Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. III. Elemental content of cells in the rat renal papillary tip.

The electrolyte and water content of cellular and interstitial compartments in the renal papilla of the rat was determined by x-ray microanalysis of frozen-hydrated tissue sections. Papillae from rats on ad libitum water were rapidly frozen in a slush of Freon 12, and sectioned in a cryomicrotome at -30 to -40 degrees C. Frozen 0.5-micrometer sections were mounted on carbon-coated nylon film over a Be grid, transferred cold to the scanning microscope, and maintained at -175 degrees C during analysis. The scanning transmission mode was used for imaging. Structural preservation was of good quality and allowed identification of tissue compartments. Tissue mass (solutes + water) was determined by continuum radiation from regions of interest. After drying in the SEM, elemental composition of morphologically defined compartments (solutes) was determined by analysis of specific x-rays, and total dry mass by continuum. Na, K, Cl, and H2O contents in collecting-duct cells (CDC), papillary epithelial cells (PEC), and interstitial cells (IC) and space were measured. Cells had lower water content (mean 58.7%) than interstitium (77.5%). Intracellular K concentrations (millimoles per kilogram wet weight) were unremarkable (79-156 mm/kg wet weight); P was markedly higher in cells than in interstitium. S was the same in all compartments. Intracellular Na levels were extremely high (CDC, 344 +/- 127 SD mm/kg wet weight; PEC, 287 +/- 105; IC, 898 +/- 194). Mean interstitial Na was 590 +/- 119 mm/kg wet weight. CI values paralleled those for Na. If this Na is unbound, then these data suggest that renal papillary interstitial cells adapt to their hyperosmotic environment by a Na-uptake process.

Pathogenic role of cyclic AMP in the impairment of urinary concentrating ability in acute hypercalcemia.

A possible association between the impairment of urinary concentrating ability and an impairment of the vasopressin-dependent cyclic AMP system in hypercalcemia was investigated in rat kidneys both in vivo and in vitro. The increases of urinary osmolality and negative free water clearance and the increase of urinary cyclic AMP excretion by vasopressin injection were significantly less in the hypercalcemic rats than in the control rats. The increase of cyclic AMP concentration by vasopressin in renal medullary tissue was significantly less in the slices obtained from the hypercalcem'c rats than in those obtained from the control rats. The activation of adenylate cyclase by vasopressin was significantly less in the group with an increased concentration of calcium in media than the control group, but phosphodiesterase activity was not affected by calcium concentration in the media. These data suggest that the impaired urinary concentrating ability in hypercalcemic kidneys is due at least in part to the direct inhibitory effect of calcium on the vasopressin-dependent cyclic AMP system at the level of adenylate cyclase in renal medulla.

Modulation of the cyclic AMP content of rat renal inner medulla by oxygen: possible role of local prostaglandins.

The lower O2 tension and more active anerobic metabolism that pertain in the inner medulla (IM) of kidney relative to cortex (C) are well recognized, but there is no evidence that O2 availability constitutes a limiting or regulatory factor in IM metabolism or function. In the present in vitro study, we examined the effects of O2 on adenosine 3',5'-monophosphate (cAMP) metabolism in slices of rat renal C and IM. After a 20-min incubation of slices in Krebs Ringer bicarbonate buffer with 95% O2 + 5% CO2 serving as the gas phase, the cAMP content of IM was 6-10 fold higher than that of C in either the presence or absence of 2 mM 1-methyl-3-isobutylxanthine in the incubation media. In slices of IM incubated for 20 min with 1-methyl-3-isobutylxanthine, cAMP was 22.5+/-SE 2.48 pmol/mg wet weight at 95% O2 and 4.37 without O2. Oxygenation of O2-deprived IM increased cAMP twofold in 2 min, an effect fully expressed in 5 min (fivefold increase). Further, cAMP of IM rose progressively and significantly over a range of atmospheric O2 content from 0 to 50% conditions which should reproduce and encompass O2 tensions that pertain in tissues in vivo. By contrast, basal cAMP content of C varied less than twofold in the presence of 95% versus no O2, implying that O2 modulation of cAMP was specific for IM. Indomethacin and meclofenamate, structurally distinct inhibitors of prostaglandin synthesis, both significantly decreased basal cAMP accumulation in oxygenated slices of IM but not of C. Meclofenamate also reduced basal adenylate cyclase activity determined in homogenates prepared from slices of IM which had been incubated at 95% O2. In slices of IM previously exposed to indomethacin or meclofenamate at 95% O2, a maximally effective concentration of exogenous prostaglandin E1 restored cAMP and adenylate cyclase activity to levels which approximated those observed at 95% O2 in the absence of an inhibitor of prostaglandin synthesis. These results suggest that O2 enhancement of cAMP content in IM may be mediated at least in part by local prostaglandins.

The urinary concentrating defect in the Gunn strain of rat. Role of bilirubin.

The role of high serum and tissue levels of unconjegated bilirubin in the pathogenesis of the impaired urinary concentrating ability was investigated in homozygous (jj) Gunn rats with the congenital absence of hepatic glucuronyl transferase. Continuous phototherapy with blue fluorescent lights at a wave length of 460 nm or oral cholestyramine feeding or both reduced serum levels of unconjugated hilirubin to levels consistently below 3.0 mg/100 ml for several weeks in both weanling and adult jj Gunn rats. The renal concentrating defect was already present in weanling jj Gunn rats by 21 days of age. In treated weanling jj animals, maximum concentrating ability and the concentration of urea and nonurea solutes in the papilla and medulla, determined after 24 h of fluid deprivation, were normal when compared to unaffected heterozygous (Jj) littermates. Solute-free water reabsorption which is reduced in jaundiced jj Gunn rats was restored to normal in treated weanling jj rats. The tissue concentration of unconjugated bilirubin was reduced throughout the papilla and inner and outer medulla in the treated jj rats in comparison with untreated jj littermates. The defect in urinary concentrating ability was only partially reversible and sometimes irreversible in adult jj rats, probably because of permanent renal parenchymal damage occurring secondary to massive crystalline deposits in the papilla and medulla. It is concluded that unconjugated bilirubin is directly involved in the pathogenesis of the concentrating defect in jaundiced jj Gunn rats.

Tryptic and chymotryptic cleavage sites in sequence of alpha-subunit of (Na+ + K+)-ATPase from outer medulla of mammalian kidney.

Localization of selective proteolytic splits in alpha-subunit of (Na+ + K+)-ATPase is important for understanding the mechanism of active Na+,K+-transport. Proteolytic fragments of alpha-subunit from pig kidney were purified by chromatography in NaDodSO4 on TSK 3000 SW columns. NH2-terminal amino acid sequences of fragments as determined in a gas phase sequenator were unambiguously located within the total sequence of alpha-subunit from sheep kidney (Shull, C.E., et al. (1985) Nature 316, 691-695) and pig kidney (Ovchinnikov, Y.A., et al. (1985) Proc. Acad. Sci. USSR 285, 1490-1495). The primary chymotryptic split in the E1-form is located between Leu-266 and Ala-267 while the tryptic cleavage site appears to be between Arg-262 and Ile-263 (Bond 3). Tryptic cleavage in the initial fast phase of inactivation of the E1-form is located between Lys-30 and Glu-31 (Bond 2). In the E2-form, primary tryptic cleavage is between Arg-438 and Ala-439 (Bond 1). Chymotryptic cleavage between Leu-266 and Ala-267 stabilizes the E1-form of the protein without affecting the sites for binding of cations or nucleotides. Titration of fluorescence responses demonstrates the importance of the NH2-terminal for E1-E2 transition. Protonation of His-13 facilitates transition from E1- to E2-forms of the protein. Removal of His-13 after cleavage of bond 2 can explain the increase in apparent affinity of the cleaved enzyme for Na+ and the shift in poise of E1-E2 equilibrium in direction of E1-forms. The NH2-terminal sequence in renal alpha-subunit is not conserved in alpha + from rat neurolemma or in alpha-subunit from Torpedo or brine shrimp. A regulatory function of the NH2-terminal part of the alpha-subunit may thus be a unique feature of the alpha-subunit in (Na+ + K+)-ATPase from mammalian kidney.

Studies on in vitro polymerization of tubulin from renal medullary extracts.

Previous in vivo studies showed that microtubules are involved in the cellular action of vasopressin. In order to analyze the role of renal medullary microtubules, a system was developed which would allow the study of the assembly of tubulin in renal medulla extracts into microtubules in vitro. The assembly of tubulin into microtubules occurred in renal medullary cytosol (100 000 times g supernatant) under specific conditions which include pre-concentration of cytosol by ultrafiltration, the presence of ethylene glycol bis(2-aminoethyl)ether tetraacetic acid (EGTA) and 4 M glycerol, and warming at 37 degrees C. Formation of microtubules, which sedimented at 100 000 times g, was proved by (a) an increase in the apparent [3H]colchicine-binding activity of depolymerized pellets, (b) appearance of typical microtubules as shown by electron microscopy, and (c) by the increase in the quantity of microtubular protein analyzed by polyacrylamide gel electrophoresis. Vinblastine at a concentrationof 10(-6) M completely blocked formation of microtubules. A slight increase of ionized calcium in the polymerization mixture also prevented microtubule assembly; this inhibitory effect of ionized calcium was present at concentrations as low as 10(-4) M. Blockade of microtubule assembly by the increase in concentration of ionized calcium or by vinblastine may be the basis of known inhibitory effect of these two agents upon the hydroosmotic effect of vasopressin in vivo.

The volume and ionic composition of cells in incubated slices of rat renal cortex, medulla and papilla.

The apparent extracellular space in incubated slices of rat renal cortex, medulla and papilla has been measured using three differently sized marker molecules, mannitol, sucrose and inulin. Cellular volumes have been estimated by following the efflux of 3-O-methyl-D-glucose from equilibrated slices. Sucrose appears to be the most accurate extracellular marker in each of the regions examined, in that the sum of its volume of distribution plus cellular volume approximates most closely to the total slice fluid volume. Inulin has the same volume of distribution as sucrose in cortical slices, but under-penetrates medullary and papillary tissue. Mannitol overestimates the extracellular space in all three regions, although its larger volume of distribution, relative to that of sucrose, was not statistically significant in papillary slices. When cell volume and composition are estimated (a) using sucrose as extracellular marker and (b) making appropriate allowance for the presence of bound tissue electrolytes, it is found that cells in each region have low Na+ and high K+ concentrations and contents. When papillary slices are incubated in medium of very high osmolality (NaCl plus urea, 2000 mosmol/kg H2O) there is a moderate (approx. 23%) decrease in cell volume and an increase in cell fluid Na+ and Cl- concentrations equal to approx. 50% of the increase in the extracellular concentrations. Cell K+ concentrations remain unchanged. The results show that cells in renal slices are able to maintain high K+-to-Na+ ratios when incubated in isosmotic (cortex) or moderately hyperosmotic media (medulla and papilla), and suggest that regulation of papillary cell volume following hyperosmotic shock can only partly be ascribed to uptake of extracellular electrolytes.

The influence of age and sex on erythropoietin titers in the plasma and tissue homogenates of hypoxic rats.

Experiments were performed to determine the erythropoietin (Ep) content of homogenates of kidneys and livers of male and female rats of various ages. In all studies, homogenates were adjusted to a concentration of 4 g of tissue per 12 ml of phosphate-buffered-saline, and the stimulus to Ep production consisted of exposure to 0.42 atmosphere for 4 h. The concentration of Ep in kidneys of male rats was about three times that found in those of females and was contained predominantly in the cortical portion of the kidneys. Ep was not detectable in kidneys of rats younger than 3 weeks of age, and reached a maximum concentration after 4 weeks of age. The Ep content of the liver was barely detectable regardless of the age of the rat or its plasma Ep titer; and did not increase significantly by administering angiotensin II or CCl4 (substances which increase extrarenal Ep production).

Glycocalyx heterogeneity of rat kidney urinary tubule: demonstration with a lectin-gold technique specific for sialic acid.

The distribution of sialic acid residues in rat kidney urinary tubule was investigated by light and electron microscopy with a lectin-gold technique. The application of the sialic acid specific Limax flavus lectin resulted in intense plasma membrane labeling of the epithelium of the entire proximal tubule and thin limbs of loop of Henle. In contrast, the plasma membrane of the epithelium lining the medullary portion of the thick ascending limb of Henle was not labeled. In the cortical portion, however, microvilli-bearing positive and smooth-surfaced negative cells were present. Moreover, all cells of the convoluted distal tubules were labeled along their plasma membrane. These data demonstrate the existence of a gross difference in glycocalyx composition between proximal tubules and thin limbs of loop of Henle on one hand and thick ascending limbs on the other. In addition, fine heterogeneity in glycocalyx composition between medullary and cortical portion of thick ascending limb exists. It is concluded that the differences in sialic acid content of the glycocalyx may be related to the functional diversity exhibited by these tubular regions.

Catecholamines in discrete kidney regions. Changes in salt-sensitive Dahl hypertensive rats.

Steady state levels of catecholamines (dopamine, norepinephrine, and epinephrine) were measured by the use of radioenzymatic techniques in discrete areas of the kidney (outer and inner cortex, outer and inner medulla) dissected by a "punch" technique from frozen kidney sections of salt-sensitive (DS) and salt-resistant (DR) Dahl rats fed a low or high salt diet. All three catecholamines were present in all areas of the kidney examined. There were gradients of concentrations of each catecholamine in different kidney areas. Renal medullary areas contained proportionally more dopamine than cortical areas. The proportion of epinephrine with respect to the total catecholamine content was relatively high in the inner medulla. Genetic factors and the amount of dietary salt influenced the catecholamine content in specific kidney areas, and these changes were different according to the area considered. DS rats when fed a high salt diet presented increased systolic blood pressure but no increased levels of dopamine in the inner medulla nor of norepinephrine in the outer medulla and outer cortex. Results suggest that either the uptake, release, storage, synthesis, or catabolism of kidney catecholamines is altered in Dahl salt-sensitive (DS) hypertensive rats and suggest specific roles for each catecholamine in discrete areas of the kidney.

Antihypertensive polar and neutral renopapillary lipids. Which is a hormone?

Two antihypertensive lipids can be derived from the renal papilla, the antihypertensive polar (APRL) and the antihypertensive neutral (ANRL) renomedullary lipid. The renal venous effluent of the unclipped kidney contains both ANRL and APRL. This effluent lowers the arterial pressure (AP) of the normal rat when infused i.v. As it lowers the AP the heart rate (HR) and sympathetic nerve activity (SNA) are depressed. ANRL infused i.v. also lowers HR and SNA as it depresses the AP. Conversely, APRL elevates HR and SNA as it lowers the AP. Thus, of the two lipids in the renal venous effluent after unclipping, ANRL appears to be dominant. APRL, however, in the renal venous effluent could potentiate the action of ANRL. The net effect of these observations is to support the view that ANRL is an antihypertensive hormone liberated by the kidney after unclipping. The renomedullary interstitial cells (RIC) degranulate after unclipping. ANRL can be derived from these cells. Thus, the RIC, cells known to exert an endocrine-type antihypertensive function, may well be the source of ANRL in the renal venous effluent after unclipping. The hormonal action of ANRL appears as a major cause of the lowering of the AP after unclipping. It is not known what factors modulate the RIC endocrine system. There is a suggestion that angiotensin may be one of these factors based on the ineffectiveness of these cells toward retarding hypertension when the circulating plasma angiotensin level is high, and their effectiveness when the circulating plasma angiotensin level is low.

Analogs of phosphatidylcholine: alpha-adrenergic antagonists from the renal medulla.

Antihypertensive polar renomedullary lipid (APRL), a conglomerate of 1-0-alkyl-2-acetoyl-glycero-3-phosphocholine analogs, ws tested in 4- to 6-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats using microcirculatory techniques. APRL (0.5 ug/ml), when added to the solution bathing the cremaster muscle, caused significant changes in the diameter, red blood cell velocity, and blood flow in both groups of rats, for arterioles and venules. Arteriolar changes in diameter were significantly greater (p less than 0.05) in SHR than in WKY. Micropipette application of APRL indicated a dose-dependent response for arterioles and venules in both groups. Moreover, the potent nature of this compound was demonstrated. Relative potency of APRL given intravenously was tested in 10- to 12-week-old SHR and WKY. The response curve was shifted significantly to the left for SHR (p less than 0.01). APRL interaction with known controllers of blood flow was tested in SHR. Blockade of cholinergic, beta-adrenergic, or histaminergic receptors did not inhibit APRL action. blockade of prostaglandin or bradykinin synthesis did not prevent depression of blood pressure by APRL. APRL (40 ug/kg) inhibited (p less than 0.001) the pressor response to norepinephrine (1-10 ug/kg) but not to angiotensin II (4 ug/kg). The present study provides direct evidence that APRL is a vasodilator with increased potency in SHR hypertension. The acute vascular response may be mediated by alpha-adrenergic antagonism.

Catecholamines in kidneys of normotensive and genetically hypertensive rats. Effects of salt load.

The tissue content of norepinephrine, dopamine, and epinephrine was determined in different zones of the kidney in normotensive Sprague-Dawley and Otago Wistar rats and in genetically hypertensive Otago Wistar rats. One kidney in each animal was chronically denervated to allow estimation of the neuronal contribution to renal catecholamine content. In all strains, the renal cortex contained negligible amounts of nonneuronal norepinephrine and dopamine, while outer and inner medullary layers contained progressively larger amounts. Nonneuronal epinephrine was distributed fairly evenly through cortex and medulla. Neuronal norepinephrine content was similar in inner and outer cortex, substantially less in outer medulla, and not discernible in inner medulla. The amounts of neuronal dopamine were consistent with its localization predominantly in noradrenergic nerves. The renal cortices of normotensive Wistar rats contained more neuronal norepinephrine and less neuronal dopamine than those of Sprague-Dawley rats, and the cortices of hypertensive Wistar rats contained slightly more norepinephrine than those of normotensive Wistar rats. In both normotensive strains, long-term salt loading decreased selectively the neuronal norepinephrine in renal cortex. By contrast, in hypertensive animals, cortical norepinephrine was not reduced by salt loading. These results indicate that the genetically hypertensive rat may have an abnormal sympathetic reflex response to increased blood volume.

Purification of Ca2+-activated K+ channel protein on calmodulin affinity columns after detergent solubilization of luminal membranes from outer renal medulla.

A method is developed for purification of the protein of the Ca2+-activated K+ channel from outer renal medulla of pig kidney. The response of this K+ channel to physiological concentrations of Ca2+ is important for regulation of transtubular NaCl transport. In reconstituted vesicles direct addition of calmodulin doubles Ca2+ activation with sufficient affinity (K1/2 0.1 nM) for chromatographic purification of the protein. For purification luminal plasma membrane vesicles are isolated on metrizamide density gradients and solubilized in CHAPS. The fraction of soluble protein retained on calmodulin-Sepharose 4B columns in the presence of Ca2+ and eluted by EGTA is 0.7%. The purified protein has high Ca2+-activated K+ channel activity after reconstitution into phospholipid vesicles. It distributes on two bands of 51 and 36 kDa after gel electrophoresis in SDS. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation of the channel. Phosphorylation from cAMP-dependent protein kinase strongly stimulates Ca2+-activated K+ channel activity and labels the 51 kDa band suggesting that this protein is involved in regulation of K+ channel opening.

Localization of 125I-atrial natriuretic factor (ANF)-binding sites in rat renal medulla. A light and electron microscope autoradiographic study.

Using light and electron microscope autoradiography in vivo, the localization of 125I-(Arg 101-Tyr 126) atrial natriuretic factor (ANF)-binding sites was studied in the renal medulla of rats. At the light microscopic level, the autoradiographic reaction was mainly distributed in patches in the outer medulla, and followed the tubular architecture in the innermost part of the inner medulla. At the electron microscopic level, binding sites were mainly found in the outer medullary descending vasa recta and inner medullary collecting ducts. These results suggest that, in rats, the renal medulla may participate in the natriuresis and diuresis produced by ANF through vascular and tubular effects; the former by changing medullary blood flow at the level of descending vasa recta and the latter by acting on electrolyte and water transport at the level of collecting ducts.

Immunocytochemical localization of epidermal growth factor in mouse kidney.

Epidermal growth factor (EGF) was originally isolated from mouse submandibular glands (SMG). However, SMG removal failed to lower circulating EGF, and large amounts of EGF have been found in mouse urine. In addition, the presence of pre-pro-EGF mRNA in mouse kidney has recently been reported by others. Kidneys may therefore represent an alternate source of EGF. In the present study, we investigated the immunocytochemical localization of EGF in mouse kidney. Male and female adult Swiss Webster mice were fixed by perfusion with 4% paraformaldehyde or Zamboni's fixative, the kidneys were frozen, and serial sections were obtained. Rabbit EGF antiserum was used for the primary incubation and the avidin-biotin complex immunoperoxidase procedure was utilized for immunostaining. EGF was immunolocalized in the apical portion of the cells lining the thick ascending limb of Henle (TALH) and the distal convoluted tubule (DCT). The macula densa, in contrast, lacked EGF immunoreactivity. No sex differences were observed in the distribution pattern or intensity of immunostaining. Infusion of EGF into sheep renal artery has been reported to induce changes in urine flow and ionic composition. Immunolocalization of EGF in the TALH and DCT documented here supports a regulatory role for EGF in the function of the mouse distal nephron.

Immunocytochemical demonstration of vasopressin binding in rat kidney.

We investigated the immunoperoxidase demonstration of vasopressin (VSP) bound to paraffin-embedded sections of rat kidney and the effects of various fixatives. Slices of rat kidney from normal and 4-day water-deprived rats were incubated with 10(-7) M VSP, fixed, and embedded in paraffin. Hydrated sections of these tissues were again incubated with 10(-7) M VSP or 10(-7) M VSP and 10(-5) M oxytocin (OXY). VSP bound to the sections was demonstrated using rabbit anti-Arg8 VSP antiserum and peroxidase-labeled second antibody. In sections of kidney from both normal and water-deprived rats, immunoperoxidase labeling was most intense in the renal papilla and was restricted to the cells of the ducts of Bellini and loops of Henle. In the medulla, the collecting ducts and medullary thick ascending limbs of Henle were moderately stained. In the normal kidney sections there was no staining of the proximal tubules, distal convoluted tubules (DCT), and only slight staining of the cortical collecting ducts (CCD). However, in the water-deprived rats there was a considerable increase in the staining of the DCT and CCD. Simultaneous incubation in OXY and VSP resulted in reduced immunoperoxidase labeling of the tubules. Omission of VSP incubation led to a similar decrease in stain intensity, indicating a specificity for the sites of VSP binding. This technique allows the identification of cells responsible for the binding of VSP in the kidney.

Immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues.

Epidermal growth factor (EGF) stimulates the growth of many tissues and inhibits stimulated gastric acid secretion. Its primary tissue of origin in man is still unknown. We used polyclonal anti-human EGF sera in the peroxidase-antiperoxidase immunocytochemical staining technique to identify immunoreactive human EGF (ihEGF) in tissue sections from 29 subjects ranging from fetuses to 63 years in age. In addition to acinar cells in the submandibular salivary glands and cells of Brunner's duodenal glands, previously reported to contain ihEGF, we found ihEGF in most anterior pituitary glycopeptide hormone-secreting cells, in gastric and pyloric gland cells of the stomach, and in bone marrow cells that resembled mononuclear phagocytes in subjects of all ages. The eccrine sweat glands in the skin of adults also contained ihEGF. Cells containing ihEGF were found singly or in clusters in the trachea of the fetus only. No fetal pancreatic islet cells stained, but occasional cells in neonates and a majority of islet cells in older subjects contained ihEGF; there was no constant association with insulin, glucagon, or somatostatin. Only the lactating breast contained ihEGF. In adults, outer adrenomedullary cells contained ihEGF. Intense immunostaining was observed in the renal medulla, apparently limited to the extracellular area between the renal tubules, and increased with age; the cortex was devoid of ihEGF. No ihEGF was detected in posterior pituitary gland, thyroid gland, heart, lung, or liver at any age. An adult prostate contained ihEGF only in an area of local injury, and some primordial follicles from the ovary of a newborn appeared to contain ihEGF. Thus, many tissues appear to synthesize hEGF, which may exert exocrine, endocrine, or paracrine functions in different tissues and at different ages.

Prostaglandin E2 binding sites in bovine iris-ciliary body.

In the eye, prostaglandins (PGs), in particular PGE2 and PGF2 alpha, may induce vasodilation, disruption of the blood-aqueous barrier, and biphasic effects on intraocular pressure, depending on the species. The initial event leading to many of these physiologic responses is the interaction between the PG and a receptor. We have explored the specificity and selectivity of PGE2 receptors in bovine iris-ciliary body (ICB) membrane preparations. Pigment-free bovine ICB membranes were prepared by high-speed sucrose density-gradient centrifugation. Membranes were incubated with 1 nM 3H-PGE2 in the presence or absence of varying concentrations of unlabeled PGE2 or F2 alpha. Binding of 3H-PGE2 to membranes at 37 degrees C increased linearly with protein concentration, and binding reached equilibrium in 30 min. Specific PGE2 binding represented 80% of total 3H-PGE2 binding. Studies with unlabeled PGE2 or F2 alpha, as competing ligands, showed a dose-dependent inhibition of 3H-PGE2 specific binding. The IC50 for unlabeled PGE2 and F2 alpha was 3 and 379 nM, respectively, which suggests a 100-fold greater selectivity of the binding sites for PGE2 over F2 alpha. Scatchard analysis of saturation data revealed a mean Kd value of 13.3 nM with a Bmax of 156 fmoles bound/mg protein. The general linearity of our Scatchard plots tends to suggests a single class of binding sites for PGE2, although more than a single binding site could be present. These results indicate that binding sites selective for PGE2 exist in the bovine ICB.

Effect of protein intake and urea on sodium excretion during inappropriate antidiuresis in rats.

Administration of urea to patients with the syndrome of inappropriate antidiuresis (SIAD) is thought to ameliorate hyponatremia by both producing an osmotic diuresis and diminishing ongoing natriuresis. The present study evaluated these effects in a rat model of SIAD utilizing dilutional hyponatremia induced by continuous infusion of 1-deamino-[8-D-arginine] vasopressin. Following 48 hours of sustained hyponatremia, separate groups of rats were then refed with either: (1) 5% dextrose alone, (2) a 20% protein chow, (3) an isocaloric protein deficient (0%) chow, or (4) the isocaloric protein-deficient chow supplemented with oral urea. Our results demonstrate that rats refed a 20% protein diet significantly improved their plasma [Na+] as compared to rats refed protein deficient diets, and this improvement was accompanied by decreases in natriuresis despite an increased glomerular filtration rate and an unchanged negative free water clearance. Identical effects were observed in rats refed a protein deficient diet but supplemented with oral urea, suggesting that urea generation from catabolism of dietary protein is responsible for the effect of protein refeeding to decrease urinary sodium excretion. Both the protein and urea refed rats had significantly higher inner medullary urea contents and concentrations compared to rats refed protein-deficient diets and also to rats studied immediately before protein refeeding, supporting the hypothesis that urea and dietary protein decrease natriuresis in patients with SIAD in association with increased inner medullary urea concentrations.