Rat Renal Tubules Research Articles

Age-associated changes in ammoniagenesis in isolated rat renal tubule segments.

We investigated renal ammoniagenesis in isolated nephron segments from control and acidotic senescent and young adult rats. When young (6 mo) and senescent (24 mo) control groups were compared, there was no significant difference in glutamine-dependent ammonia production in any nephron tubule segments. However, ammonia production rates in glomeruli from old rats were significantly greater than the rate from young rats and were correlated with the serum creatinine and blood urea nitrogen (BUN) levels. After giving young and old rats an equivalent acid load (by gavage) of ammonia chloride solutions (6 mo, blood pH 7.34; 24 mo, blood pH 7.07), we measured a significant increase in ammoniagenesis on the S1 and S2 segments of proximal tubules and the distal convoluted tubules from old rats, and no increase in any segment from young rats. When we increased the acid load in young rats to an equivalent severity of acidosis (blood pH 7.07), we found significant increases in ammonia production in the S1, S2, S3, and distal convoluted tubule. With comparable blood pH values, ammoniagenesis in S1, S2, and S3 segments from young rats was about double the values measured in segments from senescent rats. The severity of the acidosis in the 24-mo-old rats was related to serum creatinine and BUN. Our findings show that ammoniagenesis in isolated segments from senescent rats is qualitatively similar to their younger counterparts but that this maximum capacity to generate ammonia is reduced. The change in nephron capacity to synthesize ammonia may be the result of age-associated physiological changes and/or the chronic renal failure exhibited in old rats.

Biosynthesis of guanidinoacetic acid in isolated renal tubules.

Guanidinoacetic acid, a precursor of creatine, is an essential substrate for muscle energy metabolism. Since guanidinoacetic acid has been reported to be synthesized from arginine and glycine by glycine amidinotransferase (transamidinase) in kidney homogenates or slices, the purpose of this study was to provide evidence of guanidinoacetic acid synthesis in isolated tubules from rat kidneys, and to clarify the mechanism regulating it. Isolated rat tubules were incubated with various substrates. Guanidinoacetic acid was separated by high performance liquid chromatography and measured fluorometrically. Results obtained were as follows: (1) Guanidinoacetic acid was synthesized from arginine or canavanine and glycine in isolated rat tubules. (2) D,L-Norvaline, ornithine and methionine suppressed guanidinoacetic acid synthesis. (3) Creatine suppressed guanidinoacetic acid synthesis, i.e. creatine was a negative feedback inhibitor of guanidinoacetic acid synthesis in this in vitro system. (4) Guanidinoacetic acid was not synthesized from hydroxyurea, citrulline, argininosuccinic acid or canaline. These data demonstrate that guanidinoacetic acid is synthesized only from arginine or canavanine and glycine, and that the guanidine cycle may not function fully in the rat renal tubule.

Renal heme metabolism in hereditary tyrosinemia: use of succinylacetone in rat renal tubules

Succinylacetone (SA), a metabolic end-product found in urine from individuals with hereditary tyrosinemia and associated renal Fanconi syndrome and a known inhibitor of hepatic 5-aminolevulinic acid dehydratase (ALAD), has been used to study heme metabolism in isolated rate renal tubules. Heme biosynthetic porphyrin precursors are increased selectively in the presence of 4 mmol/1 SA. Total porphyrin content of the tubules are increased approximately 2-fold, while both ferrochelatase and heme oxygenase activities remain unaffected by SA. Nonetheless, total heme content is reduced, as was incorporation of radioactive label from amino[ 14C]levulinic acid. Cytochrome P-450 content remained unaffected. Impairment of iron uptake and/or transport within the cell or enhancement of heme catabolism via a non-heme oxygenase-dependent pathway could explain the observations.

Effect of Na-channel blockers and lumen Ca on K secretion by rat renal distal tubule

In previous studies the effectiveness of amiloride (AML) in reducing K secretion has been variable. Based on studies by Cuthbert and Wong (Mol. Pharmacol. 8: 222-229, 1972) in which the Na-channel-blocking action of AML in frog skin was found to require the availability of Ca ions in extracellular fluid, we postulated that the ability of AML and its analogue, benzamil (BZA), to inhibit distal tubule K secretion depends on the presence of Ca in luminal fluid. We found that addition of Ca to a perfusion solution containing 50 microM BZA did reduce K secretion more than BZA alone. Maximal inhibition was observed with 2.5 mM free ionic Ca. Graded increases in luminal Ca in presence of AML or BZA reduced K transport in a dose-dependent manner. The decrease in K secretion with increasing luminal Ca was paralleled by a decrease in transepithelial voltage. These results support our hypothesis that the effectiveness of Na-channel blockers to reduce K secretion by the rat distal tubule depends on presence of luminal Ca and suggest an interaction between luminal Ca and Na-channel blockers on the Na channel.

Inhibition by Phorbol Ester of Cellular Adenosine 3′,5′-Monophosphate Production and Cellular Free Calcium Mobilization in Response to Arginine Vasopressin in Rat Renal Papillary Collecting Tubule Cells in Culture*

We determined whether tumor-promoting factor phorbol ester modulates cellular cAMP production and the cellular free calcium concentration ([Ca2+]i) in response to arginine vasopressin (AVP) in rat renal papillary collecting tubule cells in culture. In the presence of 5 x 10(-4) M 3-isobutyl-1-methylxanthine, AVP increased cellular cAMP production in a dose-dependent manner. A 1-h exposure to 3 x 10(-7)-3 x 10(-6) M phorbol-12-myristate-13-acetate (PMA) significantly attenuated the cAMP response to AVP (1 x 10(-9) M AVP; 474.9 +/- 24.8 vs. 368.1 +/- 22.8 fmol/microgram protein; P less than 0.01). The dose-response relation with AVP thus shifted to the right. Such an inhibition was totally reversed in the presence of 2 x 10(-6) M 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase-C. Also, 1 x 10(-7) M AVP produced an increase in [Ca2+]i from 99.4 +/- 3.3 to 200.0 +/- 8.6 nM. When cells were preexposed to 1 x 10(-6) M PMA, an increase in [Ca2+]i in response to 1 x 10(-7) M AVP was significantly diminished (75.5 +/- 4.6 to 101.4 +/- 4.3 nM). The inhibition by PMA of AVP-induced increment in [Ca2+]i was significantly attenuated in the presence of 2 x 10(-5) M H-7 compared to that in its absence. Prolonged exposure to PMA did not alter the AVP-induced increases in cAMP production and [Ca2+]i. These results indicate that phorbol ester inhibits the cellular action of AVP mediated through the activation of protein kinase-C and suggest that there is an interaction between cAMP and phosphatidylinositol systems in modulating the AVP action in renal papillary collecting tubule cells.

Effect of lindane upon the β-adrenergic stimulation of cyclic AMP accumulation in rat renal cortical tubules caused by alterations in membrane fluidity

The influence of lindane (gamma-hexachlorocyclohexane) on fludity and lipid composition in rat renal cortical tubules has been investigated. Lindane increased membrane fluidity as measured by a fluorescence polarization technique using the probe diphenylhexatriene. This effect was dose-dependent and was accompanied by a 70% inhibition of the β-adrenergic stimulatory activity upon cyclic AMP accumulation after 30 min of preincubation with lindane at 25°C. Experiments with increasing concentrations of isoproterenol indicated that the efficacy, but not the potency, of the β-adrenergic effect upon cyclic AMP accumulation was affected by lindane. Lindane toxicity could also be associated with variations in the incorporation of acetate into various lipid classes. Lindane increased acetate incorporation into phospholipids and decreased that into cholestorol.

Biological Properties of a Renotropic Protein Present in Plasma of Uninephrectomized Rats

The biological characteristics of a kidney growth factor (KGF) from uninephrectomized rat plasma have been studied. A crude preparation of this factor [Nephrol. Dial. Transplant. 4: 334-338, 1989] was further purified by hydrophobic interaction HPLC and gel filtration. KGF was found to be a heat- and trypsin-resistant protein. This factor stimulated dose-dependently DNA synthesis by the mouse kidney in vivo, and by either rat renal tubules or serum-deprived LLC-PK1 cells, in vitro. KGF also increased protein synthesis in these cells, in a dose-dependent manner. Moreover, KGF stimulated sodium uptake by these cells, associated with the maximal increase of protein synthesis. Our findings indicate that KGF is a potent renotropic protein which can play a key role in the renal compensatory growth after uninephrectomy.

Phosphate-dependent glutaminase activity in rat renal cortical and medullary tubule segments

To determine whether local production of ammonium by medullary renal tubule segments may contribute to medullary ammonium accumulation, we measured activities of phosphate-dependent glutaminase (PDG) in microdissected tubule segments from rat medulla and cortex. PDG activities were very low in medullary loop of Henle segments but surprisingly high in inner medullary collecting duct (IMCD). In cortex, PDG levels were highest in distal convoluted tubule and cortical thick ascending limb, but substantial levels were also found in proximal segments, as reported previously. To determine effects of acid loading and alkali loading on PDG activity, 0.28 M NH4Cl (acid) or 0.28 M NaHCO3 (alkali) was added to rats' drinking water for 7 days. PDG activities in medullary segments were not affected by acid or alkali intake. Acid intake by rats increased PDG activity in S1 and S2 proximal convoluted tubules severalfold but did not affect the other cortical segments. We conclude that medullary loop of Henle segments probably contribute relatively little to medullary ammonium accumulation because of their low activities. The high PDG activity in IMCD suggests that ammonium could be produced and secreted by this segment. However, because total tubule length of IMCD is very low compared with proximal tubules, it appears unlikely that IMCD contributes substantially to overall renal ammonium production. PDG activity is regulated only in S1 and S2 proximal tubules, consistent with the view that the proximal tubule is the major site of regulation of renal ammonium production.

Catabolism of Cyst(e)ine by Rat Renal Cortical Tubules

Both cysteinesulfinate-independent and cysteinesulfinate-dependent pathways are involved in the catabolism of cyst(e)ine by freshly isolated rat renal cortical tubules. Sulfate and thiosulfate were shown to be the major sulfur-containing products that accumulated in incubations of renal tubules with 1 mmol/L or 25 mmol/L [35S]cyst(e)ine. Thiosulfate is an intermediate in the oxidation of the sulfide produced by the cysteinesulfinate-independent catabolism of cyst(e)ine by desulfhydration pathway(s), whereas sulfate is formed both by further oxidation of thiosulfate and by oxidation of the sulfite formed by the cysteinesulfinate-transamination pathway. Incubation of renal tubules with propargylglycine inhibited gamma-cystathionase activity by 85%, and this resulted in a 46% decrease in sulfate production and a 68% decrease in thiosulfate production when the treated renal tubules were incubated with 1 mmol/L [35S]cyst(e)ine. Addition of 25 mmol/L unlabeled cysteinesulfinate to create a diluting/trapping pool for [35S]cysteinesulfinate formed from [35S]cysteine resulted in a 53% decrease in [35S]sulfate production in incubations with 1 mmol/L cysteine. Thus, some cyst(e)ine catabolism probably occurred by a cysteinesulfinate-dependent pathway. No production of taurine or hypotaurine was detected in incubations with cyst(e)ine. Thus, cysteinesulfinate formed from cysteine was further catabolized primarily to sulfate instead of to taurine and hypotaurine. Most cyst(e)ine catabolism by the epithelial cells of the renal tubule probably can be accounted for by two pathways: 1) the beta-cleavage of cystine catalyzed by lambda-cystathionase and 2) the formation and transamination of cysteinesulfinate catalyzed by cysteine dioxygenase and aspartate aminotransferase.

High protein intake accelerates the maturation of Na, K‐ATPase in rat renal tubules

We have examined the effect of a high protein intake on renal maturation by studying the Na,K-ATPase activity in proximal convoluted tubules (PCT) and medullary thick ascending limb of Henle (TAL) in kidneys from 16- to 24-day-old rats. The rats received an isocaloric diet containing 21% (normal protein, NP) and 50% (high protein, HP) protein from 16 days of age. HP diet significantly increased the nephron filtration rate after 2 days. There was a proportionate increase in proximal tubular reabsorption. HP diet significantly increased Na,K-ATPase in both PCT and TAL after 4 days, but not after 2 days. Although HP diet caused a similar increase in Na delivery to PCT and TAL, the increase in Na,K-ATPase activity after 8 days was more pronounced in TAL than in PCT. To determine whether stimulation of Na,K-ATPase required the presence of adrenal corticoid hormones, rats adrenalectomized at 16 days of age were studied. HP diet significantly increased Na,K-ATPase in both PCT and TAL from adrenalectomized rats. To determine whether stimulation of Na,K-ATPase in PCT was mediated by increased Na entry via the amiloride-sensitive Na/H exchanger, rats were treated with amiloride via an osmotic minipump from 16 to 20 days of age. Amiloride treatment significantly retarded the normal increase of Na,K-ATPase in NP rats but had no effect on ouabain-insensitive ATPase. HP diet significantly increased PCT Na,K-ATPase, in spite of chronic amiloride treatment. HP intake accelerates maturation of Na,K-ATPase in PCT and TAL regardless of the presence of adrenal corticoid hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon flux through tricarboxylic acid cycle in rat renal tubules

Our aim was to delineate the effect(s) of chronic metabolic acidosis on renal TCA-cycle metabolism. Renal tubules isolated from control and chronically acidotic rats were incubated at pH 7.4 with either 2 mM [2,3- 13C]pyruvate or [2- 13C]acetate. GC-MS and/or 13C-NMR were utilized to monitor the flux of 13C through pyruvate dehydrogenase, pyruvate carboxylase and the TCA-cycle. With either, precursor acidosis was associated with significantly decreased formation of 13C-labelled citrate, malate, aspartate and alanine and increase formation of glucose, lactate and acetyl-CoA as compared with the control. The results indicate that adaptation of renal metabolism to chronic metabolic acidosis is associated with diminished flux through citrate synthetase and concomitantly increased flux through pyruvate carboxylase. The data suggest that depletion of TCA-cycle intermediates and enhanced ammoniagenesis in the kidney of chronically acidotic rats may be regulated at the site of mitochondrial citrate-condensing enzyme.

Effect of cystine loading on substrate oxidation by rat renal tubules

Isolated rat renal tubules were loaded with cystine by incubating them with 2 mM cystine dimethylester. The oxidation of 1 mM glucose and lactate was significantly decreased after 20 and 30 min of incubation, in the cystine-loaded tubules compared with control tubules. The oxidation of 1 mM butyrate was significantly decreased after 10 and 30 min of incubation in the cystine-loaded tubules. Cystine loading decreased the oxidation of 1 mM succinate at all time points examined. The O2 consumption of renal tubules was reduced 59% with cystine loading by the addition of 2 mM cystine dimethylester, and by 37% with 1 mM cystine dimethylester. These data indicate that loading normal renal tubule cells with cystine impairs their ability to oxidize metabolic fuels. This impairment in metabolism may explain the decreased transport observed previously in cystine-loaded tubules and may have implications for the human disorder, cystinosis.

Vasopressin-Induced Increases in Cellular Free Calcium Concentration Measured in Single Cells of Rat Renal Papillary Collecting Tubule.

We determined the cellular free calcium concentration [Ca2+]i in response to arginine vasopressin (AVP) using single cells of cultured rat renal papillary collecting tubule cells. AVP at a concentration of 1 x 10(-10) M or higher significantly increased [Ca2+]i in a dose-dependent manner. The prompt increase in [Ca2+]i induced by AVP was completely blocked by the V1V2 antagonist, but not by the V1 antagonist. Also, an antidiuretic agonist of 1-deamino-8-D-arginine vasopressin (dDAVP) increased [Ca2+]i, which was blocked by the pretreatment with the V1 V2 antagonist. An AVP-induced increase in [Ca2+]i was still demonstrable in cells pretreated with Ca2(+)-free medium containing 1 x 10(-3) M EGTA, or a blocker of cellular Ca2+ uptake, 5 x 10(-5) M verapamil. These results indicate that AVP increases [Ca2+]i through the V2 receptor in renal papillary collecting tubule cells where cAMP is a well-known second messenger for AVP, and that cellular free Ca2+ mobilization depends on both the intracellular and extracellular Ca2+.

Immunoenzyme localization of cathepsins in the Golgi region of rat hepatocytes and renal tubule cells

Localization of carboxyl proteinase (cathepsin D) and cysteine proteinases (cathepsins B, H, and L) in Golgi region was studied using an immunoenzyme technique. Rat livers and kidneys were used. The results obtained from the livers were similar to those from the kidneys. All cathepsins were detected in lysosomal compartments such as secondary lysosomes, multivesicular bodies (endosomes), and autophagosomes. Rough endoplasmic reticulum (rER), including nuclear envelope was focally stained. Most of Golgi cisternae were negative, but sometimes only one cisterna or the terminal portion of the cisterna were stained focally. Rarely, the trans Golgi network (TGN) was positive for the proteinases. Among numerous Golgi vesicles, only a few of them were stained. The positive vesicles were divided into two groups, one had a bristle coat and heavily stained, and other were smaller than 40 nm in diameter and weakly stained. The small vesicles seemed to bud from the ER and to fuse with the Golgi cisternae, while the large clathrin-coated vesicles seem to bud from the TGN. The results suggests that cathepsins are transported by vesicular system from the rER to lysosomes via Golgi apparatus. In addition, it is suggested that the small vesicles transport the proteinases from the ER to the Golgi cisternae and the large clathrin-coated vesicles from the Golgi cisternae to the lysosomes.

Proton compartmentation in rat renal cortical tubules

To study the control of renal ammoniagenesis, a technique was developed to estimate simultaneously intracellular (pHi) and intramitochondrial (pHm) pH in suspensions of rat renal cortical tubules. pHi was estimated with the fluorescent probe 2',7'biscarboxyethyl-5(6)-carboxy-fluorescein (BCECF). The intracellular distribution of the weak acid 5,5-dimethyloxazolidine-2,4-dione (DMO) allowed calculation of pHm with the use of values of pHi obtained with BCECF and tubule mitochondrial content. At medium pH (pHe) 7.4, pHi was 7.08 +/- 0.02. Over the pHe range 7.0-7.7, pHi was linearly related to pHe, but the pH gradient across the cell membrane decreased as pHe was lowered. No difference in the relationship between pHe and pHi was obtained when tubules were incubated in the presence of a nonbicarbonate or bicarbonate-buffered medium. Changes in pHe with bicarbonate-buffered media resulted in identical pHi values, whether the changes were induced by altered bicarbonate or CO2 content. At pHe 7.4, pHm was 7.78 +/- 0.6 in bicarbonate-buffered medium but was higher (0.2-0.3 pH units) when tubules were bathed in nonbicarbonate-buffered medium. pHm was linearly related to pHi in either buffer. The pH gradient across the inner mitochondrial membrane was also positively correlated with pHe. The present studies indicate the suitability of the techniques for estimating pHi and pHm simultaneously in suspensions of rat renal cortical tubules. Parallel changes occur in both intracellular compartments when pHe is altered. pHm, which is approximately 0.7 pH units greater than pHi, decreases in acute acidosis. This decrease may be important in stimulating renal ammoniagenesis, possibly by activation of alpha-ketoglutarate dehydrogenase.

Effect of Valproate on Lactate and Glutamine Metabolism by Rat Renal Cortical Tubules

The metabolic effects of sodium valproate (VPA) on rat renal cortical tubules have been examined. When 1 or 5 mM lactate was used as substrate in the incubation medium, VPA decreased markedly the lactate uptake by the tubules. When 1 or 5 mM glutamine was used, the addition of VPA accelerated glutamine uptake, ammoniagenesis, but also stimulated markedly the accumulation of lactate and pyruvate produced from glutamine. VPA had a dose-dependent inhibitory effect on gluconeogenesis from both glutamine and lactate. With 5 mM glutamine, VPA also induced a significant accumulation of glutamate in the medium. The oxygen consumption by the tubules was diminished by 40% following VPA addition. It is concluded that VPA modifies the metabolism of rat cortical tubules by interfering with the oxidation of natural substrates and stimulates in this fashion the production of ammonia by kidney tubules.

Fate of cadmium in rat renal tubules: A micropuncture study

Free-flow micropuncture was carried out in superficial nephrons of Munich-Wistar type rats infused acutely with Cd acetate (CdA) or Cd-DTPA (141 μ m Cd). Fluid obtained from Bowman's space (BS) or end-proximal tubule sites was analyzed for Cd and inulin. The fluid/plasma Cd concentration ratio in BS averaged 0.2 and 1.0 during CdA and Cd-DTPA infusions, respectively. End-proximal tubule fractional excretion of Cd during CdA infusion averaged 0.34. Previous administration of CdA (1.0 mg/kg, 48 hr before micropuncture) increased the level of circulating Cd-metallothioneins, as measured by radioimmunoassay, but did not affect the luminal tubular uptake of Cd during CdA infusion. No net transepithelial movement of Cd-DTPA was measured. It is concluded that Cd ultrafiltered during inorganic Cd administration is taken up to a large extent by the convoluted part of proximal tubules.

Aldose reductase activities in microdissected rat renal tubule segments

Osmoregulation in inner medullary cells depends in part on cellular accumulation of sorbitol, the production of which from glucose is catalyzed by aldose reductase. To identify nephron segments that contain aldose reductase, we developed a fluorometric ultramicroassay to measure aldose reductase activity in microdissected nephron segments from collagenase-treated kidneys of Sprague-Dawley rats. DL-Glyceraldehyde (10 mM) was used as a substrate. Substantial aldose reductase activities were found in all three inner medullary renal tubule segments: thin descending limbs, thin ascending limbs, and inner medullary collecting ducts. Activity increased with depth into the inner medulla in all three segments. When aldose reductase activities were normalized by cell volume the activities in the three inner medullary segments were similar. Little or no aldose reductase activity was measured in glomeruli or any cortical or outer medullary nephron segment. Both proximal convoluted and proximal straight tubules were found to have a substantial capacity to reduce DL-glyceraldehyde, but the finding of greater reductase activity with D-glucuronate (10 mM) than with D-xylose (10 mM) indicated that the activity was due to aldehyde reductase. Sorbitol dehydrogenase (measured by a similar ultramicroassay method) was present in substantial amounts in proximal tubules, but not in inner medullary collecting ducts. The overall pattern of enzyme activities is consistent with the proposed osmoregulatory role for sorbitol in all three inner medullary renal tubule segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal gluconeogenic response to different diuretics. II. Glucose production in the early hours after mersalyl, furosemide and ethacrynic acid administration

The effect of mersalyl, furosemide and ethacrynic acid administration on renal glucose production was investigated in rat renal tubules. Administration of ethacrynic acid at a dose of 50 mg/Kg animal weight provoked a transient inhibition of gluconeogenesis which became significant 3 1 2 hours after injection. Administration of an identical dose of furosemide did not affect glucose production whereas mersalyl at a dose of 15 mg/Kg exerted a severe impairment as early as 30 minutes after injection, which lasted for a long period of time. The in vivo effects of diuretics on gluconeogenesis correlate well with the previously observed in vitro effect, although ethacrynic acid showed less potency as an inhibitor in the former, probably because of its rapid clearance.

Enhancement by Veratridine and 60 mM KC1 of Vasopressin-Induced 3′,5′-Cyclic Adenosine Monophosphate Production and Cellular Free Calcium Concentration in Rat Renal Papillary Collecting Tubule Cells in Culture*

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using depolarizing agents in rat renal papillary collecting tubule cells in culture. One-hour exposure of cells to veratridine enhanced AVP-induced cAMP production in a dose-dependent manner. This enhancement by veratridine of cellular cAMP production in response to AVP was totally blunted by cotreatment with 5 X 10(-4) M verapamil, 3 X 10(-3) M cobalt, or Ca2+-free medium containing 1 X 10(-3) M EGTA. These agents block cellular Ca2+ uptake by different mechanisms. Similarly, 60 mM KCl enhanced AVP-induced cAMP production, and this effect was blocked by pretreatment with verapamil, cobalt or Ca2+-free medium containing 1 X 10(-3) M EGTA. When cellular free Ca2+ concentrations [Ca2+]i were measured by the fluorescence dye fura-2, both 1 X 10(-4) M veratridine and 60 mM KCl significantly increased [Ca2+]i from 67.6 to 141.8 nM and from 74.6 to 166.2 nM, respectively. Such rises in [Ca2+]i depended on extracellular Ca2+ since the increase in [Ca2+]i was completely blocked in Ca2+-free medium or in the presence of 3 X 10(-3) M cobalt. In addition, veratridine and 60 mM KCl significantly augmented the AVP-induced increase in [Ca2+]i. The possible mechanisms by which depolarizing agents induce cellular Ca2+ mobilization include the opening of voltage-sensitive Ca2+ channels in the plasma membrane. The present results indicate that veratridine and 60 mM KCl enhance AVP-induced cAMP production and cellular free Ca2+ concentration through cellular Ca2+ uptake in renal papillary collecting tubule.