

Isolation and fractionation in percoll gradient of guinea pig adrenal cortex cells and their functional characteristics

Suspension of isolated adrenal cells from the guinea pig adrenals was prepared using two modifications of collagenase dispersion. The cells separated by percoll density gradient gave 4 bands. The fraction of adrenocorticocytes has buoyant density of 1.03-1.05 g/ml. The lower bands comprise blood cells with density of 1.07-1.10 g/ml. The aldosterone content in the adrenocorticocyte fraction is 1200-1300 pg/10(5) cells. Intensity of labelling of cellular proteins increases only in the adrenocorticocyte fraction in response to a rise in K+ concentration in incubation medium. The Scatchard plot method was used to characterize binding of 125I-ACTH to dispersed adrenocortical cells. Binding association constant (Ka) and binding capacity (Bmax) are 1.3 X 10(8) M-1 and 2.0 X 10(-9) mol/l, respectively. A relation between functional activity of adrenocortical cells, their ultrastructural features and density is discussed.     

Renal 11-beta-hydroxysteroid dehydrogenase: a mechanism ensuring mineralocorticoid specificity

In vitro studies with mineralocorticoid receptors (MR) have shown that they are non-specific and do not distinguish between glucocorticoids (cortisol in man, corticosterone in rodents) and aldosterone. These findings contrast with in vivo aldosterone selectivity. Our studies on the congenital deficiency of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD; which converts cortisol to cortisone or corticosterone to 11-dehydrocorticosterone) and acquired deficiency secondary to liquorice or carbenoxolone indicate that this enzyme plays a crucial role in protecting the MR from glucocorticoid exposure. The localisation of 11 beta-OHSD in both the proximal and distal nephron suggests that it has both an autocrine and a paracrine role. The presence of this protective mechanism in the toad bladder suggests that it is at least 300 million years old.     

Factors involved in the uptake of corticosterone by rat liver cells

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent Kt values of the saturable systems were 64 +/- 40 nM (n = 3) and 1085 +/- 313 nM (n = 12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluoro-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35--45%. In the presence of cortisone, cortisol, dexamethasone, aldosterone, testosterone, estradiol-17beta and estrone (2 muM each) the uptake decreased 30--50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.     

Evaluation of hepatic 11 beta-hydroxysteroid dehydrogenase activity by cortisone acetate test in young adults with diabetes mellitus type 1

Cortisone acetate test was performed in twelve young adult patients with diabetes mellitus type 1, after dexamethasone administration to suppress endogenous cortisol production. Previous screening revealed that all of the subjects had peak cortisol responses in the range from subnormal to normal, as determined by a low-dose Synacthen test. The aim was to find out whether these patients would exhibit different conversion of cortisone to cortisol by 11beta-hydroxysteroid dehydrogenase. Using multifactorial ANOVA the following significant relationships were obtained between cortisol or cortisol/cortisone ratio measured during the test and other parameters examined a) before dexamethasone suppression and b) during the test: a) Cortisol at 120(th) minute negatively correlated with daily insulin dose and positively with basal aldosterone. Cortisol/cortisone ratio at 60(th), 120(th), 180(th), and 240(th) minute negatively correlated with basal aldosterone/plasma renin activity ratio, urinary free cortisol/24 hours and positively with basal dehydroepindrosterone sulphate. b) Cortisol at 120(th) minute negatively correlated with suppressed basal serum glycemia; cortisol/cortisone ratio during the whole test negatively correlated with supressed basal ACTH. The examination of peripheral metabolism of cortisol using cortisone acetate test in patients with diabetes mellitus type 1 showed adaptive changes of 11beta-hydroxysteroid dehydrogenace activity associated with altered cortisol tissue supply.     

Evidence for a tonic inhibitory role of nifedipine-sensitive calcium channels in aldosterone biosynthesis.

This study investigated the effects of the calcium channel blockers nifedipine (a dihydropyridine) and verapamil (a papaverine derivative), on aldosterone production utilizing isolation of the early and late phases of aldosterone biosynthesis. Pregnenolone production (the early phase of aldosterone biosynthesis) was assessed in trilostane-treated bovine glomerulosa cells, used to inhibit the conversion of pregnenolone onwards to aldosterone. Conversion of exogenous corticosterone to aldosterone, an index of late phase activity, was assessed using aminoglutethimide to inhibit endogenous aldosterone production. Low concentrations of nifedipine, 10(-11)-10(-9) M, stimulated basal total aldosterone biosynthesis by enhancing the late phase although the early phase was inhibited. In the presence of 12 mM potassium (K+), which is less effective in stimulating aldosterone production than lower K+ concentrations, aldosterone production was enhanced by nifedipine, 10(-8) M, by an effect on the late phase. At K+ 6 and 8 mM, nifedipine, 10(-4) M, inhibited the early phase. Nifedipine 10(-5) inhibited angiotensin II (AII)-stimulated total aldosterone biosynthesis by independent effects on the early and late phases. Verapamil, 10(-4) M, inhibited total and early phase aldosterone production at K+, 4 mM and inhibited both phases at K+, 8 mM, stimulation was not observed using verapamil. Verapamil, 10(-4) M, also inhibited AII-stimulated aldosterone production. Basal and AII-stimulated pregnenolone production were inhibited by verapamil, 10(-4) M (basal) and 10(-6) M (AII-stimulated). These studies using nifedipine have revealed subtle calcium-dependent mechanisms involved in the tonic inhibition of activity in the late phase of aldosterone biosynthesis and the reversal of the inhibitory effect of high K+ concentrations also on the late phase. In addition, the data reported indicate that both AII and K+ independently enhance activity in the early and late phases of aldosterone production by calcium-dependent mechanisms.     

Melatonin down-regulates steroidal hormones,thymocyte apoptosis and inflammatory cytokines in middle-aged T. cruzi infected rats

Chagas disease, triggered by the flagellate protozoan Trypanosoma cruzi (T. cruzi) plays a potentially threat to historically non-endemic areas. Considerable evidence established that the immuno-endocrine balance could deeply influence the experimental T. cruzi progression inside the host's body. A high-resolution multiple reaction monitoring approach (MRM^HR) was used to study the influence of melatonin on adrenal and plasma steroidal hormones profile of T. cruzi infected Wistar rats. Young (5 weeks) and middle-aged (18 months) male Wistar rats received melatonin (5 mg/Kg, orally) during the acute Chagas disease. Corticosterone, 11-dehydrocorticosterone (11-DHC), cortisol, cortisone, aldosterone, progesterone and melatonin concentration were evaluated. Interleukin-1 alpha and β (IL-1α and β), IL-6 and transforming growth factor beta (TGF-β) were also analyzed. Our results revealed an increased production of corticosterone, cortisone, cortisol and aldosterone in middle-aged control animals, thus confirming the aging effects on the steroidal hormone profile. Serum melatonin levels were reduced with age and predominantly higher in young and middle-aged infected rats. Melatonin treatment reduced the corticosterone, 11-DHC, cortisol, cortisone, aldosterone and progesterone in response to T. cruzi infection. Decreased IL-1 α and β concentrations were also found in melatonin treated middle-aged infected animals. Melatonin treated middle-aged control rats displayed reduced concentrations of TGF-β. Melatonin levels were significantly higher in all middle-aged rats treated animals. Reduced percentages of early and late thymocyte apoptosis was found for young and middle-aged melatonin supplemented rats. Finally, our results show a link between the therapeutic and biological effects of melatonin controlling steroidal hormones pathways as well as inflammatory mediators.     

Ion channels in human endothelial cells.

Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.     

Influence of potassium and calcium ions on the phosphatidylinositol and phosphatidylcholine metabolism in rat fibroblasts after growth stimulation by calf serum.

In secondary cultures of embryonic rat fibroblasts which were arrested in G1 (G0) by serum depletion and subsequently triggered into the cell cycle by readdition of growth factors isolated from fetal calf serum the influence of the potassium and calcium concentrations in the medium on phosphatidylinositol and phosphatidylcholine metabolism was investigated. The incorporation of inorganic [32P]phosphate into phosphatidylinositol is dependent on the potassium content of the culture medium. The specific activity of 32P in phosphatidylinositol is increased at K+ concentrations between 0.1 and 1 mM. Also calcium (between 0.01 and 2 mM) slightly stimulates phosphatidylinositol metabolism. Also the incorporation of myo-[3H]inositol is increased at potassium concentrations between 0.2 and 1 mM, whereas calcium is slightly inhibitory. The labelling of phosphatidylcholine with either [32P]phosphate or [3H]choline is not dependent on the potassium and calcium concentrations of the culture medium. Moreover, the phospholipid metabolism of permanently growing epithelioid and fibroblastoid cells lines, which were investigated, is considerably less dependent on the K+ and Ca2+ ions.     

Production of proliferation inhibitors by mature granulocytes

The aim of the experiments was to find ways of increasing the yield of small molecular weight inhibitors of cell proliferation released by granulocytes. Almost pure populations of granulocytes from pig or human blood, or from sterile inflammatory exudates in rats were treated in various ways and then spun down. Molecules below approximately 10 000 dalton (Diaflo ultrafiltration or Sephadex G 25 filtration) in the supernatants were tested for inhibitory activity by measuring 3H-thymidine incorporation in 5 to 6-h coverslip cultures of rat bone marrow cells. The different granulocyte treatments were: Freeze-thawing, sonication, incubation (at +4 degrees -37 degrees C) in hypotonic media (0-200 mosm/kg), storage in vitro overnight (at +4 degrees C) before incubation, incubation at 37 degrees C in complete and buffered tissue culture medium (Fischer's with 10 mmol/1 HEPES), incubation in saline only (2-h periods, approximately 70 X 10(6) cells/ml), or with lidocaine added, with Ca++ and the Ca++ ionophore A-23187, with K+ and the K+ ionophore Valinomycin, with a high K+ concentration (50 mmol/1), with arachidonic acid, with a cAMP analogue, or with a protease inhibitor added during or at the end of the incubation. On a per cell basis rat peritonitis granulocytes released more inhibitor than pig blood granulocytes, whereas human blood granulocytes were not detectably inhibitory at all. Arachidonic acid was the most promising agent tested to increase inhibitor release above that occurring spontaneously from granulocytes incubated in saline.