Atrial natriuretic peptide inhibits mineralocorticoid receptor function in rat colonic surface cells.

Atrial natriuretic peptide (ANP) inhibits and aldosterone (ALDO) stimulates Na conductive transport. Therefore, the effects of ANP and its second messenger cGMP on mineralocorticoid receptor (MR) function in rat colon surface and crypt cells were examined. 100 nM 8-Br-cGMP decreased surface [3H]ALDO binding by 42 +/- 4% but increased crypt [3HvALDO binding by 52+/-16%. ANP decreased surface [3H]ALDO binding by approximately 50% after a 2.5-h lag period but had no effect on crypt ALDO binding. ANP and cGMP rapidly (< 15 min) inhibited surface cell ALDO-induced MR nuclear translocation but did not affect crypt MR nuclear translocation. Inhibition of cGMP-dependent protein kinase with KT5823 blocked the inhibitory effects of ANP and 8-Br-cGMP on surface cell ALDO binding and MR nuclear translocation. In crypt, KT5823 increased baseline [3H]ALDO binding but did not inhibit the stimulatory effect of exogenous cGMP. DEAE-cellulose chromatography and gel mobility shift assay showed that ANP did not inhibit surface MR activation. ANP inhibited ALDO stimulated short circuit current in distal colon. These data demonstrate cell-specific regulation of MR function. In surface cells, ANP rapidly inhibits MR nuclear translocation and ALDO-induced short circuit current. ANP inhibition of MR function may be an additional mechanism of ANP antagonism of Na reabsorption.

Mineralocorticoid and glucocorticoid receptor steroid binding and localization in colonic cells.

In rat colon epithelium glucocorticoids and mineralocorticoids regulate Na transport by binding to distinct receptors and stimulating different pathways. The distribution and intracellular localization of mineralocorticoid (MR) and glucocorticoid (GR) receptors in colonic Na-absorbing surface cells and Cl-secreting crypt cells is unknown. Surface and crypt cells were sequentially isolated from rat distal colon by EDTA chelation and mechanical dissociation. Cell viability was confirmed by trypan blue exclusion and low rates of 2',7'-bis(2-carboxyethyl)-5(6)-carboxylfluorescein leak. Histologic examination, alkaline phosphatase activity, and rates of [3H]leucine incorporation confirmed separation of surface from crypt cells. Scatchard analysis of [3H]aldosterone and [3H]triamcinolone acetonide binding demonstrated that the number of MR decreased from 7,228 +/- 1,067 in surface to 2,299 +/- 434 receptors/cell in crypt cells, whereas the number of GR increased from 20,857 +/- 4,241 in surface to 58,598 +/- 8,207 receptors/cell in crypt cells. The dissociation constants were 2.8 +/- 0.4 nM for the MR and 12 +/- 3 nM for the GR. Indirect immunofluorescence using the specific anti-MR antibody hMRsN and the anti-GR antibody BuGR-2 demonstrated that both unliganded receptors were cytoplasmic and translocated to the nucleus after hormone binding. These data indicate that both surface and crypt cells are potentially responsive to mineralocorticoids and glucocorticoids and that both the MR and GR require hormone for nuclear translocation.

Glucocorticoids inhibit colonic aldosterone-induced conductive Na+ absorption in adrenalectomized rat.

Low-dose glucocorticoids induce only electroneutral Na absorption in rat colon. High-dose dexamethasone induces both electroneutral and electrogenic Na absorption. Aldosterone induces only electrogenic Na absorption and inhibits basal electroneutral transport in distal colon. To define the interrelationship between glucocorticoid-mediated and aldosterone-mediated Na absorption, adrenalectomized rats were treated with aldosterone and either glucocorticoid-receptor-specific doses of dexamethasone or the specific glucocorticoid RU26988. In combination the steroids did not increase Na and Cl absorption in proximal and distal colon and transmural potential difference (PD) and K secretion in distal colon as much as aldosterone alone. Na absorption was not inhibited by spironolactone or amiloride (10(-4) M). Transport in both segments was by Na-H exchange as demonstrated by marked inhibition by amiloride (10(-3) M) and the Na-H antiport inhibitor 5-(N-ethyl-N-isopropyl)amiloride. Thus glucocorticoids not only decreased Na absorption but also produced a marked qualitative change in the mode of Na absorption in aldosterone-treated rats. Acute dexamethasone infusion in rats pretreated with aldosterone decreased Na absorption and transmural PD within 30 min, suggesting inhibition of electrogenic Na absorption at a step distal to synthesis of aldosterone-induced proteins. These findings suggest that upregulation of one Na absorptive mechanism downregulates the other. This may explain why in intact unstressed rats there is little or no conductive Na absorption, despite sufficient endogenous steroid to occupy the aldosterone receptor. It may also explain why in proximal colon of intact rats, despite the presence of aldosterone receptors, even prolonged aldosterone exposure does not induce significant conductive Na absorption.

Low-dose glucocorticoids maintain Na-H exchange in distal colon of adrenalectomized rats.

With in vivo perfusion we demonstrated that physiological doses of glucocorticoids restore Na and Cl absorption in adrenalectomized rat colon. The absorption is spironolactone and amiloride resistant and is inhibited by the Na-H inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), suggesting that glucocorticoids modulate Na-H antiport. The present in vitro study examines pathways mediated by glucocorticoids in adrenalectomized rat distal colon and rectum. In vivo administration of 2.5 micrograms/100 g body wt dexamethasone did not alter serosal-to-mucosal flux or tissue electrical parameters but restored mucosal-to-serosal flux and net Na and Cl absorption within 2-3 h of administration to levels found in intact rat colon. Transport was not inhibited by 10(-5) M amiloride but was eliminated by 10(-5) M EIPA. After 26 h of dexamethasone, an amiloride-resistant short-circuit current was stimulated, accompanied by increased residual ion flux in rectum, but not distal colon, suggesting that a delayed or secondary effect of glucocorticoids is stimulation of electrogenic anion secretion. Thus adrenalectomy reduces net ion flux in distal colon by its effect on electroneutral mucosal-to-serosal NaCl flux. Small doses of glucocorticoids completely ameliorate this effect via stimulation of the Na-H antiport. Glucocorticoids maintain basal electroneutral NaCl absorption in distal rat colon.

Low-dose glucocorticoids stimulate electroneutral NaCl absorption in rat colon.

Glucocorticoids, not aldosterone, may regulate basal colonic NaCl transport. Aldosterone induces spironolactone-inhibitable, amiloride-inhibitable conductive Na absorption but basal transport is electroneutral and amiloride and spironolactone resistant. We examined in vivo the Na absorptive pathway induced by glucocorticoid receptor specific doses of glucocorticoid using various amiloride analogues. Doses of dexamethasone sufficient to co-occupy aldosterone receptors produced amiloride-sensitive Na absorption in proximal and distal colon. Low doses of dexamethasone or the specific glucocorticoid RU26988 markedly stimulated Na absorption but did so by an amiloride-resistant mechanism. The Na-H antiport inhibitor, 5-N-ethyl-N-isopropylamiloride (NENIA) eliminated glucocorticoid-induced Na and Cl absorption without changing transmural potential difference (PD) in proximal and distal colon (Ki = 0.7 x 10(-7) M). NENIA had no effect on aldosterone-induced transport. NENIA (10(-5) M) almost eliminated Na absorption in adrenal intact animals if infused early in the experimental protocol. With time, NENIA resistance developed, corresponding with the previously documented rise in endogenous aldosterone. Thus glucocorticoids induce an electroneutral Na absorptive pathway that may be the luminal Na-H antiport, suggesting that glucocorticoids regulate adrenal-dependent electroneutral Na absorption in rat colon.

Effect of spironolactone on glucocorticoid-induced colonic cation transport.

Glucocorticoids stimulate colonic sodium absorption. Whether this stimulation is mediated by crossover binding to the aldosterone receptor has not been established. The effect of the aldosterone receptor antagonist, spironolactone, on dexamethasone-mediated transport was investigated in proximal and distal colon of adrenalectomized rats. In adrenal-intact control rats spironolactone minimally affected sodium absorption but markedly decreased the transmural potential difference (PD). In adrenalectomized, aldosterone-replaced rats spironolactone clearly decreased sodium absorption and transmural PD in both proximal and distal colon. In adrenalectomized rats treated with 5 micrograms/100 g body wt of dexamethasone twice daily spironolactone did not decrease sodium absorption or transmural PD. However, when adrenalectomized rats were treated with 25 micrograms/100 g body wt of dexamethasone, a dose estimated from plasma levels to occupy 80% of colonic aldosterone receptors, spironolactone inhibited 20% of proximal colon and 47% of distal colon sodium absorption. Thus lower doses of glucocorticoid which predominantly occupy glucocorticoid receptors stimulate sodium absorption by a spironolactone independent mechanism. When the levels of glucocorticoid are high enough to also occupy the majority of aldosterone receptors, sodium absorption is stimulated by an aldosterone receptor mediated mechanism.

Regulation of cation transport by low doses of glucocorticoids in in vivo adrenalectomized rat colon.

A dose response curve for glucocorticoid-induced proximal and distal colonic cation transport in vivo was established in adrenalectomized rats. All doses (0.5-50 nmol/100 g body wt) stimulated sodium absorption. Distal sodium absorption did not saturate at dexamethasone levels that saturate the glucocorticoid receptor but also bind to greater than 35% of aldosterone receptors. Saturation of the pure glucocorticoid response occurred in both segments with RU26988, a synthetic glucocorticoid that does not occupy aldosterone receptors. Maximum velocities for pure glucocorticoid-induced sodium absorption were 15 and 16 mu eq/min per g dry tissue, and Michaelis constants (Km) were 4.2 and 4.6 X 10(-9) mol/liter for proximal and distal colon. Kms are similar to the dissociation constant for the colonic glucocorticoid receptor and too low for significant aldosterone receptor occupancy. Dexamethasone increased sodium absorption significantly within 30 min of injection, suggesting the response is not dependent on new protein synthesis. Similar time and dose responses in proximal and distal colon suggest glucocorticoids stimulate the same pathway in both segments.

Characterization of the rat colonic aldosterone receptor and its activation process.

Aldosterone increases sodium absorption, short circuit current, and transmural potential difference in rat colon. We studied the rat colonic aldosterone receptor using the synthetic glucocorticoid, 11 beta, 17 beta-dihydroxy-17 alpha-propynylandrosta-1,4,6-triene-3-one, to prevent binding to the glucocorticoid receptor. Specific aldosterone binding was found in proximal and distal colon. Heating to 25 degrees C decreased binding within 15 min, but the protease inhibitor, phenylmethylsulfonyl fluoride, stabilized binding. Binding was highest in terminal distal colon. Competitive binding assay showed aldosterone specificity compared to other competitors was greater at 30 than at 4 degrees C, suggesting temperature-sensitive changes in receptor specificity. Scatchard analysis revealed a straight line with a KD of 2.5 nM at 0 degrees C and 4.1 nM at 30 degrees C. Bmax was higher in distal than in proximal colon (30 degrees C, 156 +/- 33 versus 65 +/- 9 fmol/mg protein) and increased by 36% in proximal and 180% in distal colon at 30 degrees C compared to 0 degrees C. DEAE-cellulose chromatography of unactivated receptor demonstrated a single peak eluting at 200-250 mM KCl. Heat, ATP, and gel filtration did not activate the receptor, whereas increasing cytosolic salt concentration to 300 mM KCl, raising the pH to 8, or adding EGTA and EDTA caused increased DNA-cellulose binding and a new peak eluting at 30-80 mM KCl on DEAE-cellulose chromatography. There is a specific aldosterone receptor in colon with increasing number of binding sites from proximal to most distal segments paralleling aldosterone's physiological effects. Absence of receptor activation with heat, gel filtration, or ATP suggests differences between activation of the aldosterone receptor and other steroid hormone receptors.

Glucocorticoid stimulation of sodium absorption in colon epithelia is mediated by corticosteroid IB receptor.

Studies with RU26988, a synthetic glucocorticoid which does not bind to aldosterone receptors, suggest glucocorticoid-induced colonic cation transport is affected through glucocorticoid-specific receptors. RU26988 produced a 700% increase in sodium absorption and doubled transmural potential difference in proximal and distal colon of adrenalectomized rats. Scatchard analysis suggested a single class of receptors with a KD of approximately 10(-9) M. Competition of unlabeled steroids for [3H]triamcinolone acetonide-binding sites paralleled the steroids' biologic potency as glucocorticoids. Heat treatment (25 degrees C, 30 min) markedly enhanced binding of the glucocorticoid-receptor complexes to DNA-cellulose. The activated receptor from both proximal and distal colon was eluted in the prewash from DEAE-Sephadex A-50 anion exchange columns both in the presence and absence of protease inhibitors and has an estimated molecular weight (Stokes radius) of 33,000-37,000 (25-26 A). These results identify the colonic receptor as glucocorticoid binder IB, a receptor previously identified as the major binder only in kidney cortex. The finding of an apparently unique receptor in the two tissues where glucocorticoids stimulate cation transport suggests that the phenotypic response mediated by glucocorticoids in different tissues might be determined by the structure of the receptor and that glucocorticoid binder IB is the glucocorticoid cation transport receptor.

Diagnostic strategies in disorders of fluid, electrolyte and acid-base homeostasis.

Our understanding of the physiology and biochemistry of acid-base and fluid-electrolyte regulations has greatly expanded in recent years. Key physiologic principles have emerged that now permit rational diagnosis and therapy of clinical disorders of serum electrolyte concentration. This paper describes diagnostic strategies based upon these principles. The etiology of the myriad factors in hyponatremia is best derived by first measuring serum tonicity and then assessing extracellular fluid volume. The hyper-, iso- and hypotonic hyponatremia are defined, and the hypotonic group is subclassified into hypo-, iso- and hyper volemic forms. The hypernatremias are best categorized by their state of volume expansion. Classification into the hypo-, hyper- and isovolemic hypernatremias simplifies their diagnosis. Metabolic acidoses are classified in terms of the anion gap. Clinical and chemical aspects of increased and normal anion gap acidoses are described. Metabolic alkaloses require a source of new bicarbonate and its retention by the kidney. The means by which new alkali is synthesized and urinary loss prevented serve to effectively classify the alkaloses. Hypokalemic syndromes are defined in terms of associated changes in body potassium. The potassium-depleted states are further subclassified by whether normotension or hypertension is associated. Hyperkalemia is produced by redistribution of cellular and extracellular potassium or by increased body potassium. Defects in the renin-angiotensin-aldosterone-distal renal tubule effector arm usually underlie hyperkalemic states, which are than classified in terms of this regulatory hormonal cascade. Classifications for disordered serum concentrations of calcium, magnesium, phosphorus and uric acid are presented. Hormonal, metabolic and renal regulatory factors form the basis for an organized approach to these disorders.

Role of kidney in the catabolic clearance of human platelet antiheparin proteins from rat circulation.

UNLABELLED: Stimulated platelets release at least two antiheparin proteins: platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) from which beta-thromboglobulin (beta TG) is derived. We have found previously marked elevation of LA-PF4/beta TG antigen in platelet poor plasma of patients with chronic renal failure, whereas levels of PF4 remained normal. Therefore, we examined the role of the kidneys in the metabolic clearance of LA-PF4/beta TG and PF4. The supernates of aggregates of thrombin-stimulated human platelets were injected into sham operated control rats, nephrectomized rats, and into rats with acute ureteral ligation. The disappearance of human LA-PF4/beta TG antigen and PF4 in rat plasma determined by specific radioimmunoassays followed biphasic exponential curves. The half-lives (t1/2) for the fast and slow components of LA-PF4 in control rats were 6.4 and 68.4 min. Nephrectomy significantly increased these times to 9.7 and 144 min, while ureteral ligation resulted in no significant change. Comparison of the level of LA-PF4/beta TG antigen and of creatinine in aorta and in renal vein showed 25%-30% extraction of these compounds by the kidney. Less than 0.1% of the total LA-PF4 antigen injected was recovered in the urine of control rats. In contrast to these results, the clearance of PF4 was not affected by nephrectomy. IN CONCLUSION: (1) functional renal tissue is necessary for normal clearance of LA-PF4/beta TG, but renal excretion does not play a major role in its elimination suggesting that the protein is catabolized by the kidney; and (2) catabolic clearance of PF4 does not depend on functioning kidney tissue.

The kidney in health and disease: XVIII: lactic acidosis and the elevated anion gap (II).

This concluding article of a two-part discussion deals with clinical presentations and management of the basic types of lactic acidosis - with and without appaent hypoxia - and the various complicating conditions that may coexist with them. Although symptomatic treatment is essential, cure can be effected only by seeking and treating the underlying cause of the electrolyte derangement.

Role of glucocorticoids and aldosterone in maintenance of colonic cation transport.

Adrenalectomized rats were maintained on physiologic replacement doses of aldosterone or dexamethasone for 24 h after adrenalectomy. Net cation movement and transmural potential difference were determined during in vivo perfusion of the colon. Adrenalectomy without replacement steroids resulted in marked reduction of sodium and water absorption, potassium secretion, and transmural potential difference (PD). Aldosterone 10 microgram . 100 g body wt-1 . 24 h-1 significantly increased net potassium secretion above adrenalectomized levels but did not restore transport to control levels. Sodium and water absorption and transmural PD were not affected. Aldosterone 30 microgram . 100 g body wt-1 . 24 h-1 increased but did not restore net movement of sodium or potassium to control levels. In contrast to aldosterone, physiologic amounts of dexamethasone, 10 microgram . 100 g body wt-1 . 24 h-1, preserved normal electrolyte movement and electrical properties in adrenalectomized rats. In additional experiments the aldosterone antagonist spironolactone was administered for 3 days to rats with intact adrenal function. Net sodium absorption fell only 22% below control with insigificant decreases in potassium secretion and transmural PD. These data suggest that glucocorticoid hormones exert regulatory control of basal colonic fluid and electrolyte function.

Dopamine-related polyuria in patients with gram-negative infection.

Two patients developed polyuria with natriuresis while receiving intravenous dopamine hydrochloride for the treatment of hypotension. Both patients had Gram-negative pneumonia and both had evidence of extracellular volume depletion that was considered to be a result of the dopamine-induced polyuria. Following restoration of extracellular volume and withdrawal of dopamine therapy, both patients became normotensive and required no vasopressors. The diuretic action of dopamine, which persisted despite the severe intravascular volume contraction and perpetuated the hypotensive state in these two patients, is not well understood. The Gram-negative infection could play an important role possibly by enhancing the effect of dopamine on the renal vasculature.

Leukocyte responses to acute renal transplant rejection.

A retrospective study of the response of the leukocyte count to renal transplant rejection was performed in 159 rejection episodes. Results of the study showed that 1. contrary to what is commonly thought leukocytosis is the least common response to acute rejection and a decrease in leukocyte count is far more common, 2. a sudden decrease in leukocyte count in a previously stable patient can be an early sign of rejection, 3. there is a significantly greater incidence of graft loss in rejection episodes characterized by leukopenia and 4. the risk of infection following rejection is greatest in patients with rejection characterized by leukopenia.