Biomedical subjects
Publications and source records attributed to D D Fanestil.
Many commercial preparations of [32P]orthophosphate contain radioactive impurities that interfere with binding and transport studies in biological systems. One type of impurity is micro-particulate whereas another may be pyrophosphate. Methods of removing these impurities from radiolabeled orthophosphate solutions are described.
[3H]Aldosterone binds with high affinity to Type I corticosteroid receptors in cytosols from adrenalectomized rat forebrains. Physicochemical parameters of these receptors were determined in the presence of molybdate, which stabilized receptors and maintained them in a presumably untransformed state. The Stokes' radius of the molybdate-stabilized receptor was 8.1 nm, as determined by gel filtration on Sephacryl S-300. Its sedimentation coefficient was 9.1S in linear sucrose density gradients. The receptor is asymmetric, with an axial ratio of 8-10 and an apparent mol. wt of 303,000 dalton. The [3H]aldosterone-receptor complex is anionic and elutes from DEAE-Trisacryl in a single peak with a maximum at 160 mM KCl. Exposure to heat or salt in the absence of molybdate, conditions which transform other steroid receptors to smaller DNA-binding forms, causes marked instability of the [3H]aldosterone-receptor complex. The [3H]aldosterone-binding protein of rat forebrain, which displays the binding characteristics of a renal Type I (mineralocorticoid) receptor, is similar in size, shape and charge to the molybdate-stabilized oligomeric forms of other steroid hormone receptors.
Although synthetic atrial natriuretic peptides (ANP) have potent natriuretic/diuretic properties, the renal mechanisms underlying these effects are not yet clearly defined. To determine the possible sites of action for ANP within the kidney, we have localized 125I-ANP binding sites by in vitro autoradiography. Slices of rat kidney were incubated with 150 pM 125I-ANP [alpha-rat ANP (alpha-rANP), 28 amino acids] alone or in the presence of 10 nM or 1 microM unlabeled ANP. Autoradiography was performed using LKB Ultrofilm or emulsion-coated cover slips together with histochemical/immunohistochemical staining of specific tubular segments. High-affinity 125I-ANP binding sites were concentrated over glomeruli and to a lesser extent over the arterial vasculature. Lower-affinity binding sites were seen over proximal tubules and inner medullary collecting ducts. The remainder of the nephron was unlabeled. The localization of high-affinity 125I-ANP binding sites in rat kidney is consistent with a glomerular filtration/hemodynamic mechanism as the principal means by which ANP promotes natriuresis/diuresis.
Proteins from brush-border membrane vesicles of rabbit kidney cortex were solubilized with 1% octylglucoside (protein to detergent ratio, 1:4 (w/w). The solubilized proteins (80.2 +/- 2.3% of the original brush-border proteins, n = 10, mean +/- S.E.) were reconstituted into artificial lipid vesicles or liposomes prepared from purified egg yolk phosphatidylcholine (80%) and cholesterol (20%). Transport of Pi into the proteoliposomes was measured by rapid filtration in the presence of a Na+ or a K+ gradient (out greater than in). In the presence of a Na+ gradient, the uptake of Pi was significantly faster than in the presence of a K+ gradient. Na+ dependency of Pi uptake was not observed when the liposomes were reconstituted with proteins extracted from brush-border membrane vesicles which had been previously treated with papain, a procedure that destroys Pi transport activity. Measurement of Pi uptake in media containing increasing amounts of sucrose indicated that Pi was transported into an intravesicular (osmotically sensitive) space, although about 70% of the Pi uptake appeared to be the result of adsorption or binding of Pi. However, this binding of Pi was not dependent upon the presence of Na+. Both Na+-dependent transport and the Na+-independent binding of Pi were inhibited by arsenate. The initial Na+-dependent Pi transport rate in control liposomes of 0.354 nmol Pi/mg protein per min was reduced to 0.108 and 0 nmol Pi/mg protein per min in the presence of 1 and 10 mM arsenate, respectively. Future studies on reconstitution of Pi transport systems must analyze and correct for the binding of Pi by the lipids used in the formation of the proteoliposomes.
Madin-Darby canine kidney (MDCK) cells express a high density of binding sites (Bmax = 0.67 pmol/10(6) cells) for [3H]RO 5-4864, a peripheral-type benzodiazepine (BZD) receptor ligand. Receptor affinity (Kd = 45 nM) in MDCK cells is 30-50 fold lower than in rat kidney, but its pharmacological specificity is identical to that of peripheral-type BZD receptors in the rat kidney (PK 11195 greater than RO 5-4864 greater than diazepam = flunitrazepam greater than clonazepam). The MDCK cell line should provide a useful model system for studying the role of peripheral type BZD receptors in renal function.
Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.
The localization of benzodiazepine (BZD) receptors in the rat kidney was studied by autoradiography after in vitro labeling of kidney slices with flunitrazepam. The affinity, density, and rank order of displacement of [3H]-flunitrazepam by several BZDs (RO 5-4864 greater than diazepam greater than clonazepam) demonstrated that binding was to BZD receptors of the peripheral type. In autoradiograms obtained with tritium-sensitive film, a high density of silver grains was obtained in the outer medulla, with lower densities in the cortex. Binding was absent from the inner medulla (papilla). In higher resolution autoradiograms obtained with an emulsion-coated cover slip procedure, silver grains were seen to be concentrated over a tubular element in both outer medulla and cortex, identifiable by morphology and distribution as the thick ascending limb of the loop of Henle and the distal convoluted tubule. The identity of the labeled tubules was confirmed by immunofluorescent localization in adjacent slices of Tamm-Horsfall protein, a specific marker for these segments of tubules. Investigation of the effects of peripherally specific BZDs such as RO 5-4864 on distal tubule function is indicated.
Several endogenous substances that inhibit central-type benzodiazepine (BZD) receptor binding have recently been identified. We have found that ultrafiltrates of human uremic plasma, normal plasma, and urine contain competitive inhibitors of peripheral-type benzodiazepine receptors. Using urine as source, we have partially purified a peripheral-type BZD receptor inhibitor(s) by adsorption to and selective elution from small octadecyl-silane (Sep-pak) columns and thin layer chromatography. The inhibitor has a 125-fold greater affinity for peripheral-type than central-type BZD receptors and has been purified 8000-fold from urine.
The acute effects of mineralo- and glucocorticoids on urinary electrolyte excretion were studied in the conscious, acutely potassium deprived, adrenalectomized rat. Sodium, potassium, and creatinine were measured in the urine excreted from 2.5 to 5.5 h after injection of one or more of the following steroids: aldosterone (Aldo), 9-alpha fluorocortisol (FC), deoxycorticosterone (DOC), dexamethasone (Dex), and spironolactone (Spiro). The hierarchy (a) for increasing creatinine excretion was Dex greater than FC greater than Aldo greater than DOC greater than Spiro greater than none, a hierarchy consistent with glucocorticoid potency; and (b) for producing anti-natriuresis was Aldo greater than DOC greater than or equal to FC greater than or equal to none = Spiro greater than Dex, a hierarchy consistent with mineralocorticoid potency. In contrast, the kaliuresis produced by mineralo- and glucocorticoids appears different. A "mineralocorticoid" kaliuresis is 1) elicited by anti-natriuretic doses of Aldo and FC, 2) approximately twice control UKV, 3) unrelated to changes in glomerular filtration rate (GFR), and 4) inhibited by Spiro. A "glucocorticoid" kaliuresis is 1) elicited by Dex and high doses of Aldo and FC, 2) about seven to twenty-fold greater than control UKV, 3) possibly dependent, in part, on changes in GFR, and, 4) not inhibited by Spiro. DOC was not kaliuretic at anti-natriuretic doses. The urinary Na/K ratio was an unreliable index of mineralocorticoid action.
Mucosal addition of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and some lipid-soluble carbodiimides, agents which are selective for carboxyl groups, irreversibly inhibited Na+ transport as measured by short-circuit current (SCC) in the urinary bladder of the toad. The inhibition of Na+ transport by EEDQ had the following characteristics: 1) the inhibition was accompanied by a significant increase in the transepithelial electrical resistance; 2) the decrease in SCC was accounted for by a comparable decrease in 22Na+ influx without effect on Na+ efflux; 3) amphotericin B produced complete recovery of SCC inhibited with EEDQ but not with antimycin A or ouabain; 4) mucosal EEDQ decreased the amiloride-sensitive reversal of Na+ current that is induced by serosal nystatin in the absence of mucosal Na+; 5) vasopressin and acid mucosal pH caused an increase in SCC in proportion to the SCC remaining after EEDQ inhibition; and 6) Vmax of the SCC was decreased without alteration in the apparent Km for Na+. Based on these characteristics of EEDQ inhibition of Na+ transport, we infer that a carboxyl group of the Na+ channel is involved in the Na+-entry step across the apical membrane of "tight" epithelia. The inhibition of Na+ transport with EEDQ most likely involves closing the Na+ channel through a chemical reaction involving a carboxyl group of the channel.
Two titratable groups, with effect on Na+ transport and with apparent acid dissociation constants (pKaS) of 4.2 and 6.7, were found in the apical membrane of toad urinary bladder and are tentatively identified as a carboxyl and an imidazole. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a reagent selective for carboxyl residues, inhibits Na+ transport in the urinary bladder of toads. The underlying chemical reaction whereby EEDQ produces inhibition through potential modification of carboxyl residues was studied. The inhibitory action of EEDQ on Na+ transport was dependent on pH of reaction media and availability of nucleophile, indicating that formation of a covalent acyl-nucleophile bond is probably involved in the irreversible inhibition of Na+ transport. The kinetics of the inhibition showed a stoichiometry of formation of one acyl-nucleophile bond per closure of one Na+ transport site, presumably the Na+ channel. The nucleophile that appears to be involved in the formation of the acyl-nucleophile bond was tentatively identified as having an apparent pKa of 6.7. Amiloride and two analogues of amiloride added to the mucosal Ringer solution (but not serosal amiloride) protected against inhibition of Na+ transport by EEDQ--a finding consistent with the hypothesis that the EEDQ-activated carboxyl group undergoes reaction with a nucleophile at or near the site of specific binding of amiloride onto the apical membrane, most likely at the Na+ channel. Our findings led us to postulate that amiloride must interact with at least two sites on the Na+ channel in order to block the channel. One of the two sites appears to be an ionic interaction between the anionic carboxyl group at the Na+ channel and the cationic guanidinium group of amiloride.
We investigated the effect of the carboxyl-selective reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on aldosterone stimulation of Na+ transport in the urinary bladder of the toad. Na+ transport, measured as the short-circuit current (SCC), was irreversibly inhibited by EEDQ in a dose- and time-dependent manner prior to addition of aldosterone. The greater the percentage inhibition by EEDQ (X), the smaller was the maximal increase of SCC after aldosterone (Y). This relationship gave the regression equation Y = 128.41 - 1.73X, r = -0.99 (n = 35). Evidence that the inhibition of SCC produced by EEDQ was limited to effects at the mucosal membrane was attested by the following: 1) EEDQ did not alter the stimulation by aldosterone of the osmotic water flow response to antidiuretic hormone; 2) whereas inhibition of protein synthesis by cycloheximide prevented this effect of aldosterone; 3) amphotericin B fully restored SCC previously inhibited by EEDQ to the level produced in tissues not inhibited by EEDQ; 4) comparison of the effects of amiloride vs. EEDQ pretreatment on the SCC response to aldosterone and amphotericin B revealed nearly identical characteristics; 5) in contrast, amphotericin B stimulation of SCC was limited when Na+ transport was limited by antimycin A (an inhibitor of energy production) or by ouabain. The findings fail to provide positive evidence for the hypothesis that aldosterone induces the synthesis of new Na+ channels but are consistent with hormonal activation of previously existing but nonfunctioning Na+ channels.
The two [3H]aldosterone-binding proteins of rat brain cytosol were characterized by a dextran-coated charcoal method. With molybdate present to stabilize receptors, the affinities of the two sites for [3H]aldosterone in adrenalectomized perfused rat brain cytosols were 0.28 and 18.0 nM at 4 C. High affinity sites comprised 15% of the total receptor number. A small contamination of perfused brain cytosol preparations with corticosteroid-binding globulin (CBG) was found. However, due to the very high affinity of CBG for corticosterone at 4 C, this slight contamination resulted in significant alterations in the apparent affinity of steroids competing for aldosterone-binding sites. Selective precipitation of cytosol receptors with 36% (NH4)2SO4 reduced CBG concentrations to negligible levels. After blockade of low affinity sites with a highly selective glucocorticoid (RU 26988), the order of steroids in competing for the high affinity receptor was desoxycorticosterone greater than fludrocortisone greater than corticosterone greater than aldosterone greater than progesterone greater than dexamethasone. Readdition of a small quantity of dialyzed serum to cytosol preparations yielded a profile of steroid binding similar to that of the kidney mineralocorticoid receptor (aldosterone greater than desoxycorticosterone greater than corticosterone). The distribution of both receptors in brain regions of adrenalectomized rats was determined. Both receptors were at greatest density in the hippocampus and lowest density in the hypothalamus. The high affinity site was at greatest density in limbic regions, whereas the low affinity receptor, apparently identical to the glucocorticoid type II receptor, was at greatest density in cortex and cerebellum. It is concluded that the high affinity aldosterone receptor of rat brain, which had been identified in preliminary studies as a mineralocorticoid receptor, may bind either corticosterone or aldosterone in vivo.
A proteolipid that binds inorganic phosphate with high affinity and specificity has been extracted from rabbit kidney brush-border membranes. This proteolipid has been partially purified by chromatography on LH-20. The molecular weight of the proteolipid is approximately 3000 as determined by urea-sodium dodecyl sulfate gel electrophoresis. This proteolipid can bind and transport phosphate into an organic phase. The K0.5 for phosphate binding is 8 microM with a Hill coefficient of 1.92. Arsenate inhibits phosphate binding in a competitive manner with a KI of 27.5 microM. The aminoreactive reagent 2,4-dinitrofluorobenzene inhibits phosphate binding to the proteolipid. Similarly, 2,4-dinitrofluorobenzene inhibited Na+-driven Pi uptake in renal brush-border membrane vesicles. In contrast to the mitochondrial phosphate binder, this proteolipid is not inhibited by sulfhydryl reagents. We suggest that this molecular species is a likely candidate for involvement in phosphate uptake in the renal tubule.
Aldosterone stimulates the reabsorption of sodium across epithelial cells of various target tissues. The initial events in the molecular action of the mineralocorticoid are the following: (1) Diffusion of the steroid across the cellular (baso-lateral, serosal) plasma membrane into the cytoplasmic compartment. (2) Binding of the steroid to a receptor protein specific for the class of steroid and activation of this steroid-receptor-complex. (3) Translocation of the activated aldosterone-receptor complex to the nucleus and stimulation of RNA synthesis (including the synthesis of messenger RNA and ribosomal RNA). (4) Translation of the steroid-induced messenger RNAs at the ribosomal level into the aldosterone-induced proteins (AIP) within the cytoplasmic compartment. Whereas these induction steps are uniformly accepted, the mechanisms by which the AIPs increase the activity of a rate-limiting step in the sodium transport process are still object of debate. In this paper we discuss the initial events in the mode of action of aldosterone and the biochemical and physiological approaches to the aldosterone-induced proteins with special reference to the "sodium permease", the "energy", and the "sodium pump" theory. Our analysis shows that despite serious efforts by multiple laboratories, the first AIP with an established relationship to the mineralocorticoid actions of aldosterone is yet to be identified.
Explore the source record for details and available documents.
Rat alpha-fetoprotein (AFP) contains a site that both binds the protease substrate tryptophan methyl ester (TrpOMe) and influences estrogen binding. We have studied the effect of changing the amino acid and ester portions of this compound on its binding to AFP, as measured by the ability of the ester to inhibit binding of [3H]-estrone to AFP. We find that: (1) AFP binds tryptophan esters better than phenylalanine or tyrosine esters, (2) substitution of butyl or benzyl for the methyl group in TrpOMe increases binding for AFP by 30- to 100-fold, (3) substitution of p-nitrophenol at the ester position increases binding for AFP by 10(5), (4) p-nitrophenyl substitution in the ester position of tyrosine or phenylalanine methyl ester increases the affinity for AFP by 10(3) to 10(4), (5) inhibition of estrogen binding to AFP by tryptophan p-nitrophenyl esters is reversible and competitive, and (6) the hydrolysis products of tryptophan p-nitrophenyl ester are ineffective in inhibiting estrogen binding to AFP. Based on these results, we suggest that AFP contains a tryptophan ester recognition site which binds p-nitrophenyl esters with high affinity and influences estrogen binding. The ester binding site may be located spatially near the estrogen binding site.
1 The effects of u.v. irradiation at 254 nm and 350 nm on sodium transport across frog skin epithelium have been investigated. 2 Irradiation at 254 nm but not at 350 nm produces a dose-dependent, functionally selective blockade of sodium transport. The effect is apparently due to the irreversible closure of apical sodium channels. 3 The amiloride-sensitive conductance was directly related to sodium transport as measured by short circuit current (SCC) both in normal and irradiated tissues, although both conductance and current were reduced in irradiated tissues. 4 The sensitivity of epithelia to irradiation at 254 nm was defined from the rate constants for the decline in SCC during three 2 min periods of irradiation at 1850 microW cm-2. The rate constant for the initial 2 min irradiation was 0.093 +/- 0.008 min-1. 5 Lowering the sodium concentration to 5.5 mM from 110 mM increased the rate constant to 0.141 +/- 0.014 min-1, consistent with the view that more functional sodium channels exist at lowered sodium concentration. 6 Lowering the temperature to 7 degrees C from 23 degrees C reduced the rate constant to 0.032 +/- 0.007 min-1 suggesting that blockade of channels is not due to a direct interaction with photons. 7 Using a variety of experimental protocols we were unable to demonstrate that bromamiloride or iodoamiloride can act as photoligands for sodium channels in the epithelium of Rana temporaria. This is in contrast to earlier reports with other epithelia.