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D G Warnock

Publications and source records attributed to D G Warnock.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cytosolic pH sensitivity of an expressed human NHE-1 Na(+)-H+ exchanger.

These studies examined the effects of protein kinase C activation and calmodulin inhibition on the amiloride-sensitive NHE-1 isoform of the Na(+)-H+ exchanger in defined host cells. Our objective was to define differences in the cellular regulatory responses using a specified isoform of the Na(+)-H+ exchanger. Suspended cells were loaded with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and preacidified to a cytosolic pH of 6.2. Wild-type mouse Ltk- cells, human A-431 cells, and mutant mouse fibroblasts stably transfected with the human NHE-1 isoform (LAP+ cells) were examined to define the maximal rate of transport (Vmax) in response to 140 mM external Na+, the Hill stoichiometric coefficient, and the cytosolic pH at which the NHE-1 isoform was half-maximally stimulated (pH50). The mouse NHE-1 isoform had a greater affinity for cytosolic H+ than the human NHE-1 isoforms. Calmodulin antagonism with N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide reduced the Vmax and shifted the pH50 in the acidic direction, especially in the A-431 cells. Protein kinase C stimulation had a similar effect in A-431 cells and little effect in the wild-type (Ltk-) and transfected (LAP+) mouse cells. While the NHE-1 isoform contains several potential phosphorylation sites, the cellular milieu in which the isoform is expressed has an important effect on the modulation of NHE-1 activity.

Amiloride↗
PubMed1993

Amiloride-sensitive sodium conductance in human B lymphoid cells.

The Na+ conductance of RPMI 8226 human B lymphoblastoid cells was examined using whole cell patch clamp. When the bath solution contained RPMI 1640 and the pipette solution contained (in mM) 100 potassium gluconate, 30 KCl, 10 NaCl, 0.5 ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), as well as < 10 nM free Ca2+, pH 7.25, the mean membrane potential was -58 +/- 4.5 mV (n = 18). Cells were voltage clamped from -160 to +40 mV in 20-mV increments. The inward conductance was 767 +/- 221 pS/10 pF, and the outward conductance was 1,212 +/- 272 pS/10 pF (n = 12). Superfusion with 2 microM amiloride significantly hyperpolarized the cells by 7.4 +/- 2.2 mV (P = 0.004), significantly reduced the inward conductance to 221 +/- 65 pS/10 pF (P = 0.028), but had no effect on the outward conductance (1,294 +/- 236 pS/10 pF, P = 0.820, after amiloride). Next, the pipette and bath solutions were changed to (in mM) 150 sodium gluconate, 0.5 EGTA, and 20 HEPES, as well as < 10 nM free Ca2+, pH 7.25. Under these conditions amiloride significantly reduced (50%, P < 0.05; n = 7) the whole cell currents. When potassium gluconate was substituted for sodium gluconate, amiloride had no effect. Thus amiloride inhibited a Na(+)-specific conductance expressed by B lymphoid cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1992

Cloning, sequencing, and expression of Na(+)-H+ antiporter cDNAs from human tissues.

Two types of Na(+)-H+ antiporter with different sensitivities to amiloride analogues have been identified in mammalian plasma membranes. A human Na(+)-H+ antiporter cDNA was obtained by Sardet and co-workers (C. Sardet, L. Counillon, A. Franchi, and J. Pouysségur. Cell 56: 271-280, 1989) using mutant mouse fibroblasts lacking Na(+)-H+ antiporter transformed with human genomic DNA. However, the amiloride sensitivity of this cloned Na(+)-H+ antiporter was not precisely determined. Furthermore, the reported cDNA sequence may be a chimera of human and mouse genes. Hence we isolated a Na(+)-H+ antiporter cDNA actually expressed in human tissues and characterized its amiloride sensitivity. Our 4 kb cDNA obtained from human kidney cortex contained the identical open reading frame to that previously reported and the entire 3' terminus, which was quite different from that reported. This discrepancy was not due to differences in tissue-specific expression because cDNAs from different human tissues were identical, and single bands were observed under high stringency on Northern blots of various human tissues. Na(+)-H+ antiporter activity of mutant mouse fibroblasts deficient in Na(+)-H+ antiporter activity transfected with the cloned cDNA was very sensitive to amiloride and 5-N substituted analogues of amiloride. Thus the cloned cDNA represents the NHE-1 isoform of the Na(+)-H+ antiporter.

Amiloride↗
PubMed1991

IgA nephropathy in blacks: studies of IgA2 allotypes and clinical course.

The prevalence of IgA nephropathy (IgAN) varies among racial groups, being most common among Caucasians and Orientals and rare in Blacks. Other investigators have hypothesized that the risk for IgAN may be influenced by the IgA2 allotype. It has been suggested that the rare Black patients with IgAN may be homozygous for the A2m(1) allele which predominates in Whites, but is less common in Blacks. In a multicenter study, 27 Black IgAN patients were enrolled to investigate this hypothesis and analyze the clinical course of disease in Blacks. The IgA2 allotypes of 18 Black patients and 14 controls were determined using restriction fragment length polymorphism analysis. Three patients were homozygous for the A2m(1) allele, four were homozygous for A2m(2) and 11 were heterozygous. The respective allelic frequencies of A2m(1) and A2m(2) were 0.47 and 0.53 and did not differ significantly from Black controls. Most clinical manifestations of disease did not significantly differ with respect to distribution of the two alleles, although the gender ratio differed between the homozygous A2m(1) and heterozygous patients. The presence of the A2m(1) allele did not increase the risk for IgAN, and the presence of the A2m(2) allele or homozygosity for this allele did not protect Blacks from the development of IgAN.

Adult↗
PubMed1989

Pharmacokinetics of esmolol and ASL-8123 in renal failure.

The effect of renal function on the pharmacokinetics of esmolol, an ultra-short-acting beta-adrenergic blocker, and its major metabolite, ASL-8123, was examined in six healthy control subjects, six patients maintained on hemodialysis, and six patients on continuous ambulatory peritoneal dialysis (CAPD). In addition, the impact of hemodialysis and CAPD on removal of esmolol and ASL-8123 was determined. Multiple blood, urine, and dialysate samples were collected during a 72-hour period and assayed for esmolol and ASL-8123 by HPLC. The pharmacokinetic disposition of esmolol was not significantly altered by renal failure. Mean (+/- SD) total body clearance for esmolol was 171.4 +/- 69.8, 249.8 +/- 176.3, and 265.3 +/- 143.1 ml/min/kg for the control, hemodialysis, and CAPD patients, respectively. Mean elimination half-life (t1/2) was 7.2 minutes in control subjects compared with 7.1 and 8.0 minutes for the hemodialysis and CAPD groups, respectively. The apparent volume of distribution of esmolol did not differ significantly among the three groups. ASL-8123 was shown to accumulate in patients with renal failure, as evidenced by a mean maximum blood concentration of 42.8 +/- 12.2 micrograms/ml in the control group compared with 76.1 +/- 23.9 and 87.1 +/- 20.4 micrograms/ml in the hemodialysis and CAPD groups, respectively (p less than 0.05). The elimination t1/2 of ASL-8123 was prolonged in patients with renal failure, averaging more than 42 hours compared with only 4 hours in the control subjects. Approximately 20% of the esmolol dose as ASL-8123, was removed by either hemodialysis or CAPD, contributing minimally to the elimination of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1989

K-Cl cotransport systems.

The KCl cotransporter in the basolateral membrane of renal tubules may play a central role in the transcellular transport of NaCl. Because this transporter is electrically neutral, and also functions in parallel to the electrogenic Na,K-ATPase, there is an imbalance in charge which must be expressed as a cationic current across the basolateral membrane. Therefore, other pathways must also function in the basolateral membrane which permit the conductive exit of K+ in addition to the electrically-neutral KCl cotransporter. Another functional role for the KCl cotransporter is manifest during the cell volume regulatory response to cell swelling. In this setting (regulatory volume decrease), it appears that both electrically-neutral and electrically-coupled KCl efflux pathways are acutely activated. Very little is known at present about the mechanisms of short and long term regulation of the KCl cotransporter. A major obstacle at this point is the lack of a suitable, potent (that is, microM range) specific inhibitor of this transporter. It also appears that the chloride transport systems in basolateral membrane vesicles may be greatly influenced by the precise details of the method of preparation. Once these experimental details are mastered, and a suitable high affinity inhibitor is identified, then the detailed characterization and identification of the KCl cotransporter can be undertaken.

Animals↗
PubMed1989

Distinct epitopes on amiloride.

Most Na(+)-selective transport proteins are inhibited by the drug amiloride. Studies using amiloride analogues suggest that specific regions of amiloride might participate in binding to receptors on these transport proteins. To determine whether certain domains of this drug are recognized as distinct epitopes, amiloride was coupled to albumin through either its C-5 NH2-group on the pyrazine ring or through a terminal NH2-group of the guanidino moiety, and antibodies were raised against these amiloride-albumin conjugates. Studies of antibody binding to amiloride analogues identified the 3,5-diaminopyrazinyl, the guanidinocarbonyl, and the C-6 halo moieties as distinct epitopes, although the antibodies required the presence of both the 3,5-diaminopyrazinyl as well as the guanidinocarbonyl moiety for binding.

Amiloride↗
PubMed1989

Accumulation of weak base in gastric mucosa provides evidence for an acidic storage compartment.

Uptake and release of acridine orange (AO), a fluorescent weak base that accumulates in acidic spaces, were studied in perfused frog gastric mucosa. Tissue was mounted between two flow-through chambers and loaded with AO on the mucosal side. AO washout and acid secretion rate were monitored simultaneously by a flow-through fluorescence detector and a pH-stat, respectively. Data were displayed on a computer screen, stored, and analyzed. AO, in concentrations as high as 0.02 mM, does not affect the acid secretion rate. Nonlinear least-squares analysis of AO washout curves resolved two exponential components: a faster component associated mainly with AO washout from the chamber and a slower component reflecting primarily AO washout from the tissue. The slower exponential declines more slowly at higher concentrations and/or longer duration of AO loading, whereas the faster exponential is unaffected. AO washout is unaffected by the level of the steady-state acid secretion rate. Nitrite inhibits the acid secretion rate but does not affect the AO washout. When nitrite is removed, acid secretion rate and fluorescence (AO concentration in the mucosal medium) increase simultaneously and transiently. The net amount of AO released from the tissue is proportional to the net amount of acid released. Stimulation by secretagogue in basally secreting tissue causes synchronous transient increases in acid secretion rate and fluorescence. We conclude that accumulation of AO provides evidence for the existence of an intracellular storage pool of free protons within the transporting epithelium.

Acridine Orange↗
PubMed1988

Interactions of chloride and amiloride with the renal Na+/H/ antiporter.

Amiloride is a reversible inhibitor of the Na+/H+ antiporter which acts at the external aspect of the transport system. The kinetics of inhibition of the Na+/H+ antiporter with amiloride have been controversial, with the usual finding of simple competitive inhibition, but with other reports of mixed and noncompetitive inhibition of the transporter by amiloride. The present experiments demonstrate that the chloride content of the external transport buffer affects the kinetics of amiloride inhibition. Either simple competitive or mixed inhibition by amiloride was observed in the same vesicle preparations depending on the presence of chloride or gluconate in the buffer. The effect of chloride on the inhibitory effect of amiloride was dependent on the concentration of chloride and amiloride. Similar effects were observed with more potent analogues of amiloride. These findings suggest that the external aspect of the antiporter has a site or sites at which the inhibitory effects of amiloride on the Na+/H+ antiporter can be modified by chloride, even though chloride has only slight effects on the kinetics of the Na+/H+ antiporter in the absence of amiloride.

Amiloride↗
PubMed1988

Uremic acidosis.

Explore the source record for details and available documents.

Acidosis↗
PubMed1987

The effects of cycloheximide on Na+/H+ antiporter activity in cultured opossum kidney cells.

These studies examined the effects of cycloheximide on the Na+/H+ antiporter in cultured opossum kidney cells. The effects of cycloheximide on antiporter activity depended on the basal level of activity. These data suggest that the Na+/H+ antiporter may be regulated by several processes which are sensitive to protein synthesis inhibition.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Activation of ion transport systems during cell volume regulation.

This review discusses the activation of transport pathways during volume regulation, including their characteristics, the possible biochemical pathways that may mediate the activation of transport pathways, and the relations between volume regulation and transepithelial transport in renal cells. Many cells regulate their volume when exposed to an anisotonic medium. The changes in cell volume are caused by activation of ion transport pathways, plus the accompanying osmotically driven water movement such that cell volume returns toward normal levels. The swelling of hypertonically shrunken cells is termed regulatory volume increase (RVI) and involves an influx of NaCl into the cell via either activation of Na-Cl, Na-K-2Cl cotransport systems, or Na+-H+ and Cl(-)-HCO3- exchangers. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease (RVD) and involves an efflux of KCl and water from the cell by activation of either separate K+ and Cl-conductances, a K-Cl cotransport system, or parallel K+-H+ and Cl(-)-HCO3- exchangers. The biochemical mechanisms involved in the activation of transport systems are largely unknown, however, the phosphoinositide pathway may be implicated in RVI; phorbol esters, cGMP, and Ca2+ affect the process of volume regulation. Renal tubular cells, as well as the blood cells that traverse the medulla, are subjected to increasing osmotic gradients from the corticomedullary junction to the papillary tip, as well as changing interstitial and tubule fluid osmolarity, depending on the diuretic state of the animal. Medullary cells from the loop of Henle and the papilla can volume regulate by activating Na-K-2Cl cotransport or Na+-H+ and Cl(-)-HCO3- exchange systems. Both Na-Cl and Na-K-2Cl cotransport systems have been identified in the medullary Loop of Henle and it is postulated that the Na-K-2Cl cotransport system predominates during RVI and affects transepithelial NaCl transport while the Na-Cl cotransport system may function during RVD in these cells.

Animals↗
PubMed1987

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, amiloride analogues, and renal Na+/H+ antiporter.

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a carboxyl-activating agent and has been shown to inhibit the renal Na+/H+ antiporter. The purposes of the present studies were to characterize the kinetics of inhibition of the Na+/H+ antiporter by EEDQ and to determine whether amiloride analogues affect the ability of EEDQ to inhibit the rate of Na+/H+ exchange. Brush-border membrane vesicles (BBMV) were prepared from rabbit kidneys; Na+/H+ exchange rate was assessed by the fluorescence quenching of acridine orange. EEDQ produced a concentration-dependent inhibition of Na+/H+ exchange; the effect was to decrease the maximum activity (Vmax) from 5.51 to 2.03 fluorescence units X mg protein-1 X S-1) and Km (from 14.1 to 8.7 mM) compared with control BBMV. Pretreatment of BBMV with amiloride before the addition of EEDQ maintained both Vmax and Km at values that were not significantly different from those for control BBMV. Compared with a series of analogues, amiloride was only the third most potent inhibitor of the rabbit renal Na+/H+ antiporter; amiloride, however, provided the greatest protection against inhibition of the antiporter by the subsequent addition of EEDQ. These findings suggest that the 2-carbonylguanidininum moiety and 6-chloro atom are important for binding of amiloride to sites at or near the antiporter; the group at position 5 is important in determining the ability of amiloride to protect against inhibition of the Na+/H+ antiporter by EEDQ. Finally, the ability of amiloride to protect against inactivation of the renal Na+/H+ antiporter by EEDQ is reversible.

Amiloride↗
PubMed1987

K-Cl transport systems in rabbit renal basolateral membrane vesicles.

The transport pathways for chloride in basolateral membrane vesicles from the rabbit renal cortex were investigated. 36Cl uptake was stimulated by the presence of potassium in the uptake media compared with sodium or N-methyl-D-glucamine. In addition, potassium (86Rb) uptake was stimulated more by chloride than by nitrate or gluconate. Neither of these processes was further stimulated by potassium gradients plus valinomycin, suggesting the presence of an electrically neutral K-Cl cotransport system. A magnesium-induced chloride conductance was also found in the basolateral membrane vesicles. In the absence of magnesium, the chloride conductance was low; valinomycin and an inwardly directed potassium gradient did not stimulate 36Cl uptake, anthracene-9-carboxylic acid did not inhibit 36Cl uptake, and valinomycin did not stimulate chloride-dependent 86Rb uptake. However, in the presence of 1 mM magnesium, opposite results were obtained; valinomycin and an inwardly directed potassium gradient stimulated 36Cl uptake, anthracene-9-carboxylic acid inhibited 36Cl uptake, and valinomycin stimulated chloride-dependent 86Rb uptake. Therefore, an electrically neutral K-Cl cotransport and magnesium-induced chloride conductance were found in renal cortical basolateral membrane vesicles prepared from the rabbit renal cortex.

Animals↗
PubMed1987

Parallel adaptation of the rabbit renal cortical sodium/proton antiporter and sodium/bicarbonate cotransporter in metabolic acidosis and alkalosis.

Recent studies have shown that the bicarbonate reabsorptive capacity of the proximal tubule is increased in metabolic acidosis. For net bicarbonate reabsorption to be regulated, there may be changes in the rate of apical H+ secretion as well as in the basolateral base exit step. The present studies examined the rate of Na+/H+ exchange (acridine orange method) and Na+/HCO3 cotransport (22Na uptake) in apical and basolateral membranes prepared from the rabbit renal cortex by sucrose density gradient centrifugation. NH4Cl loading was used to produce acidosis (arterial pH, 7.27 +/- 0.03), and Cl-deficient diet with furosemide was used to produce alkalosis (arterial pH, 7.51 +/- 0.02). Maximal transport rate (Vmax) of Na+/H+ antiporter and Na+/HCO3 cotransporter were inversely related with plasma bicarbonate concentration from 6 to 39 mM. Furthermore, the maximal transport rates of both systems varied in parallel; when Vmax for the Na+/HCO3 cotransporter was plotted against Vmax for the Na+/H+ antiporter for each of the 24 groups of rabbits, the regression coefficient (r) was 0.648 (P less than 0.001). There was no effect of acidosis or alkalosis on affinity for Na+ of either transporter. We conclude that both apical and basolateral H+/HCO3 transporters adapt during acid-base disturbances, and that the maximal transport rates of both systems vary in parallel during such acid-base perturbations.

Acidosis↗
PubMed1986

Parathyroid hormone inhibition of Na+-H+ antiporter activity in a cultured renal cell line.

The renal effects of parathyroid hormone (PTH) include a decreased rate of acidification by the proximal tubule. To determine whether this effect represented a PTH action on the Na+-H+ antiporter, we investigated the effect of PTH on the established opossum kidney (OK) cell line. This cell line retains several features characteristic of proximal tubule cells, including an amiloride-sensitive Na+-H+ antiporter and high-affinity PTH receptors with a coupled cAMP response. We measured steady-state intracellular pH and amiloride-sensitive 22Na+ uptake as a reflection of the activity of the Na+-H+ antiporter. Under bicarbonate and CO2-free conditions, the steady-state intracellular pH of OK cell cultures was modified by altering the rate of Na+-H+ exchange. When Na+-H+ exchange was inhibited by amiloride, intracellular pH fell. Conversely, augmenting antiporter activity by addition of monensin, a Na+-H+ exchange ionophore, raised intracellular pH. PTH (2.5 X 10(-8) M) lowered intracellular pH by up to 0.17 pH units, and half of the maximum PTH effect was present at a concentration of 10(-12) M. This effect was not seen in the presence of amiloride or in the absence of sodium, suggesting that a functional Na+-H+ antiporter is necessary for its expression. The decrease in intracellular pH was reproduced by forskolin and 8-bromo-cAMP, suggesting that this is a cAMP-mediated effect. PTH, forskolin, and 8-bromo-cAMP also decreased the amiloride-sensitive component of 22Na+ uptake in OK cells by up to 64%, whereas the amiloride-insensitive component was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗