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Biomedical subjects

D G Warnock

Publications and source records attributed to D G Warnock.

At least 55 records · Page 3Linked to original sources
PubMed1996

Liddle's disease: abnormal regulation of amiloride-sensitive Na+ channels by beta-subunit mutation.

Liddle's disease is an autosomal dominant genetic disorder characterized by severe low renin hypertension ("pseudoaldosteronism") that has been genetically linked to a locus on chromosome 16 encoding the beta-subunit of an amiloride-sensitive Na+ channel (ASSC) (15). Peripheral blood lymphocytes (PBL) express ASSC that are functionally indistinguishable from those expressed by Na(+)-reabsorbing renal epithelial cells (3, 5). The amiloride-sensitive Na+ conductance in PBL from affected and unaffected individuals from the original Liddle's pedigree was examined using whole cell patch clamp. Typically, the basal Na+ currents in cells from affected individuals were maximally activated. Basal Na+ currents in cells from unaffected individuals were minimal and could be maximally activated by superfusion with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Affected cells could not be further stimulated with CPT-cAMP. Superfusion with a supermaximal concentration of amiloride (2 microM) inhibited both the cAMP-activated Na+ conductance in unaffected cells and the constitutively activated inward conductance in affected cells. Cytosolic addition of a peptide identical to the terminal 10 amino acids of the truncated beta-subunit normalized the cAMP-mediated but not the pertussis toxin-induced regulation of the mutant ASSC. The findings show that lymphocyte ASSC are constitutively activated in affected individuals, that a mutation of the beta-subunit alters ASSC responsiveness to specific regulatory effectors, and that the cellular mechanism responsible for the pathophysiology of Liddle's disease is abnormal regulation of Na+ channel activity. These findings have important diagnostic and therapeutic implications and provide a cellular phenotype for the diagnosis of pseudoaldosteronism.

Amiloride↗
PubMed1996

Peptide block of constitutively activated Na+ channels in Liddle's disease.

Hypertension is a multifactorial disorder that results in an increased risk of cardiovascular and end-stage renal disease. Liddle's disease represents a specific hypertensive disease and expresses itself in the human population as an autosomal dominant trait. Recent experimental evidence indicates that patients with Liddle's disease have constitutively active amiloride-sensitive Na+ channels and that these channels are phenotypically expressed in lymphocytes obtained from normal and affected members of the original Liddle's kindred. Linkage analysis indicates that this disease results from a deletion of the carboxy-terminal region of the beta-subunit of a recently cloned epithelial Na+ channel (ENaC). We report the successful immunopurification and reconstitution of both normal and constitutively active lymphocyte Na+ channels into planar lipid bilayers. These channels display all of the characteristics typical of renal Na+ channels, including sensitivity to protein kinase A phosphorylation. We demonstrate that gating of normal Na+ channels is removed by cytoplasmic trypsin digestion and that the constitutively active Liddle's Na+ channels are blocked by a beta- or gamma-ENaC carboxy-terminal peptide in a GTP-dependent fashion.

Amiloride↗
PubMed1996

Stretch modulates amiloride sensitivity and cation selectivity of sodium channels in human B lymphocytes.

Stretch-mediated regulation of amiloride-sensitive Na+ channels was examined in Epstein-Barr virus-transformed human B lymphocytes. Cation conductances were measured using whole cell patch-clamp techniques. Stretch activation, induced by increasing the hydrostatic pressure of the bath solution, immediately and reversibly increased both inward and outward ionic conductances once a threshold of 2-5 mmH2O was reached. Ionic substitutions confirmed that stretch enhanced membrane conductivity for both Na+ and K+. Amiloride (2 microM) completely prevented the response to elevated hydrostatic pressure; however, when amiloride was applied after stretch-induced activation, the sensitivity to amiloride was dramatically decreased (inhibitor concentration that reduces whole cell current by 50% of approximately 20 microM). Evidence that the currents induced by stretch were mediated by Na+ channels was provided by the lack of response to stretch in lymphocytes from patients with Liddle's syndrome, which is caused by expression of a truncated mutant of the beta-subunit of the amiloride-sensitive Na+ channel. Pretreatment with colchicine (0.5 mM, 30 min) prevented stretch-induced activation, which shows evidence of the involvement of the cytoskeleton. These data indicate that stretch regulates the conductance of amiloride-sensitive Na+ channels in immortalized human B lymphocytes and also alters its cationic selectivity and its sensitivity to amiloride.

Amiloride↗
PubMed1996

Renal Na+/H+ exchanger isoforms and their regulation by thyroid hormone.

Na+ crosses the luminal membrane of the proximal tubule primarily via Na+/H+ exchange (NHE), and NHE activity is influenced by thyroid status. Pharmacological, immunological, and kinetic studies indicate multiple isoforms of NHE, and four full-length cDNAs have been cloned to date. The aims of this study were to determine which NHE mRNAs (NHE1, -2, -3, and -4) were expressed in the rat proximal tubule, the relative abundance of each in the renal cortex, and the effect of thyroid status on their expression. By blot hybridization of poly(A)+ RNA, all NHE isoform mRNAs were detected in the rat renal cortex; NHE1, -2, and -3 in the proximal tubule; and NHE1 and -3 in LLC-PK1 cells. NHE3 mRNA abundance was fourfold higher than the other three isoforms in renal cortex. The effect of thyroid status was assessed in renal cortex from euthyroid, hypothyroid, and hyperthyroid rats. Although none of the NHE mRNA levels was altered in the transition from euthyroid to hypothyroid states, both NHE2 and NHE3 mRNA levels increased 1.5-fold in the transition from hypo- to hyperthyroidism. NHE3 protein, measured by immunoblot with the use of an NHE3-specific antibody, was detected at 83-85 kDa in renal cortex and codistributed on sorbitol gradients with the brush-border marker alkaline phosphatase. No significant difference in NHE3 protein abundance was detected between hypothyroid and hyperthyroid rats. In conclusion, in the renal cortex, the NHE3 isoform predominates at the mRNA level, is expressed in apical membranes, and increases at the mRNA but not the protein levels in response to thyroid hormone treatment, suggesting parallel changes in synthesis and turnover of NHE3 by thyroid hormone.

Animals↗
PubMed1996

Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis.

Acute arterial hypertension provokes a rapid decrease in proximal tubule (PT) Na+ reabsorption, increasing flow to the macula densa, the signal for tubuloglomerular feedback. We tested the hypothesis, in rats, that Na+ transport is decreased due to rapid redistribution of apical Na+/H+ exchangers and basolateral Na+ pumps to internal membranes. Arterial pressure was increased 50 mmHg by constricting various arteries. We also tested whether transporter internalization occurred when PT Na+ reabsorption was inhibited with the carbonic anhydrase inhibitor benzolamide. Five minutes after initiating either natriuretic stimuli, cortex was removed, and membranes were fractionated by density gradient centrifugation. Urine output and endogenous lithium clearance increased threefold in response to either stimuli. Acute hypertension provoked a redistribution of apical Na+/H+ exchanger NHE3, alkaline phosphatase, and dipeptidyl peptidase IV to higher density membranes enriched in the intracellular membrane markers. Basolateral membrane Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity decreased 50%, 25-30% of the alpha 1-and beta 1-subunits redistributed to higher density membranes, and the remainder is attributed to decreased activity of the transporters. Benzolamide did not alter Na+ transporter activity or distribution, implying that decreasing apical Na+ uptake does not initiate redistribution or inhibition of basolateral Na(+)-K(+)-ATPase. We conclude that PT natriuresis provoked by acute arterial pressure is mediated by both endocytic removal of apical Na+/H+ exchangers and basolateral Na+ pumps as well as decreased total Na+ pump activity.

Acute Disease↗
PubMed1996

Genetic association of 11 beta-hydroxysteroid dehydrogenase type 2 (HSD11B2) flanking microsatellites with essential hypertension in blacks.

11 beta-Hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) specifically modulates access of the mineralocorticoid aldosterone to the kidney mineralocorticoid type 1 receptors in a physiological environment in which there is a molar excess of cortisol. Cortisol and aldosterone have similar affinities for mineralocorticoid receptors. Mechanistically, 11 beta-HSD2 converts cortisol to cortisone. The other known isoform, 11 beta-HSD1, not only catalyzes the cortisol to cortisone reaction but also the reverse reaction, making it unlikely to play an important role in modulating the access of aldosterone to mineralocorticoid receptors. Mutations in the HSD11B2 gene (both exonic and intronic) have been demonstrated to cause reduced activity of this enzyme in the syndrome of apparent mineralocorticoid excess, a rare autosomal recessive disorder. We hypothesized that this locus is also involved in the etiology of essential hypertension. To test this locus and flanking chromosomal regions for allelic association and genetic linkage to essential hypertension, it is necessary to have informative genetic markers. To this end, we have refined the localization of 11 beta-HSD2 to 16q22.1. We genotyped subjects using the nearest flanking microsatellites (D16S301 and D16S496). We conducted an association study using black subjects with hypertensive end-stage renal disease, black normotensive control subjects, and black and white individuals from the general population. We used chi 2 analysis and Fisher's exact test to test for association with these candidate gene markers. No significant association was found between D16S301 and hypertension. However, a positive association with hypertension was found at the D16S496 microsatellite locus (chi 2 = 6.98, df = 1, P < or = .008). Our data suggest that HSD11B2 is associated with hypertension in our black subjects with hypertensive end-stage renal disease. The 16q22.1 chromosome region potentially harbors a candidate gene for essential hypertension. Confirmation of our findings in another independently ascertained group of hypertensive subjects will provide a basis for proceeding with sib-pair linkage analyses.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Polymorphism in the beta subunit and Na+ transport.

Primary dysregulation of the epithelial sodium channel, manifested by continuing Na+ channel activity despite dietary salt excess, would cause inappropriate renal sodium reabsorption, blunted sodium excretion, and low-renin hypertension. There are now well-characterized genetic causes of hypertension in human pedigrees, which are explained by inappropriate and/or constitutive activation of the epithelial Na+ channel. Although Liddle's syndrome has been the most thoroughly investigated, the incidence of such activating mutations in the subunits of the Na+ channel appears to be relatively rare. Of continuing interest is the possibility that polymorphisms in the channel subunits could result in activation of the channel in response to normal regulatory influences. One such example is provided by a provocative report by Su et al. In this issue of the journal (J Am Soc Nephrol 1996;7:2543-2549). Despite several important technical limitations of the findings presented in the article, the suggestion that polymorphisms found in defined human populations would affect the regulation of sodium channel activity in response to environmental variables is worthy of serious consideration, and serves as a further stimulus to defining the functional significance of the various polymorphisms described in the subunits of the amiloride-sensitive sodium channel.

Amino Acid Sequence↗
PubMed1995

Mutational analysis of transmembrane histidines in the amiloride-sensitive Na+/H+ exchanger.

The histidine-reactive reagent, diethyl pyrocarbonate (DEPC) inhibits the human amiloride-sensitive Na+/H+ exchanger (NHE1) in stably transfected fibroblasts. NHE1 was protected by cimetidine and amiloride from DEPC, and DEPC inhibition was reversed with hydroxylamine, suggesting a role for critical histidine groups in NHE activity. We replaced the histidines (H) in putative transmembrane domains (H35, H120, H349) with glycine (G) using site-directed mutagenesis. There was no significant change in NHE activity of the H120G; H349G; H120,349G; and H35,120,349G mutants compared with wild type. The 50% inhibition concentration values for amiloride, ethyl isopropyl amiloride (EIPA), and cimetidine of the H349G mutant were significantly increased compared with the wild-type NHE1. We also examined the DEPC effect on the transport activity of the triple histidine mutant (H35,120,349G) and found that NHE1 activity was still inhibited by DEPC with reversal by hydroxylamine and protected by amiloride and cimetidine. Kinetic analysis of DEPC inhibition indicated that two "critical" histidine residues are required for NHE transport activity. Substitutions of H349 with asparagine (N), glutamine (Q), serine (S), tyrosine (Y), valine (V), leucine (L), and phenylalanine (F) were also examined. There were no changes in NHE activity of these mutants compared with wild type. The H349G and H349L mutants became more resistant to amiloride, whereas the H349Y and H349F mutants became more sensitive to amiloride. The H349S (mimics NHE3) and H349Y (mimics NHE4) mutations had only modest effects on amiloride sensitivity. These results indicate that H349 affects the interaction of NHE1 with its inhibitors, even though substitutions at this site, per se, do not appear to explain the differences in amiloride sensitivity between different NHE isoforms. Despite clear-cut effects of the H349G mutation on the competitive interaction of NHE1 with cimetidine and EIPA, this mutation did not affect the affinity of NHE1 for its cationic substrates (Na+, Li+).

Amiloride↗
PubMed1995

Immunopurification and functional reconstitution of a Na+ channel complex from rat lymphocytes.

Patch-clamp experiments have demonstrated an amiloride-sensitive Na+ conductance in human B lymphoid cells. We measured whole cell currents in rat lymphocytes and observed a similar Na(+)-specific inward conductance. The presence of 400 microM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate in the bath significantly increased the inward current, and this adenosine 3',5'-cyclic monophosphate activation was abolished by 2 microM amiloride. We immunopurified a protein complex from rat lymphocyte membranes using an anti-bovine kidney Na+ channel antibody. The complex consisted of five distinct polypeptides with apparent M(r) values of 110,000, 92,000, 59,000, 48,000, and 42,000. This putative channel complex was incorporated into planar lipid bilayers, where we observed single Na+ channel activity that was blocked by amiloride in a concentration-dependent manner. The addition of protein kinase A and ATP to the "intracellular" solution elicited a twofold increase in channel activity. Reverse transcription-polymerase chain reaction analysis was used to determine if the rat lymphocytes express the message for the recently cloned Na+ channel of the rat colon (rENaC). Primers for the alpha-subunit of rENaC identified no message in the lymphocyte RNA, while primers for the beta-subunit of the clone produced low levels of the expected product. Thus it appears that a rENaC-like beta-subunit may be an essential component of the lymphocyte Na+ channel that was isolated. At the same time, this channel is different from those recently cloned in that it does not include an alpha-subunit homologous to that of rENaC.

Animals↗
PubMed1995

Cyclosporine inhibits the renal response to L-arginine in human kidney transplant recipients.

To evaluate the association of cyclosporine (CsA)-related nephrotoxicity with nitric oxide (NO) and endothelin, the effects of L-arginine (LA) and branched-chain amino acid (BCAA) infusions on renal hemodynamics in 5 normal volunteers and 12 renal transplant recipients were assessed. In normal humans, LA, but not BCAA, reduced mean arterial pressure and renal vascular resistance while increasing RPF and urinary nitrate (NO3-) excretion. Group 1 included six transplant recipients not on CsA; Group 2 subjects (N = 6) were receiving CsA. In both groups, mean arterial pressure declined during the infusion of LA (116 +/- 4 to 109 +/- 4 mm Hg; P < 0.001) but not BCAA (116 +/- 3 to 115 +/- 3; P = not significant). In Group 1, LA increased RPF 33 +/- 13% (329 +/- 48 to 436 +/- 77 mL/min per 1.73 m2; P = 0.01) and GFR 37 +/- 16% (95 +/- 7 to 130 +/- 18 mL/min per 1.73 m2; P = 0.01); renal vascular resistance declined 27 +/- 6%. In Group 2, LA did not affect renal hemodynamics. No changes occurred with BCAA in either group. LA increased urinary NO3-excretion by 27 +/- 17% in Group 1 (P < 0.05), but only by 16 +/- 13% in Group 2 (P = not significant). Urinary endothelin excretion was higher in Group 2 subjects (10.1 +/- 1.3 versus 5.3 +/- 0.8 pg/mL of GFR, P < 0.01). LA-induced renal vasodilation is associated with the increased urinary excretion of NO3-.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Liddle's syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel.

Liddle's syndrome (pseudoaldosteronism) is an autosomal dominant form of human hypertension characterized by a constellation of findings suggesting constitutive activation of the amiloride-sensitive distal renal epithelial sodium channel. We demonstrate complete linkage of the gene encoding the beta subunit of the epithelial sodium channel to Liddle's syndrome in Liddle's original kindred. Analysis of this gene reveals a premature stop codon that truncates the cytoplasmic carboxyl terminus of the encoded protein in affected subjects. Analysis of subjects with Liddle's syndrome from four additional kindreds demonstrates either premature termination or frameshift mutations in this same carboxy-terminal domain in all four. These findings demonstrate that Liddle's syndrome is caused by mutations in the beta subunit of the epithelial sodium channel and have implications for the regulation of this epithelial ion channel as well as blood pressure homeostasis.

Amino Acid Sequence↗
PubMed1994

Liddle syndrome: clinical and cellular abnormalities.

Clinical findings resemble those of primary hyperaldosteronism, except that aldosterone secretion is negligible. The fault appears to lie with continuously avid sodium channels in the distal nephron, resulting in excessive salt absorption, potassium wasting, and severe hypertension. Insights gained in this disorder may help clarify more common forms of low-renin hypertension.

Adolescent↗
PubMed1994

G-proteins modulate amiloride-sensitive sodium channels.

We examined the regulation of an amiloride-sensitive sodium conductance expressed in human B lymphoid cells. This conductance was activated by two independent pathways, one involving cyclic adenylyl monophosphate (cAMP)-dependent protein kinase and the other involving a pertussis toxin-sensitive G-protein. Cholera toxin, presumably by increasing cellular cAMP, and pertussis toxin, which ADP-ribosylates certain GTP-binding proteins, both independently increased the amiloride-sensitive sodium conductance. Simultaneous treatment with both toxins, however, failed to increase the sodium conductance, implying that a single set of sodium channels was being affected by both toxins. In cells preactivated with pertussis toxin, 8-chlorophenylthio-cAMP inhibited the activated sodium conductance back to the basal level. Thus, cyclic AMP-dependent pathways can either activate or inhibit amiloride-sensitive sodium channels, depending upon the activation state of a pertussis toxin-sensitive GTP-binding protein. These findings support a hypothesis for the regulation of amiloride-sensitive sodium channels which incorporates the independent effects of cholera and pertussis toxins, and in which cyclic AMP can play a dual role in the regulation of channel activity.

Amiloride↗
PubMed1994

Analysis of the genetic contamination of salt-sensitive Dahl/Rapp rats.

Genetic contamination of Dahl/Rapp salt-sensitive rats (SS/JrHsd) was recently discovered in animals received from Harlan Sprague Dawley, Inc, the main supplier of this strain to researchers in the United States. We were interested in determining how this problem occurred and the extent of contamination in the breeding colony in hopes of quickly correcting the problem and reestablishing the supply of this important model of genetic hypertension. DNA was extracted from whole blood obtained from each rat in the Harlan foundation colony and their offspring, the pedigree expansion colony. Several microsatellite-based genetic markers that were polymorphic between the normal and contaminant alleles were used to test these two colonies. All 10 breeder pairs in the foundation colony were tested at six different loci and found to be homozygous for the normal allele in each case. All 60 members of the pedigree expansion colony were also tested, and several rats carrying contaminant alleles were found, thus localizing the origin of the contamination to this colony. We offer several recommendations regarding precautions researchers using inbred animals should take in designing future experiments.

Alleles↗
PubMed1994

Renal apical membrane sodium-hydrogen exchange in genetic salt-sensitive hypertension.

Inbred Dahl/Rapp salt-sensitive and salt-resistant rats differ in their blood pressure response to dietary salt. We studied sodium-hydrogen (Na-H) exchanger kinetics in renal brush border membrane vesicles prepared from both strains on either a 1% or 8% NaCl diet. Kinetics measurements were made with the acridine orange fluorescence quenching technique in vesicles prepared at pH 6.0. The initial Na-H exchange rate was measured using preparations with similar initial quench values. The maximal transport rate (Vmax, fluorescence units per second per milligram protein [+/- SEM]) in salt-sensitive rats on a 1% NaCl diet was significantly lower than that in salt-resistant rats (36.9 +/- 4.4 versus 51.8 +/- 5.5, respectively, P < .0005). With the 8% NaCl diet for 1 week, the Vmax of salt-resistant rats decreased and became similar to that of salt-sensitive rats. The affinity for sodium (Km, millimoles per liter [+/- SEM]) was also lower in salt-sensitive rats than in salt-resistant rats while on a 1% NaCl diet (11.8 +/- 1.0 versus 19.6 +/- 2.3, respectively, P < .002). These values converged when both strains were fed an 8% NaCl diet for 1 week. Inhibition by 25 mumol/L amiloride was less in salt-sensitive rats than in salt-resistant rats on the 1% NaCl diet. These results show that salt-sensitive rats have lower renal apical membrane Na-H exchange activity than salt-resistant rats on a 1% NaCl diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1993

Chromosomal localization of angiotensin II type 1 receptor isoforms in the rat.

The cDNA sequences of two different isoforms of the rat angiotensin II type 1 receptors, AT1A and AT1B, have been reported. A single set of polymerase chain reaction primers was used to amplify sequence from both AT1A and AT1B from rat genomic DNA. Genomic DNA from a panel of rat x mouse somatic hybrid cell lines which had been characterized as to the rat chromosomal content was then amplified with these primers. The amplified products from rat AT1A and AT1B were distinguished from each other and those of the mouse by the use of differential restriction patterns. Using this method, AT1A was localized to rat chromosome 17 and AT1B to rat chromosome 2.

Angiotensin II↗