Potassium restriction downregulates ROMK expression in rat kidney.

The ROMK family of proteins has biophysical properties and distribution within the kidney similar to those of secretory potassium channels of the distal nephron. To study the regulation of ROMK during variations in dietary potassium, we measured the abundance of ROMK protein in rat kidney by immunoblotting. Neither 2 nor 5 days of a high-potassium diet had an effect on protein abundance in the cortex or medulla. Potassium deprivation (2 or 5 days) decreased ROMK protein content in both cortical and medullary fractions, to 51 and 40% of controls, respectively. To see whether the Na-K-2Cl cotransporter is similarly affected by potassium restriction, we analyzed immunoblots by using an antibody for the rat type 1 bumetanide-sensitive cotransporter (BSC-1). Like ROMK, BSC-1 protein content was found to decrease significantly in the renal medulla of potassium-deprived rats. In the thick ascending limb of Henle's loop, a decrease in ROMK and BSC-1 could result in decreased reabsorption of NaCl, a finding associated with hypokalemia.

Characterization and regulation of H-K-ATPase in intercalated cells of rabbit cortical collecting duct.

K-dependent H+ extrusion was investigated using fluorescence techniques in rabbit cortical collecting tubules (CCTs). Experiments were performed in split-open tubules from normal animals exposed to the intracellular pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). This preparation permitted the study of individual intercalated cells (ICs). In the ICs, partial recovery of pH(i) was observed in response to an acute acid load upon readdition of 5 mM K to the superfusate. This recovery was SCH 28080-inhibitable (10(-5) M) and ouabain-insensitive suggesting the process is mediated by a gastric-type H-K ATPase. To see if H-K ATPase plays a role in acid secretion its function was evaluated under chronic metabolic acidosis (CMA) conditions. CMA was induced by replacing drinking water with 75 mM NH4Cl in 5% sucrose for 10-14 days. The SCH 28080-inhibitable K-dependent pH(i) recovery rate was three-fold higher in CMA ICs compared to controls. To determine the location of the H-K ATPase, CCTs were microperfused and individual peanut lectin binding (PNA) ICs studied. K-dependent pH(i) recovery was measured in response to an NH4Cl pulse. An apical SCH 28080-inhibited K-dependent pH(i) recovery process was observed in control and CMA ICs. Taken together these data confirm the existence of a gastric-type H-K ATPase in ICs of rabbit CCT. Based on our findings the H-K ATPase is found on the apical side of the cell and is stimulated under conditions of CMA.

Localization of ROMK channels in the rat kidney.

Renal potassium secretion occurs in the distal segments of the nephron through apically located secretory potassium (SK) channels. SK may correspond to the ROMK channels cloned from rat kidney. In this study, the localization of ROMK at the cellular level in the rat kidney was examined using an affinity-purified polyclonal antibody raised against a C-terminal peptide of ROMK. The specificity of the antibody was demonstrated by immunoblots of membranes of Xenopus oocytes expressing ROMK2. Immunoblots of homogenates from rat renal outer medulla and cortex revealed predominant bands of 70 to 75 kD, which were ablated by preadsorption with an excess of peptide. These bands were specific for the rat kidney. Immunolocalization studies revealed that ROMK is expressed in specific nephron segments in both the cortex and medulla. In the cortex, ROMK was found in the apical domain of the thick ascending limb of Henle's loop, the connecting tubule, and in some, but not all, cells of cortical collecting tubules. In the medulla, expression in the apical membrane of the thick ascending limbs of Henle's loop was strong, whereas outer medullary collecting ducts were weakly stained. Expression in the thick ascending limb was also heterogeneous; some cells that expressed the Na-K-Cl cotransporter were weakly stained with the anti-ROMK antibody. No staining of glomeruli, proximal tubules, or inner medullary collecting ducts was found. The localization of ROMK agrees well with the findings of SK in patch-clamp studies and supports the view that ROMK is the SK channel of the distal segments of the nephron.

Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis.

Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5-trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.

Stimulation of apical H-K-ATPase in intercalated cells of cortical collecting duct with chronic metabolic acidosis.

This study evaluated the role of H-K-adenosinetriphosphatase (H-K-ATPase) with chronic metabolic acidosis (CMA) in intercalated cells (ICs) of rabbit cortical collecting duct (CCD). CMA was induced by replacing drinking water with 75 mM NH4Cl in 5% sucrose for 10-14 days. CCDs isolated from CMA and control rabbits were split open and exposed to the intracellular pH (pHi) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions, the resting pHi in ICs was similar for both groups. K-dependent pHi recovery (5 mM K, 140 mM N-methyl-D-glucamine) was monitored in response to a pulse of NH4Cl (10 mM). The K-dependent pHi recovery rate was threefold higher in CMAICs compared with controls and was abolished with the gastric H-K-ATPase inhibitor, Sch-28080 (10(-5) M). Polarity of the H-K-ATPase was studied in microperfused CMA and control CCDs. Luminal K-dependent pHi recovery was monitored in response to an acute pulse of NH4Cl in individual peanut lectin agglutinin (PNA)-binding ICs. The apical Sch-28080-inhibitable K-dependent pHi recovery rate was significantly greater in CMA ICs than control ICs. In summary, CMA enhances functional activity of an apical H-K-ATPase in PNA-binding ICs of rabbit CCD.