Biomedical subjects
Publications and source records attributed to W B Reeves.
Chloride channels are found throughout the nephron. Many chloride channels expressed in the kidney have now been cloned. The CIC family of voltage-dependent chloride channels accounts for most of the known renal chloride channels. Unfortunately, relatively little is known concerning their functional properties. The role that chloride channels may play in renal disease has only recently been addressed.
The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.
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These experiments were intended to identify candidate cDNAs which might encode basolateral membrane Cl- channels of the mTAL using a homology-based cloning strategy. We prepared a cDNA library using a 1.8 to 3.2 kb mRNA fraction from rabbit outer medulla that induces a Cl- conductance in cellular membranes of Xenopus laevis oocytes. The cDNA library was screened with two 32P-oligonucleotide probes corresponding to highly conserved sequences in other Cl- channels. We isolated two cDNAs: rbClC-Ka and rbClC-Kb. The protein sequences deduced from these two cDNAs had 99% homology. Using RT-PCR technology, cultured mouse mTAL cells were found to contain mRNA corresponding to those two cDNAs. Expression of the mRNAs corresponding to these two cDNAs was kidney-specific and was greater in rabbit renal medulla than rabbit renal cortex. Finally, by using RT-PCR technology in combination with microdissected glomeruli or tubule segments, we found mRNA for rbClC-Ka in glomeruli, proximal convoluted tubules, mTAL and cortical collecting tubules.
The inactivation of physiological glucocorticoids by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) confers mineralocorticoid specificity to certain aldosterone target tissues. However, 11 beta-HSD activity in a human mineralocorticoid-responsive tissue has never been characterized. The present studies describe the features of 11 beta-HSD in the cultured human colonic epithelial cell line, T84. The 11 beta-HSD activity of T84 cells resided in the microsomal fraction and showed a marked preference for NAD rather than NADP as cofactor. NAD or NADP (200 microM) increased the conversion of corticosterone to 11-dehydrocorticosterone by 24.1 +/- 2.1 and 0.5 +/- 0.7 pmol.mg protein-1.20 min-1, respectively, indicating a > 40-fold preference for NAD vs. NADP. The Michaelis constant values for corticosterone and cortisol were 11.3 +/- 1.5 and 79.8 +/- 10 nM, respectively. The T84 11 beta-HSD was inhibited by 11-dehydrocorticosterone in a noncompetitive fashion [inhibition constant (Ki) = 180 +/- 9.6 nM] and by carbenoxolone in a competitive fashion (Ki = 17.4 +/- 1.3 nM). The expression of mineralocorticoid receptors in these cells was demonstrated by reverse transcriptase-polymerase chain reaction of mRNA isolated from T84 cells and by [3H]aldosterone binding studies. The coexpression of this NAD-dependent isoform of 11 beta-HSD and mineralocorticoid receptors is consistent with the view that the NAD-dependent isoform is responsible for the specificity of mineralocorticoid responses.
The experiments reported herein compared Cl- channels fused into bilayers from rabbit outer medullary vesicles with Cl- channels in excised patches of basolateral membranes from cultured mouse medullary thick ascending limb (MTAL) cells and evaluated whether the latter were plausible candidates for the Cl- channels mediating net NaCl absorption in microperfused mouse MTAL segments. The unique signature characteristics of Cl- channels incorporated into lipid bilayers from outer medullary vesicles include activation of open probability (Po) by increases in the Cl- concentrations bathing intracellular faces; activation of Po by protein kinase A (PKA) + ATP, when the Cl- concentrations bathing intracellular faces are low; and no effect of PKA + ATP on Po with high cytoplasmic-face Cl- concentrations. These same properties were observed in Cl- channels studied using excised patches of basolateral membranes from mouse MTAL cells. Moreover, in both bilayers and in excised patches, the sharpest fractional increase in Cl- channel Po occurred with cytosolic-face Cl- concentration increases to values similar to the antidiuretic hormone (ADH)-dependent values of intracellular Cl- activity in microperfused mouse MTAL segments, and these fractional Po increases were adequate to account quantitatively for the ADH-dependent increase in basolateral membrane Cl- conductance in microperfused mouse MTAL segments. Thus the excised-patch basolateral Cl- channels reported here are reasonable candidates for those mediating net Cl- absorption in the MTAL.
Cl- channels fused from basolateral mTAL membranes into planar bilayers have distinctive functional characteristics which, when taken together, are unique among Cl- channels. The properties of these 50 to 60 pS channels can account for the characteristics of basolateral Cl- conductances in microperfused mTAL segments and thus may mediate net basolateral Cl- absorption in the intact mTAL. In the present studies, we solubilized basolateral membranes from rabbit mTAL. Since basolateral mTAL Cl- channels contain arginine- and lysine-rich domains, we exposed these solubilized membranes to sequential cation- and anion-exchange chromatography. The bound and unbound eluates from cation- and anion-exchange chromatography were reconstituted into proteoliposomes which, when fused into bilayers, yielded Cl- channels whose properties were virtually identical to those described above for native basolateral mTAL channels fused into bilayers. As judged by valinomycin-sensitive conductive 36Cl- uptake, proteoliposomes reconstituted from the unbound eluates after anion-exchange chromatography were enriched at least 30-fold in Cl- channel activity and had about 30% of the total Cl- channel activity solubilized in native vesicles.
Ion channels in endosomal membranes from rabbit kidney cortex were studied after reconstitution into planar lipid bilayers. The most frequently observed ion channel was anion selective (PCl/PK = 13) and had a single-channel conductance of 116 pS when the cis and trans solutions contained 410 and 150 mM KCl, respectively, and a conductance of 90 pS in symmetrical 150 mM KCl solutions. The anion selectivity sequence of the channel was NO3- > F- > Br- > Cl- >> I-. The activity of the channel was voltage dependent such that hyperpolarization of the cis, or cytoplasmic, surface of the channel increased the open probability (Po). The activity of the channel was also highly dependent on the calcium activity of the cis but not the trans solution. Channels were fully active (Po > 0.7) at Ca2+ concentration > 1 microM, but channel activity was completely absent (Po < 0.001) at Ca2+ concentration < 250 nM. The effects of calcium on Po were not voltage dependent. The Cl(-)-channel blocker 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-inden -5- yl)oxy]-acetic acid (IAA-94/95) produced a concentration-dependent reversible flickering block of the endosomal channel with a Ki of 15 microM. 4,4'-Dinitrostilbene-2,2'-disulfonic acid, a disulfonic stilbene, also produced a flickering block of the channel with a Ki of approximately 5 microM. Because endosomal Cl- channels are believed to facilitate endosomal acidification, we tested the effects of IAA-94/95 and deletion of Ca2+ on the rate of acidification of intact endosomes. Because neither maneuver affected acidification, we conclude that the 116-pS channel does not participate in endosomal acidification. This channel may be involved in other endosomal processes, e.g., cell volume regulation and control of membrane trafficking.
The surface epithelium of rabbit renal papilla was dissected free from its supporting tissue and mounted in an Ussing chamber. The conductive properties of the epithelium and of the apical and basolateral cell membranes were examined with KCl-filled microelectrodes. The transepithelial voltage was 0.07 +/- 0.15 mV, and the transepithelial resistance was 107 +/- 15 omega.cm2 (n = 29). The fractional resistance of the apical membrane (fRa) was 0.93 +/- 0.01 (n = 103 cells, 29 tissues). The apical membrane was not conductive to Na+, K+, or Cl-. An increase in the K+ concentration of the basolateral solution from 5 to 50 mM depolarized the basolateral membrane voltage (Vb) from -59 +/- 1.6 to -31.2 +/- 2.2 mV (n = 28 cells) and increased fRa from 0.935 +/- 0.01 to 0.962 +/- 0.01 (P < 0.001, n = 21 cells). Likewise, 5 mM barium in the basolateral solution depolarized Vb from -57.7 +/- 2.0 to -29.8 +/- 2.2 mV (n = 21 cells). A tenfold decrease in the Cl- concentration of the basolateral solution caused an 8.3 +/- 1.9 mV depolarization in Vb. Thus the basolateral cell membrane is conductive to K+ and Cl-. Exposure of the apical membrane to amphotericin B demonstrated that the transepithelial resistance is determined primarily by the paracellular pathway.
The mechanisms responsible for the loss of cell potassium during renal ischemia are poorly understood. The present studies examined the hypothesis that potassium channels are activated as an early response to hypoxia and contribute to potassium loss independent from an inhibition of active K+ uptake. Potassium flux in suspensions of freshly isolated rat proximal tubules was measured using an ion-selective electrode. Exposure of the tubules to hypoxia for only 2.5 min resulted in a rise in the passive leak rate of K+ but no decrease in active K+ uptake. The passive leak of K+ was associated with a 40% decrease in cell ATP content. The passive K+ efflux was inhibited by 5 mM Ba2+ (95%) and by 15 mM tetraethylammonium (85%) suggesting that K+ channels were the primary route of K+ movement. The effects of K+ channel blockade on the development of hypoxic injury were also examined. Tetraethylammonium and glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced hypoxic injury as assessed by the release of lactate dehydrogenase or measurement of DNA damage. These results suggest that activation of K+ channels is an early response to hypoxia and contributes to hypoxic renal injury.
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A unique property of basolateral membrane Cl- channels from the mTAL is that the Cl- concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (Po). The K1/2 for maximal activation of Po by Cl- facing intracellular domains of these channels is 10 mM Cl-. The present experiments evaluated the nature of these Cl(-)-interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl- channel faces, could augment Po with a K1/2 in the range of 10 mM isethionate without affecting conductance (gCl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mM phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mM), prevented the activation of Po usually seen when the Cl- concentration of solutions facing intracellular channel domains was raised from 2 to 50 mM. However, when the Cl- channel activity was increased by first raising the Cl- concentration bathing intracellular channel faces from 2 to 50 mM, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl- channel faces had no effect on Po. We conclude that the intracellular aspects of these Cl- channels contain Cl(-)-interactive loci (termed [Cl]i) which are accessible to impermeant anions in intracellular fluids and which contain arginine- and lysine-rich domains which can be inactivated, at low ambient Cl- or isethionate concentrations, by interactions with PGO or TNBS.
Cl- channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl- activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl- in the absence of PKA. The PKA requirement for activation of Cl- channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl- concentrations on the properties of Cl- channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl- channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl- concentration from 2 to 50 mM increased both channel activity (Po) and channel conductance (gCl, pS). Likewise, trans PKA increased mTALH Cl- channel activity by increasing the activity of individual channels when the trans solutions were 2 mM Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect gCl. Moreover, when mTALH Cl- channels were inactivated by reducing cis Cl- concentrations to 50 mM, cis PKA addition did not affect Po. These results are consistent with the view that these Cl- channels originated from basolateral membranes of the mTALH. Cl- channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl- concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl- channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl- channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl- channel activity by both intracellular Cl- and by PKA differ in basolateral mTALH Cl- channels compared to apical Cl- channels from either the tracheal or small intestine.
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Cl- channels from basolateral membranes of the mammalian thick ascending limb of Henle differ significantly, in certain of their functional characteristics, from Cl- channels in apical membranes of secretory epithelia such as trachea or small intestine and from certain Cl- channels in the central nervous system. Yet there is no sequence information available about basolateral thick ascending limb Cl- channels. As an initial step in the isolation of a Cl- channel from the thick ascending limb of Henle's loop, we attempted a functional expression of a Cl- conductance in oocytes from Xenopus laevis. Oocytes injected with mRNA isolated from the outer medulla of rabbit kidney had a membrane conductance, measured using a two-electrode voltage clamp, which was sixfold greater than in water-injected oocytes (9.05 +/- 1.56 vs. 1.43 +/- 0.15 microS, respectively). Ion substitution experiments showed the conductance in the RNA-injected oocytes to be Cl- selective. In addition, the Cl(-)-channel blocker diphenylamine-2-carboxylate (1 mM) almost completely inhibited (79 +/- 6%) the increased conductance seen in the RNA-injected oocytes. Fractionation of the mRNA by sucrose gradient centrifugation revealed that peak Cl- channel activity was expressed using an mRNA fraction of 1.8-3.2 kb in size. These results demonstrate that a membrane Cl- conductance can be expressed in X. laevis oocytes injected with size-selected fractions of mRNA from rabbit outer renal medulla.
We examined the interactions of cAMP-dependent protein kinase and varying aqueous Cl- concentrations in modulating the activity of Cl- channels obtained by fusing basolaterally enriched renal outer medullary vesicles into planar lipid bilayers. Under the present experimental conditions, the cis and trans solutions face the extracellular and intracellular aspects of these Cl- channels, respectively. Raising the trans Cl- concentration from 2 to 50 mM increased the channel open-time probability, raised the unit channel conductance, and affected the voltage-independent determinant (delta G) of channel activity but not the gating charge (Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990. J. Membrane Biol. 118:269-278). With 2 mM trans KCl, trans addition of the catalytic subunit of PKA (C-PKA) plus ATP increased channel open-time probability and altered the voltage-independent determinant of channel activity without affecting either unit channel conductance or gating charge. The effect was ATP specific, did not occur with (C-PKA plus ATP) addition to cis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50 mM) trans Cl- concentrations. These data indicate that (C-PKA plus ATP) might modulate Cl- channel activity by phosphorylation at or near the Cl(-)-sensitive site on the intracellular face of these channels.
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We evaluated the effects of varying aqueous Cl- concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl- channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for channel activity, and the effects of varying Cl- concentrations and/or PGO on the relation between holding voltage (VH, mV) and open-time probability (Po). Reducing cis Cl- concentrations, in the range 50-320 mM, produced a linear reduction in fractional open time (Po) with a half-maximal reduction in Po at cis Cl- approximately 170 mM. Channel activity was sustained by equimolar replacement of cis Cl- with F-, but not with impermeant isethionate. For trans solutions, the relation between Cl- concentration and Po was negatively cooperative, with 50% reduction in po at 10 mM Cl-. Reducing cis Cl- had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent energy (delta G) for channel opening. Phenylglyoxal (PGO) reduced Z and altered delta G for Cl- channel activity when added to cis, but not trans solutions. Furthermore, in the presence of cis PGO, reducing the cis Cl- concentration had no effect on Z but altered delta G. Thus we propose that cis PGO and cis Cl- concentrations affect separate sites determining channel activity at the extracellular faces of these Cl- channels.