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M L Zeidel

Publications and source records attributed to M L Zeidel.

At least 73 records · Page 4Linked to original sources
PubMed1992

Purification and partial characterization of candidate antidiuretic hormone water channel proteins of M(r) 55,000 and 53,000 from toad urinary bladder.

Antidiuretic hormone (ADH) increases toad bladder granular cell apical membrane osmotic water permeability (Pf) by insertion of cytoplasmic vesicles containing water channels into the apical membrane. Termination of ADH stimulation results in endocytosis of water channel-containing membrane. In previous work, we have purified water channel-containing vesicles and demonstrated that they contain 12 major protein bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of vectorial labeling studies of granular cells and purified vesicles, we have proposed previously that vesicle proteins of 55, 53, and 17 kDa are ADH water channel components. In this report, we have purified and analyzed these three proteins using a combination of SDS-PAGE, peptide mapping, amino acid composition, and amino-terminal analyses. The 55- and 53-kDa proteins are distinct protein species possessing a high degree of structural similarity. Both possess a large content of cysteine. The 17-kDa protein appears to be a proteolytic fragment of the 53-kDa protein. None of these three proteins is phosphorylated or contains large amounts of covalently linked carbohydrate. ADH-elicited Pf is inhibited by the organic mercurial reagent fluorescein mercuric acetate (FMA). Exposure of water channel-containing vesicles to FMA labels selectively four vesicle proteins of 92, 55, 53, and 29 kDa while reducing vesicle Pf by 82%. The combination of FMA and 2-mercaptoethanol or exposure to another mercurial reagent, n-ethylmaleimide, does not inhibit vesicle Pf. Together, these data provide additional evidence for the role of the 55- and 53-kDa proteins as components of the ADH water channel. These candidate ADH water channel proteins are distinct from a 28-kDa candidate water channel protein (CHIP 28) isolated recently from human erythrocyte membranes and kidney proximal tubule by Agre and co-workers (Preston, G. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387).

Amino Acids↗
PubMed1992

Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein.

Water rapidly crosses the plasma membranes of red blood cells (RBCs) and renal tubules through highly specialized channels. CHIP28 is an abundant integral membrane protein in RBCs and renal tubules, and Xenopus laevis oocytes injected with CHIP28 RNA exhibit high osmotic water permeability, Pf [Preston et al. (1992) Science 256, 385-387]. Purified CHIP28 from human RBCs was reconstituted into proteoliposomes in order to establish if CHIP28 is itself the functional unit of water channels and to characterize its physiological behavior. CHIP28 proteoliposomes exhibit Pf which is up to 50-fold above that of control liposomes, but permeability to urea and protons is not increased. Like intact RBC, the Pf of CHIP28 proteoliposomes is reversibly inhibited by mercurial sulfhydryl reagents and exhibits a low Arrhenius activation energy. The magnitude of CHIP28-mediated water flux (11.7 x 10(-14) cm3/s per CHIP28) corresponds to the known Pf of intact RBCs. These results demonstrate that CHIP28 protein functions as a molecular water channel and also indicate that CHIP28 is responsible for most transmembrane water movement in RBCs.

Aquaporin 1↗
PubMed1992

Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles.

Unconjugated bilirubin (bilirubin-IX alpha), the hydrophobic end product of heme degradation, is esterified in the hepatocyte endoplasmic reticulum to water-soluble conjugates prior to excretion in bile. To characterize the process of intracellular bilirubin transport, the kinetic and thermodynamic activation parameters for the spontaneous transfer of bilirubin between small unilamellar egg lecithin vesicles were determined. Bilirubin-IX alpha was added to donor vesicles labeled with the fluorescent phospholipid probe, (5-(dimethylamino)naphthalene-1-sulfonyl) dipalmitoyl-L-alpha-phosphatidylethanolamine (dansyl-PE). When bound to the donor vesicles, bilirubin quenches the dansyl probe fluorescence through resonance energy transfer. The movement of bilirubin from dansyl-labeled donor vesicles to unlabeled acceptor vesicles was monitored directly by the reemergence of dansyl fluorescence over time. Vesicle fusion and intervesicle transfer of the dansyl-PE probe were excluded by quasielastic light scattering and fluorescence resonance energy transfer studies. Stopped-flow analysis demonstrated that the transfer of bilirubin was described by a single-exponential function with a mean half-time of 2.0 +/- 0.1 ms (+/- SD) at 37 degrees C. The rate of bilirubin transfer was independent of acceptor vesicle concentration and decreased with increasing buffer ionic strength, indicating that intermembrane transfer occurred via aqueous diffusion, rather than vesicle collisions. The free energy of activation (delta G++) for the dissociation of bilirubin from donor vesicles was 14.2 kcal.mol-1. These studies suggest that bilirubin is associated with phospholipid bilayers at the membrane-water interface. We postulate that the movement of unconjugated bilirubin between intracellular membranes occurs via spontaneous transfer through the aqueous phase.

Bilirubin↗
PubMed1992

Role of glucose carrier in human erythrocyte water permeability.

Although the transport properties of human erythrocyte water channels have been well characterized, the identity of the protein(s) mediating water flow remains unclear. Recent evidence that glucose carriers can conduct water raised the possibility that the glucose carrier, which is abundant in human erythrocytes, is the water channel. To test this possibility, water permeabilities and glucose fluxes were measured in large unilamellar vesicles (LUV) containing human erythrocyte lipid alone (lipid LUV), reconstituted purified human erythrocyte glucose carrier (Glut1 LUV), or reconstituted glucose carrier in the presence of other human erythrocyte ghost proteins (ghost LUV). In glucose and ghost LUV, glucose carriers were present at 25% of the density of native erythrocytes, were oriented randomly in the bilayer, and exhibited characteristic inhibition of glucose flux when exposed to cytochalasin B. Osmotic water permeability (Pf, in centimeters per second; n = 4) averaged 0.0012 +/- 0.00033 in lipid LUV, 0.0032 +/- 0.0015 in Glut1 LUV, and 0.006 +/- 0.0014 in ghost LUV. Activation energies of water flow for the three preparations ranged between 10 and 13 kcal/mol; p-(chloromercuri)benzenesulfonate (pCMBS), an organic mercurial inhibitor of erythrocyte water channels, and cytochalasin B did not alter Pf. These results indicate that reconstitution of glucose carriers at high density increases water permeability but does not result in water channel activity. However, because the turnover number of reconstituted carriers is reduced from that of native carriers, experiments were also performed on erythrocyte ghosts with intact water channel function. In ghosts, Pf averaged 0.038 +/- 0.013 (n = 9), while the activation energy for water flow averaged 3.0 +/- 0.3 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Glucose↗
PubMed1992

Cellular NH4+/K+ transport pathways in mouse medullary thick limb of Henle. Regulation by intracellular pH.

Fluorescence and electrophysiological methods were used to determine the effects of intracellular pH (pHi) on cellular NH4+/K+ transport pathways in the renal medullary thick ascending limb of Henle (MTAL) from CD1 mice. Studies were performed in suspensions of MTAL tubules (S-MTAL) and in isolated, perfused MTAL segments (IP-MTAL). Steady-state pHi measured using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) averaged 7.42 +/- 0.02 (mean +/- SE) in S-MTAL and 7.26 +/- 0.04 in IP-MTAL. The intrinsic cellular buffering power of MTAL cells was 29.7 +/- 2.4 mM/pHi unit at pHi values between 7.0 and 7.6, but below a pHi of 7.0 the intrinsic buffering power increased linearly to approximately 50 mM/pHi unit at pHi 6.5. In IP-MTAL, NH4+ entered cells across apical membranes via both Ba(2+)-sensitive pathway and furosemide-sensitive Na+:K+(NH4+):2Cl- cotransport mechanisms. The K0.5 and maximal rate for combined apical entry were 0.5 mM and 83.3 mM/min, respectively. The apical Ba(2+)-sensitive cell conductance in IP-MTAL (Gc), which reflects the apical K+ conductance, was sensitive to pHi over a pHi range of 6.0-7.4 with an apparent K0.5 at pHi approximately 6.7. The rate of cellular NH4+ influx in IP-MTAL due to the apical Ba(2+)-sensitive NH4+ transport pathway was sensitive to reduction in cytosolic pH whether pHi was changed by acidifying the basolateral medium or by inhibition of the apical Na+:H+ exchanger with amiloride at a constant pHo of 7.4. The pHi sensitivities of Gc and apical, Ba(2+)-sensitive NH4+ influx in IP-MTAL were virtually identical. The pHi sensitivity of the Ba(2+)-sensitive NH4+ influx in S-MTAL when exposed to (apical+basolateral) NH4Cl was greater than that observed in IP-MTAL where NH4Cl was added only to apical membranes, suggesting an additional effect of intracellular NH4+/NH3 on NH4+ influx. NH4+ entry via apical Na+:K+ (NH4+):2Cl- cotransport in IP-MTAL was somewhat more sensitive to reductions in pHi than the Ba(2+)-sensitive NH4+ influx pathway; NH4+ entry decreased by 52.9 +/- 13.4% on reducing pHi from 7.31 +/- 0.17 to 6.82 +/- 0.14. These results suggest that pHi may provide a negative feedback signal for regulating the rate of apical NH4+ entry, and hence transcellular NH4+ transport, in the MTAL. A model incorporating these results is proposed which illustrates the role of both pHi and basolateral/intracellular NH4+/NH3 in regulating the rate of transcellular N H4+ transport in the MTAL.

Amiloride↗
PubMed1992

Water and nonelectrolyte permeabilities of apical membranes of toad urinary bladder granular cells.

Certain types of epithelial cells such as those lining the toad urinary bladder have been classified as "tight" because their apical membranes exhibit low permeabilities to water, ions, and small nonelectrolytes. However, the permeability properties and structural features of these specialized apical membranes remain unclear because these membranes have never been purified. To isolate toad bladder granular cell apical membranes, we derivatized the bladder apical surface with the membrane-impermeant bifunctional reagent N-hydroxysulfosuccinimydyl-S,S-biotin (NHS-SS-biotin). After cell disruption, these derivatized apical membranes were purified using streptavidin-coated magnetic beads in a magnetic field. With the use of lactoperoxidase-mediated radioiodination as a marker for apical membrane, this preparative procedure purified apical membrane 48- or 72-fold as compared with homogenate. Thin section electron microscopy revealed unilamellar vesicles with some nonvesiculated membranes, while fragments of organelles such as mitochondria were absent. Water and nonelectrolyte permeabilities of purified apical membrane vesicles were similar to those obtained in intact bladders in the absence of antidiuretic hormone stimulation. The results demonstrate that isolated apical vesicles do not contain water channels and confirm the applicability of Overton's rule to the apical membrane of the toad urinary bladder. The technique has general applicability to isolation of other plasma membranes, and the apical membranes obtained are suitable for structural analysis.

Animals↗
PubMed1992

Cytoplasmic dilution induces antidiuretic hormone water channel retrieval in toad urinary bladder.

Antidiuretic hormone (ADH) increases the osmotic water permeability (Pf) of the toad urinary bladder by insertion of water channels into the apical cell membrane. Transepithelial water flow (Jv) reduces Pf by inducing endocytosis of apical water channels despite continuous ADH stimulation. This phenomenon is termed flux inhibition. We wished to determine whether cytoplasmic dilution or transcellular Jv causes flux inhibition because both have been proposed previously as a primary regulatory mechanism for this process. Apical membrane endocytosis was quantified by monitoring the uptake of the fluid phase marker fluorescein isothiocyanate dextran (FITC-dextran). FITC-dextran fluorescence was monitored in Triton X-100 extracts of epithelial cells as the ratio of total tissue fluorescence compared with background fluorescence. The background was defined as cellular autofluorescence and nonspecific tissue staining due to the presence of small amounts of free fluorescein contaminating the FITC-dextran. FITC-dextran uptake measured under symmetric isotonic (220 mosmol/kgH2O) conditions in either the absence (1.0 +/- 0.4 SD; n = 14) or presence (1.3 +/- 0.3; n = 4) of ADH was not statistically different from that of background. In contrast, flux inhibition induced by a 180 mosmol/kgH2O apical-to-basolateral osmotic gradient increased FITC-dextran uptake to 3.4 +/- 1.3 (n = 7). FITC-dextran uptake was identical in bladders exposed to symmetric hypotonic (150 mosmol/kgH2O) solutions during ADH (3.6 +/- 0.9; n = 6) or adenosine 3',5'-cyclic monophosphate (3.1 +/- 0.4 fold; n = 3) stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Functional and structural characterization of endosomes from toad bladder epithelial cells.

Previous functional studies of toad bladder endosomes have been complicated by the presence of multiple endosome subpopulations each possessing different permeability characteristics. To identify and characterize both water channel-containing vesicles (WCV) and other endosome subpopulations, we combined flow cytometry, electron microscopy, stop-flow fluorometry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Flow cytometry of endosomes identified distinct populations of fluorescein-labeled endosomes in bladders after removal of antidiuretic hormone (ADH) stimulation (ADH withdrawal). Centrifugation separated the larger fluorescein-labeled vesicles, sedimenting at lower speed (intermediate pellet, IP), from the smaller fluorescein-labeled vesicles, sedimenting at high speed (high-speed pellet, HSP). Permeability and structural studies of these subpopulations revealed the following. 1) IP endosomes labeled 10 min after ADH withdrawal (ADH IP) represented a highly purified population of WCV with high water permeability (Pf) that exhibited a low-activation energy and sensitivity to organic mercurials. 2) IP endosomes from unstimulated bladders did not contain functional water channels. 3) HSP from either ADH withdrawal or unstimulated bladders exhibited low Pf and acidified after addition of extravesicular ATP; moreover, protein compositions of purified HSP were distinct from those of purified IP. These results suggest that HSPs represent constitutive and not ADH-sensitive endosomes. 4) High permeability to protons (PH+) was seen in ADH IP endosomes but not the other fractions, providing strong evidence that the ADH water channel conducts protons. 5) Multivesicular bodies (MVB) exhibited low Pf and PH+, indicating that they do not possess functional water channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Atrial natriuretic peptide(31-67) inhibits Na+ transport in rabbit inner medullary collecting duct cells. Role of prostaglandin E2.

Atrial natriuretic peptide (ANP)(31-67), a portion of the atrial peptide prohormone, circulates in humans, and its plasma level varies with atrial pressure. Like the more widely studied carboxy-terminal fragment ANP(99-126), ANP(31-67) stimulates natriuresis and diuresis. We examined the mechanism of this natriuresis by measuring the effects of ANP(31-67) on Na+ transport in cells of the rabbit inner medullary collecting duct (IMCD). ANP(31-67) (10(-8) M) caused a 26 +/- 4% inhibition of oxygen consumption (QO2); half-maximal inhibition occurred at 10(-11) M, suggesting a physiologic effect. This effect was not additive with either ouabain or amiloride, suggesting that it reflected inhibition of Na+ transport-dependent QO2. ANP(31-67) reduced the amphotericin-induced stimulation of QO2 consistent with inhibition by this peptide of the Na(+)-K(+)-ATPase. In addition, ANP(31-67) reduced ouabain-sensitive 86Rb+ uptake under Vmax conditions. Several lines of evidence indicated that PGE2, a known endogenous IMCD Na(+)-K(+)-ATPase inhibitor, mediates pump inhibition by ANP(31-67). Thus, ANP(31-67) inhibits Na+ transport by inhibiting the Na(+)-K(+)-ATPase of IMCD cells, an effect mediated by the generation of PGE2.

Amiloride↗
PubMed1991

Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder.

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.

Adenosine Triphosphatases↗
PubMed1991

Interleukin-1 inhibition of Na(+)-K(+)-ATPase in inner medullary collecting duct cells: role of PGE2.

Interleukin-1 (IL-1), a cytokine produced by macrophages, causes an increase in Na+ excretion in experimental animals. Micropuncture studies have determined that the natriuretic effect of IL-1 is largely due to inhibition of Na+ reabsorption in the collecting duct. The current studies made use of suspensions of rabbit inner medullary collecting duct (IMCD) cells to examine the mechanism by which IL-1 regulates Na+ transport. IL-1 reduced ouabain-sensitive 86Rb+ uptake by 48% at 10 s, 36% at 30 s, and 29% at 60 s, suggesting an inhibitory effect on Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity. IL-1 inhibition of 86Rb+ uptake occurred in a dose-dependent manner. This effect appears to be mediated by prostaglandin E2 (PGE2) because 1) ibuprofen blocks the inhibitory effect of IL-1 on IMCD Na(+)-K(+)-ATPase activity, 2) IL-1 and PGE2 cause equivalent and nonadditive inhibition of 86Rb+ uptake, 3) IL-1 causes a two- to threefold increase in PGE2 content in IMCD cells, and 4) dose-response curves were similar for IL-1 stimulation of PGE2 content and inhibition of 86Rb+ uptake in IMCD cells. Thus the natriuretic effect of IL-1 is due, at least in part, to stimulation of PGE2 production by collecting duct cells with resultant inhibition of Na(+)-K(+)-ATPase activity.

Animals↗
PubMed1990

Renal actions of atrial natriuretic peptide: regulation of collecting duct sodium and water transport.

ANP stimulates a profound natriuresis and diuresis by a series of concerted actions along the nephron, including stimulation of glomerular filtration and inhibition of net salt and water reabsorption in the cortical and inner medullary collecting ducts. Several actions of ANP contribute to its natriuretic and diuretic effects in the collecting duct. These include reductions in aldosterone secretion, increases in hydrostatic pressures opposing Na+ reabsorption, possible stimulation of medullary washout, and direct inhibition of salt and water transport. In both CCD and IMCD, ANP antagonizes the hydroosmotic actions of vasopressin, which leads to diuresis. The mechanisms by which ANP inhibits response to vasopressin remain unclear, although in IMCD, cGMP can duplicate the response to ANP. In CCD, ANP can inhibit Na+ reabsorption via cGMP; the transport pathway regulated by ANP is unknown. In IMCD, ANP acting via cGMP inhibits a conductive Na+ or cation channel, which appears to be on the luminal membrane.

Absorption↗
PubMed1990

pH regulation and response to AVP in A10 cells differ markedly in the presence vs. absence of CO2-HCO3-.

The fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to determine the effect of ambient CO2-HCO3- on the regulation of intracellular pH (pHi) and the pHi response to arginine vasopressin (AVP) in A10 vascular smooth muscle (VSM) cells. Steady-state pHi averaged 7.04 +/- 0.02 in the absence and 7.25 +/- 0.01 in the presence of CO2-HCO3-. In the absence of CO2-HCO3-, virtually all (greater than 96%) of the acid extrusion from acidification occurred by amiloride-sensitive Na(+)-H+ exchange. However, in the presence of CO2-HCO3-, acid extrusion after acidification occurred by both Na(+)-H+ exchange and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive Na(+)-dependent Cl(-)-HCO3- exchange. In CO2-HCO3(-)-containing media, amiloride-sensitive Na(+)-H+ exchange mediated 85% of acid extrusion at a pHi of 6.48, but the DIDS-sensitive acid extrusion mechanism (NA(+)-dependent Cl(-)-HCO3- exchange) was the dominant acid extrusion mechanism at a pHi of 6.94. Base exited A10 cells by a DIDS-sensitive process consistent with Na(+)-independent Cl(-)-HCO3- exchange. Both amiloride- and DIDS-sensitive processes regulated steady-state pHi in CO2-HCO3-. AVP (10(-7) M) alkalinized steady-state pHi in the absence of CO2-HCO3- (delta pHi = 0.08 +/- 0.01 pH units) by stimulating Na(+)-H+ exchange; however, AVP did not alter pHi of untreated cells in CO2-HCO3- (delta pHi = -0.01 +/- 0.01 pH units) because of concomitant stimulation of Na(+)-independent Cl(-)-HCO3-exchange. We conclude that the steady-state pHi, the mechanisms of pHi regulation, and the pHi response to AVP in A10 cells are critically influenced by the presence of extracellular CO2-HCO3-. Thus the potential contribution of pHi changes to VSM cell responses to vasoactive agents should be evaluated in the presence of CO2-HCO3-.

Amiloride↗
PubMed1990

Na(+)-H+ antiporter and Na(+)-(HCO3-)n symporter regulate intracellular pH in mouse medullary thick limbs of Henle.

To determine mechanisms of intracellular pH (pHi) regulation in mouse medullary thick limbs (MTAL), pHi was measured in MTAL suspensions and in the isolated perfused MTAL by use of 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF). A method to obtain MTAL suspensions from the mouse outer medulla is reported. Characterization of suspensions with microscopy, anti-Tamm-Horsfall antibody labeling, measurement of O2 consumption, and adenosine 3',5'-cyclic monophosphate responses to antidiuretic hormone indicated that these suspensions were highly purified for viable MTAL tubules. The resting pHi was 7.41 +/- 0.02 (means +/- SE) in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered media and 7.23 +/- 0.02 in CO2- HCO3(-)-buffered media, both at extracellular pH 7.4. MTAL tubules exhibited rapid pHi recovery from intracellular acidification. Recovery of pHi was dependent on luminal Na+ (apparent Km = 13.2 +/- 3.2 mM) and was inhibited by amiloride (apparent Ki = 10.6 microM), consistent with the activity of an apical Na(+)-H+ antiporter. Antiporter activity was enhanced by acidification and was diminished at the resting pHi. Recovery from intracellular alkalinization (rapid withdrawal of CO2- HCO3-) was sensitive to the stilbene anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, Cl(-)-insensitive, and Na(+)-sensitive, consistent with the activity of a Na(+)-(HCO3-)n symporter. Both transporters were significantly involved in steady-state pHi regulation in the presence of CO2- HCO3-. In contrast, the Na(+)-H+ antiporter played the dominant role in steady-state pHi regulation in the absence of CO2- HCO3-.

Animals↗
PubMed1990

Brain natriuretic peptide: interaction with renal ANP system.

Brain natriuretic peptide (BNP) has recently been found in porcine brain and has been shown to cause diuresis and natriuresis when injected in rats, effects similar to those caused by atrial natriuretic peptide (ANP). BNP is also synthesized in the cardiac atria and circulates in plasma. The amino acid sequence of the peptide resembles that of ANP particularly closely within the ring structure of the peptide. We examined the potential role of BNP in modulating renal function by assessing its ability to mimic the effects of ANP on rat glomeruli and in rabbit inner medullary collecting duct cells (IMCD). BNP bound with high affinity to glomeruli (Kd approximately 900 pM) and IMCD cells (Kd approximately 500 pM). In IMCD cells, BNP stimulated particulate guanylate cyclase (approximately 3-fold at maximum ligand concentration) and inhibited conductive 22Na+ uptake by 50% at concentrations at which ANP is also effective. In rat glomeruli, BNP bound with high affinity to the low-molecular-weight receptors but with lesser affinity to the higher-molecular-weight guanylate cyclase-linked receptors (Kd approximately 50 nM). In addition, the guanosine 3',5'-cyclic monophosphate accumulation response was less impressive in glomeruli than the guanylate cyclase response in IMCD tissue. Thus we conclude that BNP is of only slightly reduced affinity and potency for the ANP receptors in the kidney and probably acts through these receptors to exert its physiological effects.

Animals↗
PubMed1990

Mitochondrial injury: an early event in cisplatin toxicity to renal proximal tubules.

Oxygen consumption (QO2) and net K+ transport were studied in rabbit proximal tubule suspensions to define the early effects of cisplatin on proximal tubule function. Cisplatin caused dose-dependent inhibition of QO2, which was delayed in onset. The concentration of cisplatin required for inhibition decreased as the duration of exposure was increased [40-min exposure, threshold concentration of 10(-4) M, inhibitor constant (Ki) of 10(-3) M; 4-h exposure, threshold concentration of 3 X 10(-5) M, Ki of 10(-4) M]. Both ouabain-sensitive and ouabain-insensitive QO2 were reduced, indicating inhibition of all adenosinetriphosphatases, including Na(+)- K(+)-ATPase activity. There was a parallel fall in ouabain-sensitive net K+ transport and cytosolic K+ content, confirming the latter observation. Na(+)-K(+)-ATPase activity was unchanged in cell membranes prepared by hypotonic lysis from cisplatin-treated tubules, indicating an indirect cytosol-dependent mechanism of enzyme inhibition. Nystatin-stimulated QO2 was reduced in cisplatin-treated tubules, excluding inhibition of Na+ entry as the mechanism of injury and suggesting mitochondrial injury. The latter was confirmed by measurement of carbonylcyanide-m-chlorophenylhydrazone (CCCP)-uncoupled QO2 in intact cells and ADP-stimulated (state 3) QO2 in digitonin-permeabilized tubules. Furthermore, by maximally stimulating mitochondrial respiration with CCCP and nystatin, it was possible to demonstrate mitochondrial injury at a time when basal QO2 and K+ transport were apparently normal. These data suggest that mitochondrial injury is a central event in cisplatin toxicity to the proximal tubule.

Adenosine Diphosphate↗
PubMed1990

Kinins inhibit conductive Na+ uptake by rabbit inner medullary collecting duct cells.

Kinins promote natriuresis in vivo, at least in part by altering Na+ transport in the collecting duct. Using freshly prepared suspensions of rabbit inner medullary collecting duct (IMCD) cells, we have examined the effects of kinins on Na+ transport using measurements of oxygen consumption (QO2) and isotopic Na+ uptake. Bradykinin (BK) inhibited IMCD cell QO2 by 24.7 +/- 0.9% without significantly reducing QO2 in cells derived from the outer medullary collecting duct. BK and kallidin half-maximally inhibited QO2 at concentrations in the 10(-12)-10-(-11) M range; beta 1-receptor agonists did not alter QO2, and beta 1-receptor antagonism did not reduce the effect of kinins. These observations indicate that the actions of kinins on IMCD cells are mediated by beta 2-receptors or a distinct subclass. Several observations indicate that kinins reduce QO2 by inhibiting Na+ entry: in the absence of Na+, BK did not reduce QO2; BK inhibition of QO2 was not additive with ouabain, amiloride, atrial natriuretic peptide (ANP), or 8-bromoguanosine 3',5'-cyclic monophosphate and was abolished in the presence of the cation ionophore amphotericin B. Measurements of isotopic Na+ uptake demonstrated that BK reduced the initial rate of Na+ entry by 58%; BK inhibited the amiloride-sensitive component of conductive Na+ uptake. Because ANP inhibits conductive Na+ entry in IMCD cells via stimulation of cGMP accumulation, the effect of BK on cGMP levels was determined. Unlike ANP, BK did not increase cGMP levels, indicating that transport effects of kinins in IMCD are not mediated by cGMP. Thus kinins directly inhibit conductive Na+ entry in IMCD cells at concentrations suggestive of a physiological effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗