Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and (35)S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 +/- 2% of control and reduces the rate of H+ secretion by 77 +/- 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.     

Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells

The response ofH⁺-ATPase to lethal acid stress isunknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cyclesof lethal acid stress. Cells were grown to confluence on coverslips,loaded with2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, andmonitored for intracellular pH(pH_i) recovery from an acid load. The rate of Na⁺-independentpH_i recovery from an acid load inmutant cells was approximately fourfold higher than in parent cells(P < 0.001). TheNa⁺-independentH⁺ extrusion was ATP dependent and K⁺ independent and wascompletely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,or N-ethylmaleimide. Theseresults indicate that theNa⁺-independentH⁺ extrusion in cultured medullarycells is mediated via H⁺-ATPaseand is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H⁺-ATPase remainedunchanged in mutant cells compared with parent cells. We propose thatlethal acid stress results in increased H⁺-ATPase activity in innermedullary collecting duct cells. Upregulation ofH⁺-ATPase could play a protectiverole against cell death in severe intracellular acidosis.

     

Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells

Exocytic insertion of H⁺-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H⁺-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H⁺-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H⁺-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H⁺-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H⁺-ATPase. In a pull-down assay of H⁺-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H⁺-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H⁺-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H⁺-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H⁺-ATPase vesicles. soluble N-ethylmaleimide-sensitive factor attachment protein target receptor; cell pH; acid secretion     

Urea transport in freshly isolated and cultured cells from rat inner medullary collecting duct

Summary Regulation of urea transport by vasopressin in inner medullary collecting duct (IMCD) cells is thought to be important for the urinary concentrating mechanism. Isolated tubule perfusion studies suggest the existence of a saturable urea carrier. We have measured¹⁴C-urea efflux in IMCD cells which were freshly isolated and grown in primary culture. Cells were isolated from rat papilla by collagenase digestion and hypotonic shock. In suspended cells,¹⁴C-urea efflux (J _urea from loaded cells was exponential with time constant 59±3 sec (sem,n=6, 23°C).J _urea had an activation energy of 4.1 kcal/mole and was inhibited 42±7% by 0.25mm phloretin and 30–40% by the high affinity urea analogues dimethylurea and phenylurea.J _urea was increased 40–60% by addition of vasopressin (10^–8 m) or 8-bromo-cAMP (1mm); stimulatedJ _urea was inhibited 55±8% by the kinase A inhibitor H-8. Phorbol esters and epidermal growth factor did not alterJ _urea. IMCD cells grown in primary culture were homogeneous in appearance with>fivefold stimulation of cAMP by vasopressin. The exponential time constant for urea efflux was 610±20 sec (n=3).J _urea was not altered by vasopressin, cAMP or phloretin. Another function of in vivo IMCD cells, vasopressin-dependent formation of endosomes containing water channels, was absent in the cultured cells. These results demonstrate presence of a urea transporter on suspended IMCD cells which is activated by cAMP and inhibited by phloretin and urea analogues. The urea transporter and its regulation by cAMP, and cAMP-dependent apical membrane endocytosis, are lost after growth in primary culture.     

Regulation of cGMP production by intracellular alkalinization in cultured rat inner medullary collecting duct cells

We evaluated the relationship between cell pH and cGMP production in cultured rat renal inner medullary collecting duct cells. The cGMP level, 21 +/- 6, was not different in control vs. alkalinized cells, 49 +/- 17 fmol/mg protein (p greater than 0.5). 10(-11) M atrial natriuretic peptide (ANF) enhanced cGMP production in alkalinized cells, 426 +/- 34 vs. 141 +/- 9*. Conversely, alkalinization inhibited 10(-4)M nitroprusside (SNP) induced cGMP formation, 29 +/- 9 vs. 332 +/- 67*. Phosphodiesterase inhibition abolished the difference in cGMP production by ANF but did not reverse the inhibitory effect of alkalinization on SNP induced cGMP production. In rat renal inner medullary collecting duct cells, cellular alkalinization plays a significant role in the regulation of guanylate cyclase mediated cGMP production. * = p less than 0.05).     

19-Nordeoxycorticosterone synthesis by rat kidney inner medullary collecting duct cells

19-Nordeoxycorticosterone (19-nor-Doc), a potent mineralocorticoid, was found to be synthesized by the isolated rat kidney perfused by an adrenal precursor (19-oxo-Doc). To determine if this bioconversion is a function of renal tubular cells, various adrenal precursors of 19-nor-Doc were added separately to rat kidney inner medullary collecting duct cells culture media at a concentration of 10 nM. While 4.6% +/- 1.0% of 19-oxo-Doc (n = 3) and 14.4% +/- 1.4% of 19-oic-Doc (n = 3) were converted to 19-nor-Doc after 24 hours of incubation, Doc, and 19-OH-Doc were not converted. This represents further evidence that Doc has to be metabolized to 19-oxo-Doc or 19-oic-Doc (19-carboxy-Doc) before it can be converted by the kidney inner medullary collecting duct cells to 19-nor-Doc.     

Vasopressin-enhanced urea transport by rat inner medullary collecting duct cells in culture

The distal inner medullary collecting duct (IMCD) is critical in the urinary concentrating process, in part because it is the site of vasopressin (AVP)-regulated permeability to urea. The purpose of these experiments was to develop a cell culture model of the IMCD on permeable structure and to characterize the responsiveness to AVP. Rat IMCD cells were grown to confluence on collagen-coated Millipore filters glued onto plastic rings. To assess the time required to achieve confluence, the transepithelial resistance was measured periodically and was found to be stable after 2 weeks, at a maximal value of 595 +/- 22 omega cm2. In separate monolayers the effect of AVP on inulin and urea permeability was determined. While inulin permeability was unchanged after AVP, urea permeability increased from 6.0 +/- 0.4 to peak values of 16.0 +/- 3.8 (10 nM), 23.1 +/- 3.9 (1 microM) and 28.1 +/- 4.9 (10 microM) x 10(-6) cm s-1 (n = 24). In 10 other monolayers, after the addition of 1 mM 8-Br-cAMP, urea permeability increased from 5.1 +/- 0.3 to 8.1 +/- 1.6 x 10(-6) cm s-1 and, after 8-Br-cAMP + 3-isobutyl-1-methylxanthine, to 12.2 +/- 0.7 x 10(-6) cm s-1. We conclude that rat IMCD cells grown in culture exhibit the characteristics of a 'tight' epithelium. Inulin and urea permeability are not different in the absence of AVP, consistent with high resistance junctional complexes. Furthermore, IMCD cells retain the capacity for AVP-regulated urea permeability, a characteristic feature of this nephron segment in vivo.     

Vasopressin-enhanced urea transport by rat inner medullary collecting duct cells in culture

Abstract. The distal inner medullary collecting duct (IMCD) is critical in the urinary concentrating process, in part because it is the site of vasopressin (AVP)-regulated permeability to urea. The purpose of these experiments was to develop a cell culture model of the IMCD on permeable structure and to characterize the responsiveness to AVP. Rat IMCD cells were grown to confluence on collagen-coated Millipore filters glued onto plastic rings. To assess the time required to achieve confluence, the transepithelial resistance was measured periodically and was found to be stable after 2 weeks, at a maximal value of 595 ± 22 ω cm². In separate monolayers the effect of AVP on inulin and urea permeability was determined. While inulin permeability was unchanged after AVP, urea permeability increased from 6.0 ± 0–4 to peak values of 16.0 ± 3–8(10nM),23.1 ± 3–9(1 μM)and28 1 ± 4–9(10μM) X 10^-6cms^-1 ( n = 24). In 10 other monolayers, after the addition of 1 mM 8-Br-cAMP, urea permeability increased from 5.1 ±0–3 to 8.1 ± 1–6 times 10^-6 cm s^-1 and, after 8-Br-cAMP +3-isobutyl-l-methylxanthine, to 12.2 ± 0–7 times 10^-6 cms^-1. We conclude that rat IMCD cells grown in culture exhibit the characteristics of a 'tight' epithelium. Inulin and urea permeability are not different in the absence of AVP, consistent with high resistance junctional complexes. Furthermore, IMCD cells retain the capacity for AVP-regulated urea permeability, a characteristic feature of this nephron segment in vivo.     

A voltage-gated K(+) current in renal inner medullary collecting duct cells

We studied the K⁺-selective conductances in primary cultures of rat renal inner medullary collecting duct (IMCD) using perforated-patch and conventional whole cell techniques. Depolarizations above –20 mV induced a time-dependent outward K⁺ current (I_vto) similar to a delayed rectifier. I_vto showed a half-maximal activation around 5.6 mV with a slope factor of 6.8 mV. Its K⁺/Na⁺ selectivity ratio was 11.7. It was inhibited by tetraethylammonium, quinidine, 4-aminopyridine, and Ba²⁺ and was not Ca²⁺ dependent. The delayed rectifying characteristics of I_vto prompted us to screen the expression of Kv1 and Kv3 families by RT-PCR. Analysis of RNA isolated from cell cultures revealed the presence of three Kv -subunits (Kv1.1, Kv1.3, and Kv1.6). Western blot analysis with Kv -subunit antibodies for Kv1.1 and Kv1.3 showed labeling of 70-kDa proteins from inner medulla plasmatic and microsome membranes. Immunocytochemical analysis of cell culture and kidney inner medulla showed that Kv1.3 is colocalized with the Na⁺-K⁺-ATPase at the basolateral membrane, although it is also in the cytoplasm. This is the first evidence of recording, protein expression, and localization of a voltage-gated Kv1 in the kidney IMCD cells. kidney; Kv1.3; potassium channel; potassium transport; whole cell clamp; immunocytochemistry; confocal microscopy     

Calpain-mediated AQP2 proteolysis in inner medullary collecting duct

Vitamin D-elicited hypercalcemia/hypercalciuria is associated with polyuria in humans and in animal models. In rats, dihydrotachysterol (DHT) induces AQP2 water channel downregulation despite unaltered AQP2 mRNA expression and thus we investigated the mechanism of AQP2 degradation. Incubation of AQP2-containing inner medullary collecting duct (IMCD) endosomes with Ca(2+) or calpain elicited AQP2 proteolysis, an effect abolished by leupeptin. This endogenous, Ca(2+)-sensitive protease activity exhibited a different proteolytic digest pattern from trypsin, which also degraded AQP2 in vitro. IMCDs contain abundant micro-calpain protein and functional calpain proteolytic activity as demonstrated by immunohistochemistry, immunoblotting, and gel zymography. Furthermore, by small particle flow cytometry we demonstrated that micro-calpain colocalizes with apical IMCD endosomes. DHT does not appear to elicit general proteolysis, however, in addition to AQP2 degradation, DHT treatment also diminished micro-calpain and calpastatin expression although whether these changes contributed to the AQP2 instability remains unclear. Together, these data show for the first time that AQP2 is a substrate for calpain-mediated proteolysis and that furthermore, micro-calpain, like AQP2, is both highly expressed in renal inner medulla and localized to apical IMCD endosomes.     

Mechanisms of vasopressin-induced intracellular Ca2+ oscillations in rat inner medullary collecting duct

Arginine vasopressin (AVP) causes increase in intracellular Ca(2+) concentration with an oscillatory pattern. Ca(2+) mobilization is required for AVP-stimulated apical exocytosis in inner medullary collecting duct (IMCD). The mechanistic basis of these Ca(2+) oscillations was investigated by confocal fluorescence microscopy and flash photolysis of caged molecules in perfused IMCD. Photorelease of caged cAMP and direct activation of ryanodine receptors (RyRs) by photorelease of caged cyclic ADP-ribose (cADPR) both mimicked the AVP-induced Ca(2+) oscillations. Preincubation of IMCD with 100 μM 8-bromo-cADPR (a competitive inhibitor of cADPR) delayed the onset and attenuated the magnitude of AVP-induced Ca(2+) oscillations. These observations indicate that the cADPR/RyR pathway is capable of supporting Ca(2+) oscillations and endogenous cADPR plays a major role in the AVP-induced Ca(2+) oscillations in IMCD. In contrast, photorelease of caged inositol 1,4,5-trisphosphate (IP(3)) induced Ca(2+) release but did not maintain sustained Ca(2+) oscillations. Removal of extracellular Ca(2+) halted ongoing AVP-mediated Ca(2+) oscillation, suggesting that it requires extracellular Ca(2+) entry. AVP-induced Ca(2+) oscillation was unaffected by nifedipine. Intracellular Ca(2+) store depletion induced by 20 μM thapsigargin in Ca(2+)-free medium triggered store-operated Ca(2+) entry (SOCE) in IMCD, which was attenuated by 1 μM GdCl(3) and 50 μM SKF-96365. After incubation of IMCD with 1 nM AVP in Ca(2+)-free medium, application of extracellular Ca(2+) also triggered Ca(2+) influx, which was sensitive to GdCl(3) and SKF-96365. In summary, our observations are consistent with the notion that AVP-induced Ca(2+) oscillations in IMCD are mediated by the interplay of Ca(2+) release from RyRs and a Ca(2+) influx mechanism involving nonselective cation channels that resembles SOCE.     

Histochemical carbonic anhydrase in rat inner medullary collecting duct.

Rat inner medullary collecting duct (IMCD) secretes substantial amounts of H+. However, carbonic anhydrase (CA), a concomitant of H+ secretion, has been generally reported absent in this segment. To reexamine this problem, we investigated CA and the morphological phenotypes of cells comprising the IMCD by CA histochemistry, using a modified Hansson technique with light and electron microscopy. Throughout the medulla, tubule cells exhibit histochemical CA activity. In the initial third of the inner medulla, a small proportion have features of intercalated cells and demonstrate some degree of CA activity. However, the majority population in the early portions of the IMCD appears to consist of principal cells. These also show CA staining of widely variable intensity, both among and within cells. A third cell type, previously called "IMCD cells", appears in the middle portion of the IMCD and is the only cell type present near the papilla tip. In contrast to previous reports, these "IMCD cells" have histochemical CA staining, also of highly variable intensity. These results demonstrate that stainable carbonic anhydrase to support acidification is present throughout the rat IMCD, both in intercalated cells and in some cells clearly not of this type. Therefore, the presence of CA is not specific for the intercalated cell type and suggests that other cell types may participate in acid secretion in IMCD.     

Adenosine triphosphate inhibits endothelin-1 production by rat inner medullary collecting duct cells

Adenosine triphosphate (ATP) and endothelin (ET)-1 inhibit vasopressin-stimulated water reabsorption in the inner medullary collecting duct (IMCD). Because both ATP and ET-1 are released by the IMCD and can act in an autocrine manner to regulate IMCD water transport, we sought to determine whether these factors can modulate the other's production. To begin such studies, the effect of ATP on IMCD ET-1 production was examined. ATP caused a dose-dependent inhibition of ET-1 release and inhibited ET-1 mRNA levels in primary cultures of rat IMCD cells. This effect was first evident after 4 hrs of exposure to ATP and persisted for at least 24 hrs. The 50% inhibitory concentration for ATP inhibition of ET-1 production was approximately 1 microM, and the maximal response was observed at 25-100 microM. ATP acted, at least in part, through the P2Y2 receptor because its effect was mimicked by UTP, but not by the P2X agonist, alpha,beta-methylene-ATP. N-methyl-L-arginine, or indomethacin, did not block the ATP inhibitory effect. In summary, these data demonstrate that ATP inhibits IMCD ET-1 protein and mRNA accumulation, that this is mediated via P2Y receptors, and that the ATP effect is independent of cyclooxygenase or nitric oxide synthase metabolites. These findings suggest that although ATP and ET-1 both antagonize vasopressin action in the IMCD, they may have a complex interaction that ultimately determines the degree to which they each participate in modulating collecting duct function.     

Characterization of L-arginine transporters in rat renal inner medullary collecting duct

Previous work from our laboratory has demonstrated that the inner medullary collecting duct (IMCD) expresses a large amount of nitric oxide synthase (NOS) activity. The present study was designed to characterize the transport of NOS substrate, L-arginine, in a suspension of bulk-isolated IMCD cells from the Sprague-Dawley rat kidney. Biochemical transport studies demonstrated an L-arginine transport system in IMCD cells that was saturable and Na(+) independent (n = 6). L-Arginine uptake by IMCD cells was inhibited by the cationic amino acids L-lysine, L-homoarginine, and L-ornithine (10 mmol/l each) and unaffected by the neutral amino acids L-leucine, L-serine, and L-glutamine. Both L-ornithine (n = 6) and L-lysine (n = 6) inhibited NOS enzymatic activity in a dose-dependent manner in IMCD cells, supporting the important role of L-arginine transport for NO production by this tubular segment. Furthermore, RT-PCR of microdissected IMCD confirmed the presence of cationic amino acid transporter CAT1 mRNA, whereas CAT2A, CAT2B, and CAT3 were not detected. These results indicate that L-arginine uptake by IMCD cells occurs via system y(+), is encoded by CAT1, and may participate in the regulation of NO production in this renal segment.