Reduced Na-K pump but increased Na-K-2Cl cotransporter in aorta of streptozotocin-induced diabetic rat.

The activities of Na-K-ATPase and Na-K-2Cl cotransporter (NKCC1) were studied in the aorta, heart, and skeletal muscle of streptozotocin (STZ)-induced diabetic rats and control rats. In the aortic rings of STZ rats, the Na-K-ATPase-dependent (86)Rb/K uptake was reduced to 60.0 +/- 5.5% of the control value (P < 0.01). However, Na-K-ATPase activity in soleus skeletal muscle fibers of STZ rats and paired control rats was similar, showing that the reduction of Na-K-ATPase activity in aortas of STZ rats is tissue specific. To functionally distinguish the contributions of ouabain-resistant (alpha(1)) and ouabain-sensitive (alpha(2) and alpha(3)) isoforms to the Na-K-ATPase activity in aortic rings, we used either a high (10(-3) M) or a low (10(-5) M) ouabain concentration during (86)Rb/K uptake. We found that the reduction in total Na-K-ATPase activity resulted from a dramatic decrement in ouabain-sensitive mediated (86)Rb/K uptake (26.0 +/- 3.9% of control, P < 0.01). Western blot analysis of membrane fractions from aortas of STZ rats demonstrated a significant reduction in protein levels of alpha(1)- and alpha(2)-catalytic isoforms (alpha(1) = 71.3 +/- 9.8% of control values, P < 0.05; alpha(2) = 44.5 +/- 11.3% of control, P < 0.01). In contrast, aortic rings from the STZ rats demonstrated an increase in NKCC1 activity (172.5 +/- 9.5%, P < 0.01); however, in heart tissue no difference in NKCC1 activity was seen between control and diabetic animals. Transport studies of endothelium-denuded or intact aortic rings demonstrated that the endothelium stimulates both Na-K-ATPase and Na-K-2Cl dependent (86)Rb/K uptake. The endothelium-dependent stimulation of Na-K-ATPase and Na-K-2Cl was not hampered by diabetes. We conclude that abnormal vascular vessel tone and function, reported in STZ-induced diabetic rats, may be related to ion transport abnormalities caused by changes in Na-K-ATPase and Na-K-2Cl activities.

Role of 11beta-hydroxysteroid dehydrogenase in nongenomic aldosterone effects in human arteries.

The aim of the present study was to demonstrate rapid effects of aldosterone on the Na(+)-H(+) exchanger in strips of human vascular vessels and to determine whether 11beta-hydroxysteroid dehydrogenase enzyme (11beta-HSD) could play a protective role in this response, such as that described for the classic type I mineralocorticoid receptor (MR). The activity of 11beta-HSD isoforms 1 and 2 were measured in fetal and adult arteries. Both isoforms are present in adult and fetal vessels. However, a significant difference in the proportion of each isoform was found. Isoform 1 activity (in pmol x min(-1) x 100 mg(-1) protein) was 42+/-5 in fetal vessels and 29+/-2 in adult arteries, and isoform 2 activity was 78+/-7 in fetal and 12+/-2 in adult tissue. The nongenomic effect of aldosterone on Na(+)-H(+) exchanger activity was measured in strips of chorionic and radial uterine arteries loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Recordings of intracellular pH (pH(i)) were made by videofluorescence microscopy. Aldosterone (0.5 nmol/L) rapidly increased pH(i), with a half-maximal effect between 2 and 3 nmol/L in both fetal and adult vessels. Ethylisopropylamiloride, a specific inhibitor of the Na(+)-H(+) exchanger, inhibited this effect. The hormone-mediated increase in pH(i) was unaffected by spironolactone, a classic antagonist of MR, but was completely blocked by RU28318. Cortisol (up to 1 micromol/L) had no effect on pH(i), but when applied in the presence of carbenoxolone, a dramatic increase in Na(+)-H(+) exchanger activity was evident. The increments on pH(i) for each cortisol concentration were similar to those observed for aldosterone. These findings suggest that vascular 11beta-HSD plays an active role in maintaining the specificity of the rapid effects of aldosterone.

Adrenal-dependent modulation of the catalytic subunit isoforms of the Na+-K+-ATPase in aorta.

Na+-K+-ATPase gene expression and activity were studied in aortas from adrenalectomized (ADX) rats and ADX rats with deoxycorticosterone supplement (ADX-DOCA). Northern analysis of RNA from ADX rats revealed a significant decrease in alpha2-mRNA levels (38.5 +/- 8.3% of control, P < 0.01) that was prevented by DOCA (P < 0.05). A decrease to 55.8 +/- 7.7% in alpha2-isoform protein was observed 8 days after adrenal removal (P < 0.05); DOCA reversed this effect (90.8 +/- 10.5%). Adrenalectomy induced a decrease of 68.5 +/- 4.5% in beta1-mRNA (P < 0.01) and 52.7 +/- 8.3% in ADX-DOCA rats (P < 0.01). Also, a reduction in beta1-isoform protein that was not prevented by DOCA was detected after adrenalectomy (47.1 +/- 11%, P < 0.01). In contrast, no differences in alpha1-mRNA or -protein levels were observed. Vascular sodium pump activity was reduced to 59.8 +/- 4.6% of control values after adrenalectomy (P < 0.01); this reduction was reversed by DOCA. Our data indicate that corticosteroids regulate Na+-K+-ATPase isoform expression and activity in vascular tissue in vivo, suggesting a mineralocorticoid-dependent modulation of alpha2-Na+-K+-ATPase gene expression in aorta, with beta1-isoform expression dependent on the presence of glucocorticoids.

Increased Na,K,Cl cotransporter and Na, K-ATPase activity of vascular tissue in two-kidney Goldblatt hypertension.

The properties of the Na/K pump and Na,K,Cl cotransporter were studied in vascular tissue of two-kidney Goldblatt hypertensive rats. These transport systems were measured as ouabain-sensitive and bumetanide-sensitive 86Rb/K uptake in aortic rings, left ventricular muscle and soleus skeletal muscle fibers of control and hypertensive Sprague-Dawley rats. A dramatic increment in Na/K pump activity was observed in intact aortic rings from the hypertensive group. The same was true for the Na,K,Cl cotransporter. The transport parameters related to the left ventricular muscle and soleus skeletal muscle were not significantly altered in the hypertensive rats. Measurements of the catalytic isoforms of the Na+, K(+)-ATPase in the aortic rings indicated that both isoforms (alpha 1 and alpha 2) were elevated in the same proportion in the hypertensive rats. The results also indicate that the endothelium plays an important role in both transport systems: in the absence of endothelium, a much lower 86Rb/K uptake was observed than in intact aortic rings, either from control or hypertensive vascular tissue. Nevertheless, when the 86Rb/K transport activity was measured in denuded aortic rings, a significantly higher ouabain and bumetanide sensitive 86Rb/K uptake was also observed in the hypertensive rats. These data also show no alteration in the endothelium of the hypertensive rats as compared to control animals. The presence of endothelium had a more striking effect on the alpha 2 catalytic isoform activity than on the alpha 1 isoform. We conclude that there is a significant increment in the Na/K pump and Na,K,Cl cotransporter in two kidney-Goldblatt hypertension, that is specific for vascular smooth muscle.

[Comparison of extra renal potassium management in hypertensive, diabetic and normal subjects]. / Estudio comparativo del manejo extrarrenal de potasio en sujetos controles, pacientes hipertensos y diabéticos.

BACKGROUND: Sodium and potassium ions are involved in the regulation of blood pressure and the genesis of hypertension. AIM: To assess internal potassium balance, as a measure of sodium pump activity, in subjects with essential hypertension and diabetic patients. PATIENTS AND METHODS: Eleven hypertensive subjects, 5 non-insulin-dependent diabetics and 16 age matched controls were studied. An acute oral load of 0.8 mEq/Kg body weight of KCl was administered and blood samples were drawn every 30 min thereafter, until 120 min, to measure plasma K+ levels. Urinary K+ excretion during this period was also measured. In eight hypertensive patients, the test was repeated after two week of supplementation with 60 mEq/day of KCl. The maximal increase in plasma potassium levels and the time required to achieve the maximum concentration was recorded. RESULTS: All patients had normal serum creatinine levels. Mean fasting blood glucose of diabetic patients was 133 +/- 15.1 mg/dl. No difference between patients and controls in maximal increase plasma potassium increase, was observed. In hypertensive patients the lapse to achieve the maximal potassium concentration was longer than in controls. After the period of potassium supplementation in hypertensive patients, there was a significant increase in basal plasma K+ levels and the temporal pattern of plasma potassium increase was similar to that of controls. Between 63 and 68% of retained K+ load was translocated to the intracellular space at 120 min in all study groups. CONCLUSIONS: Internal potassium balance is not significantly altered in subjects with essential hypertension or in non-insulin-dependent diabetics.

Tissue-specific modulation of Na, K-ATPase alpha-subunit gene expression in uremic rats.

Chronic renal failure in the rat is associated with an impaired extrarenal potassium handling, whereas a renal adaptive mechanism of the remaining nephrons has been described. To understand the molecular basis of potassium homeostasis during renal failure we investigated the in vitro pump activity and the catalytic mRNA transcription in three different tissues: skeletal muscle, isolated adipocytes and kidney. The activity of the sodium pump, as measured by ouabain-sensitive 86Rb/K uptake in isolated adipocytes and skeletal muscle fibers, revealed a significant reduction of the pump activity in uremic rats. The reduction of the Na, K-ATPase activity in adipose tissue was associated with a similar decrement of both catalytic subunits (alpha 1 and alpha 2), whereas in the skeletal muscle tissue was only related to a decrease in the activity of the alpha 1 isoform. The expression of rat Na, K-ATPase catalytic isoforms mRNAs in kidney, muscle and adipose tissue from control and chronic renal failure rats was investigated at the molecular level with cDNA probes specific for the catalytic isoforms (alpha 1 and alpha 2). Northern blot analysis revealed that the respective catalytic mRNAs of uremic rats are regulated in a tissue-specific manner that are in agreement with the sodium-potassium pump activity. Muscle and adipose tissue showed a decrement in the levels of expression for the alpha 1 isoform mRNA. In contrast to these tissues, an increment in alpha 1 mRNA expression was observed in the kidney of rats with chronic renal failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chronic renal failure on Na,K-ATPase alpha 1 and alpha 2 mRNA transcription in rat skeletal muscle.

Previous studies have suggested that an alteration in the expression of the Na,K-ATPase of muscle may be an important determinant of enhanced insulin sensitivity in chronic renal failure. Therefore, in the present studies we have examined the effect of uremia on the Na,K-ATPase alpha isoforms in skeletal muscle, at the level of mRNA expression and enzymatic activity. The activity of the sodium pump, as measured ouabain-sensitive 86Rb/K uptake in soleus muscle, revealed a reduction in the activity in uremia, related to the increment in plasma creatinine values. The decrement in 86Rb uptake by the rat soleus muscle of experimental animals was associated with changes on Na,K-ATPase gene product. Northern analysis of mRNA revealed isoform-specific regulation of Na,K-ATPase by uremia in skeletal muscle: a decrease of approximately 50% in alpha 1 subunit Na,K-ATPase mRNA, as compared to controls. The decrement in alpha 1 mRNA correlates with the decreased activity of the Na,K-ATPase in uremia, under basal conditions and with the almost complete inhibition of the Na,K-ATPase, of uremic tissue by a concentration of 10(-5) M ouabain. Although the activity of the alpha 2 isoform pump was not modified by uremia, the 3.4-kb message for this enzyme was increased 2.2-fold; this discrepancy is discussed. Altogether these findings demonstrate that the defective extrarenal potassium handling in uremia is at least dependent in the expression of alpha 1 subunit of the Na,K-ATPase.

Contrasting effects of sn-1,2-dioctanoyl glycerol as compared to other protein kinase C activators in adrenal glomerulosa cells.

Angiotensin II acts on adrenal glomerulosa cells to induce the phospholipase C-mediated generation of inositol trisphosphate and sn-1,2-diacylglycerol as the major products of inositol phospholipid breakdown. This last product is known to activate protein kinase C, but its role in the action of angiotensin II on steroidogenesis has not been defined. We report herein that, in bovine adrenal glomerulosa cells, protein kinase C activators, such as phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate, mezerein and sn 1,2 oleoyl acetoylglycerol, each failed to increase steroidogenesis. These results contrast with our recent report on the enhancement of aldosterone output by sn-1,2-dioctanoylglycerol (DiC8) [J. Steroid Biochem. 35 (1990) 19-33]. In addition, the difference between DiC8 and the other protein kinase activators was also observed in the pattern of 86Rb efflux from preloaded glomerulosa cells; only DiC8 mimicked the effect of angiotensin II on ion fluxes. Furthermore, staurosporine, a potent inhibitor of protein kinase C, was capable of amplifying the aldosterone output induced by a maximally effective concentration of DiC8 or angiotensin II. These data suggest that the effect of the cell permeant DiC8 on aldosterone biosynthesis either is not mediated by protein kinase C activation, or is mediated by a phorbol ester-insensitive isoenzyme of protein kinase C.

Enhanced insulin sensitivity in extrarenal potassium handling in uremic rats.

Translocation of potassium to the intracellular compartment is impaired in advanced chronic renal failure. The purpose of this study was to evaluate the role of endogenous insulin in the disposal of an oral potassium load in uremia. Experiments were done on male Sprague-Dawley rats. Chronic renal failure (CRF) was induced by 3/4 nephrectomy. The results show that the addition of oral glucose to a potassium load was more effective in the translocation of potassium to the intracellular compartment in uremic animals. Further, suppression of endogenous insulin secretion with somatostatin caused a much higher increase in plasma potassium (K) of uremic rats (1.09 +/- 0.15 mEq/liter in CRF vs. 0.28 +/- 0.03 mEq/liter in control). Experiments to assess the activity of the Na pump were done in soleus muscles derived from these animals. Although a 50% reduction of the basal Na pump activity was found in the uremic muscles, the addition of insulin 100 mU/ml caused a relatively greater stimulation of ouabain-sensitive 86Rb uptake in the uremic muscle as compared to the control tissue (203% vs. 77% increment). These data suggest a greater sensitivity to insulin action on extrarenal potassium disposal in uremia.

sn-1,2 dioctanoylglycerol mimics the effects of angiotensin II on aldosterone production and potassium permeability in isolated bovine glomerulosa cells.

In order to elucidate the possible role in glomerulosa cells of diacylglycerol released by angiotensin II we have studied the action of a synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), on aldosterone production and potassium permeability in bovine adrenal cells. DiC8 elicited an increase in 86Rb efflux from cells previously equilibrated with the isotope. The action of DiC8 on the rate coefficient for 86Rb efflux was similar to that previously described for angiotensin II (Am. J. Physiol. 254 (1988) E144-149), i.e. DiC8 induced an immediate increase in 86Rb efflux followed by a sustained decrease in potassium permeability. This DiC8 induced inhibition was observed even in the presence of depolarizing concentrations of potassium. The effect of DiC8 on aldosterone secretion from adrenal glomerulosa cells was measured using a perifusion system. DiC8 (300 microM) caused a significant increase of aldosterone production, comparable to that seen with angiotensin II (100 nM). These results indicate that DiC8 has similar effects to angiotensin II on both potassium permeability and steroidogenesis, which suggests that activation of protein kinase C is involved in the changes of ionic permeability induced by this hormone in bovine adrenal glomerulosa cells.

Effect of a simultaneous potassium and carbohydrate load on extrarenal K homeostasis in end-stage renal failure.

Patients with chronic renal failure (CRF) are continuously exposed to hyperkalemia. In these patients the extrarenal disposal of a potassium load may be very important to determine the plasma potassium levels. We studied the effect of a combined oral load of potassium (0.5 mEq/kg body weight) and carbohydrate (0.5 g/kg body weight) to mimic normal ingestion of potassium. Eight CRF patients and 5 control subjects were studied. The maximal increase in plasma potassium levels achieved was significantly higher in the patients (1.07 +/- 0.1 mEq/l) than in controls (0.39 +/- 0.05 mEq/l). Basal insulin levels were higher in the CRF patients and increased with the oral potassium and carbohydrate load in both controls and patients. In the CRF patients only 58.9 +/- 3% of the potassium load was translocated to the intracellular space compared to 81 +/- 6% in the controls. No correlation was found between the acid base status and maximal potassium increase. We conclude that patients with CRF exhibit an impaired extrarenal handling of potassium and that this abnormality does not appear to be related to insulin secretion or acid base status.

Alpha 2-agonists block ADH action in toad bladder and this inhibition is not modified by indomethacin.

To identify the type of alpha-adrenoceptors involved in the inhibition of the hydrosmotic effect of antidiuretic hormone (ADH) on the toad bladder, we studied the effect of different alpha-adrenergic agonists and antagonists on ADH-induced water transport. Serosal addition of epinephrine (10(-6) M) and norepinephrine (10(-6) M) in the presence of 10(-4) M propranolol significantly inhibited the hydrosmotic effect of ADH (arginine vasopressin). This inhibitory effect of the catecholamines was completely reversed by 10(-5) M yohimbine but not by prazosin. Clonidine did not block ADH-induced water transport, but guanabenz, another alpha 2-agonist, inhibited water transport in response to ADH. In bladders pretreated with indomethacin to block prostaglandin synthesis, basal water permeability was increased, and even in this condition epinephrine inhibited ADH-induced water transport. These studies indicate that alpha 2-adrenergic receptors are involved in the inhibitory effect of catecholamines on ADH-mediated water permeability in the toad bladder. However, this effect was not mimicked by clonidine, as in the case of rabbit cortical collecting tubule. The inhibitory effect of epinephrine appears to be exerted independently of prostaglandin synthesis.

Steroidogenesis and ionic permeability in adrenal glomerulosa cells.

Over the past several years it has become possible to study some of the electrical properties of excitable and endocrine cells by measuring fluxes of radioactive tracer ions; 86Rb fluxes has been widely used to study potassium permeability. We have validated this approach in adrenal glomerulosa cells, in which we demonstrated the presence of a Ca-dependent K channel that is activated by angiotensin II, ATP, the ionophore A23187 and external K. Here, we present evidence that the steroidogenic response of the bovine adrenal glomerulosa cells is related, in the case of angiotensin II, to the inhibitory effect to the hormone on the coefficient rate of 86Rb efflux that occurs after the initial transient increase. This inhibition of the potassium permeability is probable responsible of the depolarization of the cells. Apamin, that blocks the initial transient raise on 86Rb efflux mediated by angiotensin II, has a minor stimulatory action on the hormone induce steroidogenesis; whereas the opposite is true for the steroidogenic action of potassium ions in the presence of apamin. The second groups of experiments examined the effect of angiotensin II on 86Rb fluxes when the Ca in the medium was increased from 0.6 to 1.25 mM in the case of bovine glomerulosa cells or angiotensin was assayed in rat glomerulosa tissue perifused with 0.6 mM Ca; in both conditions only the inhibitory effect in 86Rb efflux was observed. When the effect of external ATP on steroidogenesis was examined a significant increase on aldosterone secretion occurred probable by a similar mechanism. These results are indicative that Ca-mediated K efflux in adrenal glomerulosa cells may provide a modulatory mechanism for agonist action.

Effects of opioid peptides on aldosterone production: stimulatory effect of leu-enkephalin.

The effects of several opioid peptides (Leu-enkephalin, Met-enkephalin, D-Ala2,D-Leu5-enkephalin, and peptide E) on aldosterone secretion have been studied in isolated bovine adrenal glomerulosa cells. Leu-enkephalin significantly increased aldosterone production in a dose-dependent manner (10(-10)-10(-7) M). Similar results on steroidogenesis were obtained with the synthetic opioid D-Ala2,D-Leu5-enkephalin, whereas much higher concentrations of Met-enkephalin were required. Peptide E did not affect steroidogenesis. Naltrexone or naloxone (10(-5) M) potentiated the effect of Leu-enkephalin on aldosterone secretion. The effect of a general opioid antagonist (diprenorphine) or an agonist (etorphine) was also studied. The stimulation by Leu-enkephalin of aldosterone secretion was only partially blocked by diprenorphine. Etorphine (10(-6) and 10(-8) M) had no effect on basal steroidogenesis. It is proposed that the stimulatory effect of Leu-enkephalin on aldosterone production could be mediated by a different receptor than the classical opioid receptors presently known.

Angiotensin II causes a dual effect on potassium permeability in adrenal glomerulosa cells.

In previous studies it was shown that angiotensin II causes a Ca-dependent increase in the K permeability of bovine adrenal glomerulosa cells [Am. J. Physiol. 250 (Endocrinol. Metab. 13): E125-E130, 1986]. Here we show that angiotensin II causes a significant and prolonged reduction in the 86Rb release immediately after the transient rise in 86Rb efflux. This inhibition was dose related. Apamin (100 nM) and tetraethylammonium (10 mM) completely abolished the initial transient rise in 86Rb efflux without affecting the latter sustained phase of reduced radioisotope release. On the contrary, the effect of angiotensin II on the second phase was absent when Ca was removed from the perifusion medium or replaced with Sr, but the effect on the early transient phase of 86Rb efflux was maintained in the absence of external Ca. An additional finding was the increased coefficient rate of 86Rb efflux that occurred when the cells were depolarized with 12 mM K. However, this effect was not observed when the inhibitory phase due to angiotensin II was fully developed and Ca was present in the external media. On the other hand, the biphasic effect of angiotensin II was still present in depolarized cells. These results suggest that angiotensin II may modulate membrane potential by changes in K permeability of the bovine adrenal glomerulosa cells.

An unusual pattern of Na+ and K+ movements across the horse erythrocyte membrane.

Marked differences in the activities of three monovalent cation transport systems in horse versus human erythrocytes are reported. Whereas horse erythrocytes exhibit a 6-fold higher sodium-lithium countertransport, the unidirectional flux of potassium through the sodium pump is 3-4 times slower and the sodium-potassium cotransport system cannot be detected. In spite of this, horse and human cells are able to maintain similar Na+ and K+ gradients.

Effect of angiotensin II, ATP, and ionophore A23187 on potassium efflux in adrenal glomerulosa cells.

Angiotensin II stimulus on perifused bovine adrenal glomerulosa cells elicited an increase in 86Rb efflux from cells previously equilibrated with the radioisotope. When 45Ca fluxes were measured under similar conditions, it was observed that Ca and Rb effluxes occurred within the first 30 s of the addition of the hormone and were independent of the presence of external Ca. The 86Rb efflux due to angiotensin II was inhibited by quinine and apamin. The hypothesis that the angiotensin II response is a consequence of an increase in the K permeability of the glomerulosa cell membrane triggered by an increase in cytosolic Ca is supported by the finding that the divalent cation ionophore A23187 also initiated 86Rb or K loss (as measured by an external K electrode). This increased K conductance was also seen with 10(-4) M ATP. Quinine and apamin greatly reduced the effect of ATP or A23187 on 86Rb or K release in adrenal glomerulosa cells. The results suggest that Ca-dependent K channels or carriers are present in the membranes of bovine adrenal glomerulosa cells and are sensitive to hormonal stimulus.

Effect of corticosteroids on electrolyte transport in the descending colon of the rat.

The effect of corticosteroids on electrolyte movement was studied in an everted sac model from distal rat colon. Corticosterone had a significant stimulatory effect on sodium and fluid transfer in a dose-related manner. At the low dose of 10(-9) M corticosterone increased sodium absorption by 29.4 microEq X g-1 X h-1, a value that was statistically greater than the effect of 10(-9) M dexamethasone. At the higher concentration of 10(-7) M the effect of corticosterone on sodium movement was twice that of aldosterone or dexamethasone in equimolar concentrations. By contrast, the effect of corticosterone on potassium movement was biphasic: at lower concentration (10(-9) M) a significant net secretion occurred, whereas potassium absorption was observed at 10(-7) M corticosterone. Dexamethasone significantly increased net potassium secretion, meanwhile the effect of aldosterone on potassium movement was not significantly different from controls. These data suggest that the native glucocorticoid, corticosterone, exerts regulatory control of colonic fluid and electrolyte function.

Effect of corticosteroids on electrolyte transport in the descending colon of the rat.

The effect of corticosteroids on electrolyte movement was studied in an everted sac model from distal rat colon. Corticosterone had a significant stimulatory effect on sodium and fluid transfer in a dose-related manner. At the low dose of 10(-9) M corticosterone increased sodium absorption by 29.4 microEq X g-1 X h-1, a value that was statistically greater than the effect of 10(-9) M dexamethasone. At the higher concentration of 10(-7) M the effect of corticosterone on sodium movement was twice that of aldosterone or dexamethasone in equimolar concentrations. By contrast, the effect of corticosterone on potassium movement was biphasic: at lower concentration (10(-9) M) a significant net secretion occurred, whereas potassium absorption was observed at 10(-7) M corticosterone. Dexamethasone significantly increased net potassium secretion, meanwhile the effect of aldosterone on potassium movement was not significantly different from controls. These data suggest that the native glucocorticoid, corticosterone, exerts regulatory control of colonic fluid and electrolyte function.

Identification of mineralocorticoid binding sites in rat brain by competition studies and density gradient centrifugation.

A search for mineralocorticoid binding sites in neural tissue was carried out on the basis of competition by two mineralocorticoid antagonists (spironolactone and glycyrrhetinic acid), a mineralocorticoid agonist (9 alpha-fluorocortisol) and a pure glucocorticoid (RU 26988). The hippocampus was selected for these studies, in view of its preferential concentration of binding sites for [3H]-aldosterone (ALDO) and two glucocorticoids: [3H]-corticosterone (CORT) and [3H]-dexamethasone (DEX). Inhibition studies performed with 5 X 10(-4)-5 X 10(-9) M spironolactone and with 5 X 10(-4)-5 X 10(-8) M glycyrrhetinic acid, showed a dose-response reduction of [3H]-ALDO, [3H]-CORT and [3H]-DEX binding, implying that in brain these compounds did not behave as exclusive ALDO antagonists. Two concentrations of 9 alpha-fluorocortisol (5 X 10(-9) and 2.5 X 10(-8) M) preferentially displaced [3H]-ALDO in comparison to [3H]-DEX or [3H]-CORT. Relative binding affinity (RBA) of 9 alpha-fluorocortisol was also higher for the mineralocorticoid than for the glucocorticoids. RU 26988 (5 X 10(-6)-2.5 X 10(-8)M) gave differential inhibition of the three ligands and its RBA for [3H]-DEX site was twice as high as for [3H]-CORT, and three orders of magnitude higher than for [3H]-ALDO binding sites, thus clearly separating sites for gluco- and mineralocorticoids. Further evidence for the presence of separate binding molecules for mineralo- and glucocorticoids in hippocampal cytosol was provided by ultracentrifugation on 16-41% glycerol gradients containing molybdate, which yielded sedimentation values of 9.88 +/- 0.27 for [3H]-DEX (n = 5), 10.48 +/- 0.27 for [3H]-CORT (n = 9) and 11.3 +/- 0.13 for [3H]-ALDO (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)