The effects of genetic polymorphisms in the organic cation transporters OCT1, OCT2, and OCT3 on the renal clearance of metformin.

Organic cation transporters (OCTs) can mediate metformin transmembrane transport. We explored metformin pharmacokinetics in relation to genetic variations in OCT1, OCT2, OCT3, OCTN1, and MATE1 in 103 healthy male Caucasians. Renal clearance varied 3.8-fold and was significantly dependent on creatinine clearance (r(2) = 0.42, P < 0.0001), age (r(2) = 0.09, P = 0.002), and OCT1 polymorphisms. Carriers of zero, one, and two low-activity OCT1 alleles (Arg61Cys, Gly401Ser, 420del, or Gly465Arg) had mean renal clearances of 30.6, 33.1, and 37.1 l/h, respectively (P = 0.04, after adjustment for creatinine clearance and age). Immunohistochemical staining of human kidneys demonstrated OCT1 expression on the apical side of proximal and distal tubules. Increased renal clearance, in parallel with the known decreased hepatic uptake, may contribute to reduced metformin efficacy in low-activity genotypes. Renal OCT1 expression may be important not only in relation to metformin but with respect to other drugs as well.

Subchronic cadmium treatment affects the abundance and arrangement of cytoskeletal proteins in rat renal proximal tubule cells.

Disfunction of proximal tubules (PT) in cadmium (Cd) nephrotoxicity in mammals results from the diminished functional capacity of brush-border membrane (BBM) caused by (a) direct inhibition of BBM transporters by Cd, (b) shortening and loss of microvilli, and (c) loss of specific BBM transporters. The loss of transporters may partially result from impaired intracellular vesicle recycling due to loss or/and inhibition of vacuolar H+-ATPase in the PT cell organelles. Cytoskeleton plays an important role in vesicle-mediated recycling and processing of BBM transporters in PT cells. Experiments in vitro have indicated that Cd may affect the state of polymerization of some cytoskeletal proteins. In this work we studied the in vivo effect of CdCl2-treatment in rats (2 mg Cd/kg b. m., s.c., daily for 14 days) upon abundance and arrangement of actin filaments, actin-bundling protein villin, and microtubules (MT) in PT cells. Cd-treatment elicited a dramatic accumulation of Cd in the kidney cortex (200 microg/g tissue wet mass after 14 days) and a strongly increased abundance of metallothionein in PT cells. As revealed by immunocytochemistry in tissue cryosections, the staining intensity of actin and villin in PT cells of Cd-treated rats was generally decreased, without a marked change in their intracellular distribution, whereas MT became largely irregular, diminished in most cells, and lost in many cells. However, the immunoblots revealed an increased content of villin and alpha-tubulin in cortical tissue homogenates from Cd-treated rats, thus indicating an impaired bundling of actin and greatly depolymerized MT in cells intoxicated with Cd. The partial loss of apical actin and villin in PT cells of Cd-treated rats may reflect (or cause) shortening and loss of microvilli, whereas derangement and depolymerization of MT may contribute to the impairment of intracellular recycling of BBM proteins, and lead to the loss of BBM transporters.

Aquaporin 9 expression along the male reproductive tract.

Fluid movement across epithelia lining portions of the male reproductive tract is important for modulating the luminal environment in which sperm mature and reside, and for increasing sperm concentration. Some regions of the male reproductive tract express aquaporin (AQP) 1 and/or AQP2, but these transmembrane water channels are not detectable in the epididymis. Therefore, we used a specific antibody to map the cellular distribution of another AQP, AQP9 (which is permeable to water and to some solutes), in the male reproductive tract. AQP9 is enriched on the apical (but not basolateral) membrane of nonciliated cells in the efferent duct and principal cells of the epididymis (rat and human) and vas deferens, where it could play a role in fluid reabsorption. Western blotting revealed a strong 30-kDa band in brush-border membrane vesicles isolated from the epididymis. AQP9 is also expressed in epithelial cells of the prostate and coagulating gland where fluid transport across the epithelium is important for secretory activity. However, it was undetectable in the seminal vesicle, suggesting that an alternative fluid transport pathway may be present in this tissue. Intracellular vesicles in epithelial cells along the reproductive tract were generally poorly stained for AQP9. Furthermore, the apical membrane distribution of AQP9 was unaffected by microtubule disruption. These data suggest that AQP9 is a constitutively inserted apical membrane protein and that its cell-surface expression is not acutely regulated by vesicular trafficking. AQP9 was detectable in the epididymis and vas deferens of 1-wk postnatal rats, but its expression was comparable with adult rats only after 3--4 wk. AQP9 could provide a route via which apical fluid and solute transport occurs in several regions of the male reproductive tract. The heterogeneous and segment-specific expression of AQP9 and other aquaporins along the male reproductive tract shown in this and in our previous studies suggests that fluid reabsorption and secretion in these tissues could be locally modulated by physiological regulation of AQP expression and/or function.

Serotonin transporter in rat platelets. Level of protein expression underlies inherited differences in uptake kinetics.

By breeding selection for the extreme values of platelet serotonin level (PSL), two sublines of Wistar-derived rats, with constitutionally high or low PSL and platelet serotonin uptake (PSU), have been developed. Searching for the basis of these differences, we performed quantitative western blot analysis of serotonin transporter (5HTt) in platelet membranes isolated from both rat sublines. A polyclonal anti-5HTt antibody labeled a single, 5HTt-related 94 kDa protein band in platelet membranes, with significantly stronger intensity in membranes from rats that exhibited a high PSL. We conclude that the inherited differences in PSL and PSU in rats, following breeding selection, are determined by the level of 5HTt expression in platelet membranes.

Distribution of the vacuolar H+ atpase along the rat and human male reproductive tract.

Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.

The integrity of renal cortical brush-border and basolateral membrane vesicles is damaged in vitro by nephrotoxic heavy metals.

Poisoning of experimental animals with cadmium (Cd), mercury (Hg), lead (Pb) or cis-diamminedichloroplatinum (cis-Pt) causes shortening and focal loss of microvilli in proximal tubule (PT) cells, thus indicating that the reduced reabsorptive surface due to damaged integrity of brush-border membrane (BBM) may contribute to the reabsorptive and secretory defects in these toxic states. In addition, in in vitro studies with isolated renal cortical BBM vesicles (BBMV), heavy metals (HM) inhibit transport of various compounds, and these data were interpreted as being a result of a direct inhibition of the respective membrane transporters. In this work we used a DeltapH-driven acridine orange fluorescence quench assay to test if various divalent cations affect in vitro the integrity of BBMV and basolateral membrane vesicles (BLMV) isolated from the rat renal cortex. In Cd-treated BBMV we found that: (a) the integrity of vesicles decreased with increasing concentrations of Cd; and (b) the loss of sealed vesicles was high at 37 degrees C, intermediate at 25 degrees C, and very low at 0 degrees C. The loss of sealed BBMV was caused also by Hg, Cu, Pb and Zn (Hg>>>Cu=Cd>Pb=Zn). Cis-Pt, Al, Fe, Ba, Mg and Mn had no effect. BLMV were damaged by HM with an efficiency Hg>>>Cd=Pb=Cu, whereas other divalent cations, including Zn, were ineffective. An SH-group protector, dithiothreitol, prevented the loss of sealed vesicles in some (Hg, Pb, Cu) but not in all (Cd, Zn) cases. We conclude that the nephrotoxic HM directly damage the integrity of PT cell plasma membranes; this may cause shortening and loss of microvilli and basolateral invaginations in HM-treated experimental animals in vivo. The data also indicate that caution should be taken when effects of HM on various transports are studied in isolated membrane vesicles in vitro; an impaired transport may result from the loss of vesicle integrity, and not necessarily from the direct inhibition of a transporter.

Cadmium inhibits vacuolar H(+)ATPase-mediated acidification in the rat epididymis.

In rats, an acidic luminal pH maintains sperm quiescence during storage in the epididymis. We recently showed that vacuolar H(+)ATPase-rich cells in the epididymis and vas deferens are involved in the acidification of these segments. Treatment of rats with cadmium (Cd) leads to alkalinization of this fluid by an unknown mechanism. Because Cd may affect H(+)ATPase function, we examined 1) the in vivo effect of Cd poisoning on H(+)ATPase-rich cell morphology and on the abundance and distribution of the 31-kDa H(+)ATPase subunit in cells along the rat epididymis, and 2) the in vitro effect of Cd on H(+)ATPase activity and function in the isolated vas deferens. Immunofluorescence and immunoblotting data from rats treated with Cd for 14-15 days (2 mg Cd/kg body mass/day) showed that 1) H(+)ATPase-positive cells regressed to a prepubertal phenotype, and 2) H(+)ATPase was lost from the apical pole of the cell and was redistributed into an intracellular compartment. In experiments in vitro, Cd inhibited bafilomycin-sensitive ATPase activity in isolated total cell membranes and, as measured using a proton-selective extracellular microelectrode, inhibited proton secretion in isolated vas deferens. We conclude that alkalinization of the tubule fluid in the epididymis and vas deferens of Cd-treated rats may result from the loss of functional H(+)ATPase enzyme in the cell apical domain as well as from a direct inhibition of H(+)ATPase function by Cd.

Tetanus toxin-mediated cleavage of cellubrevin inhibits proton secretion in the male reproductive tract.

Our laboratory has previously shown that the vacuolar H(+)-ATPase, located in a subpopulation of specialized cells establishes a luminal acidic environment in the epididymis and proximal part of the vas deferens (Breton S, Smith PJS, Lui B, and Brown D. Nat Med 2: 470-472, 1996). Low luminal pH is critical for sperm maturation and maintenance of sperm in a quiescent state during storage in these organs. In the present study we examined the regulation of proton secretion in the epididymis and vas deferens. In vivo microtubule disruption by colchicine induced an almost complete loss of H(+)-ATPase apical polarity. Endocytotic vesicles, visualized by Texas red-dextran internalization, contain H(+)-ATPase, indicating active endocytosis of the pump. Cellubrevin, an analog of the vesicle soluble N-ethyl malemide-sensitive factor attachment protein (SNAP) receptor (v-SNARE) synaptobrevin, is highly enriched in H(+)-ATPase-rich cells of the epididymis and vas deferens, and tetanus toxin treatment markedly inhibited bafilomycin-sensitive proton secretion by 64.3+/-9.0% in the proximal vas deferens. Western blotting showed effective cleavage of cellubrevin by tetanus toxin in intact vas deferens, demonstrating that the toxin gained access to cellubrevin. These results suggest that H(+)-ATPase is actively endocytosed and exocytosed in proton-secreting cells of the epididymis and vas deferens and that net proton secretion requires the participation of the v-SNARE cellubrevin.

In colchicine-treated rats, cellular distribution of AQP-1 in convoluted and straight proximal tubule segments is differently affected.

In cells along the nephron, the recycling of various components between the plasma membrane and intracellular organelles by vesicle trafficking depends on intact microtubules (MT). Previous studies of rats treated with the MT-disrupting drug colchicine showed that some brush-border membrane (BBM) transporters in renal proximal convoluted tubule cells (PCTC) become internalized in numerous vesicles randomly scattered in the cytoplasm. In this study, we compare the intracellular distribution of MT and several BBM proteins [megalin, vacuolar (V)-ATPase, water channel aquaporin-1 (AQP-1)] as well as endocytosis of the in-vivo-injected fluorescent marker FITC-dextran in PCTC and proximal straight tubule cells (PSTC) in control and colchicine-treated rats. Immunoblotting and immunocytochemical data show that in the PCTC and PSTC colchicine treatment causes: (1) disappearance of MT, (2) strongly diminished endocytosis of FITC-dextran, and (3) marked loss of megalin and V-ATPase from the BBM and their redistribution into intracellular vesicles. Similar pattern was observed for the distribution of AQP-1 in the PCTC. However, in the PSTC, the staining intensity of AQP-1 in the BBM, as well as its intracellular distribution remained unaffected by colchicine treatment. We conclude that in the PSTC, either MT play a minor role in the recycling of AQP-1 between the BBM and intracellular vesicles or BBM AQP-1 turns over much more slowly than in the PCTC.

Na/K-ATPase in intercalated cells along the rat nephron revealed by antigen retrieval.

The Na/K-ATPase plays a fundamental role in the physiology of various mammalian cells. In the kidney, previous immunocytochemical studies have localized this protein to the basolateral membrane in different tubule segments. However, intercalated cells (IC) of the collecting duct (CD) in rat and mouse were unlabeled with anti-Na/K-ATPase antibodies. An antigen retrieval technique has been recently described in which tissue sections are pretreated with sodium dodecyl sulfate before immunostaining. This procedure was used to reexamine the presence of Na/K-ATPase in IC along the rat nephron using monoclonal antibodies against the Na/K-ATPase alpha-subunit. Subtypes of IC along the nephron were identified by their distinctive staining with polyclonal and monoclonal antibodies to the 31-kD vacuolar H+ -ATPase subunit, whereas principal cells (PC) were labeled with a polyclonal antibody to the water channel aquaporin-4 (AQP-4). In PC, the Na/K-ATPase and AQP-4 staining colocalized basolaterally. In contrast to previous reports, we found that IC of all types showed basolateral labeling with the anti-Na/K-ATPase antibody. The staining was quantified by fluorescence image analysis. It was weak to moderate in IC of cortical and outer medullary collecting ducts and most intense in IC of the initial inner medullary collecting duct. IC in the initial inner medulla showed a staining intensity that was equivalent or stronger to that in adjacent principal cells. Models of ion transport at the cellular and epithelial level in rat kidney, therefore, must take into account the potential role of a basolateral Na/K-ATPase in intercalated cell function.

Postnatal development of H+ ATPase (proton-pump)-rich cells in rat epididymis.

Active proton secretion and bicarbonate reabsorption by epithelial cells of the mammalian excurrent duct system maintains an acidic luminal pH that is involved in creating a suitable environment for sperm maturation and storage. Both an apical Na/H exchanger and an apical H+ ATPase have been implicated in luminal acidification. The H+ ATPase is located in apical and/or narrow cells in the caput epididymidis, and clear cells in the corpus and cauda epididymidis. As a step toward understanding the acute and chronic regulation of luminal acidification in excurrent ducts, we have followed the appearance of H+ ATPase-rich cells in rat epididymis during postnatal development, using antibodies to subunits of the H+ ATPase. In addition, we performed double staining with antibodies against carbonic anhydrase type II (CAII). H+ ATPase-rich cells were already detectable 2 weeks after birth in all regions of the epididymis, and reached maximum numbers after 3-4 weeks. CAII-rich cells followed a similar developmental pattern. In adult rats, the number of H+ ATPase/CAII-positive cells in the cauda was on average more than double the number in the caput epididymidis, although considerable intertubule variability was seen in both regions. Double immunostaining showed that CAII and H+ ATPase were colocalized in the same cells in the caput and cauda, but H+ ATPase-rich cells in the corpus contained low levels of CAII. These results demonstrate that differentiated subpopulations of proton-secreting epithelial cells appear early during epididymal development, and that the induction of H+ ATPase in these cells occurs prior to sexual maturation.

Cadmium inhibits vacuolar H(+)-ATPase and endocytosis in rat kidney cortex.

The mechanism of cadmium (Cd)-induced damage in the mammalian proximal tubule that is manifested by defects in reabsorption of various compounds, is poorly understood. A vacuolar H(+)-ATPase (V-ATPase) in proximal tubule (PT) brush border and intracellular vesicles may be affected by Cd, and this may influence intracellular vesicle trafficking and reabsorption of the filtered proteins. We studied the effects of Cd on V-ATPase and endocytosis in rat renal PT in vivo and on acidification mechanisms in isolated renal cortical organelles in vitro. The V-ATPase activity in brush border membrane (BBM) from Cd-intoxicated rats was 40% lower compared to that in control animals. Immunofluorescence studies in cortical tissue sections and Western blot studies in BBM from Cd-treated rats showed a strongly decreased abundance of the 31 kDa and 70 kDa V-ATPase subunits. Functional studies in vivo showed a dramatically diminished endocytosis of fluorescein-labeled dextran in PT cells from Cd-treated animals, whereas morphological studies revealed a loss of endocytic invaginations and subapical vesicles in the same cells. In studies in vitro, Cd inhibited V-ATPase activity in a concentration- and time-dependent manner in both BBM and endocytic vesicles, whereas in endocytic vesicles, Cd inhibited ATP-driven intravesicular acidification and accelerated the dissipation of transmembrane pH gradients. We conclude that Cd may impair acidification in cell organelles by (a) causing a loss of V-ATPase protein in their limiting membranes, (b) inhibiting the intrinsic V-ATPase activity, and (c) dissipating the transmembrane pH gradient. This may inhibit endocytosis of filtered proteins and impair vesicle-mediated recycling of some membrane transporters, thus contributing to the loss of reabsorptive capacity of the PT.

Immunolocalization of vacuolar-type H+-ATPase in rat submandibular gland and adaptive changes induced by acid-base disturbances.

Using antibodies against the 31-kD and 70-kD subunits of vacuolar type H+-ATPase (V-ATPase) and light microscopic immunocytochemistry, we have demonstrated the presence of this V-ATPase in rat submandibular gland. We have also investigated the adaptive changes of this transporter during acid-base disturbances such as acute and chronic metabolic acidosis or alkalosis. Our results show intracellularly distributed V-ATPase in striated, granular, and main excretory duct cells in controls, but no V-ATPase immunoreaction in acinar cells. Both acute and chronic metabolic acidosis caused a shift in V-ATPase away from diffuse distribution towards apical localization in striated and granular duct cells, suggesting that a V-ATPase could be involved in the regulation of acid-base homeostasis. In contrast, during acidosis the main excretory duct cells showed no changes in the V-ATPase distribution compared to controls. With acute and chronic metabolic alkalosis, no changes in the V-ATPase distribution occurred. (J Histochem Cytochem 46:91-100, 1998)

Absence of vacuolar H+-ATPases from rat small intestine brush-border membranes.

Rat small intestinal brush-border membrane (BBM) vesicles were solubilized to test for the presence of an intravesicular H+-ATPase. Several detergents that in rat renal BBM vesicles revealed a bafilomycin-sensitive H+-ATPase failed to expose a similar ATPase in small intestinal vesicles. Antibodies against the 31-kDa and 70-kDa H+-ATPase subunits labelled respective antigens in the BBM of renal proximal tubules, but not in the BBM of enterocytes. The results demonstrate that a bafilomycin-sensitive, vacuolar-type H+-ATPase is absent from the BBM of enterocytes and, hence, cannot contribute to H+ secretion.

Regulation of AE1 anion exchanger and H(+)-ATPase in rat cortex by acute metabolic acidosis and alkalosis.

The cortical collecting duct (CCD) mediates net secretion or reabsorption of protons according to systemic acid/base status. Using indirect immunofluorescence, we examined the localization and abundance of the vacuolar H(+)-ATPase and the AE1 anion exchanger in intercalated cells (IC) of rat kidney connecting segment (CNT) and CCD during acute (6 hr) metabolic (NH4Cl) acidosis and respiratory (NaHCO3) alkalosis. AE1 immunostaining intensity quantified by confocal microscopy was elevated in metabolic acidosis and substantially reduced in metabolic alkalosis. AE1 immunostaining was restricted to Type A IC in all conditions, and the fraction of AE1+IC was unchanged in CNT and CCd. Metabolic acidosis was accompanied by redistribution of H(+)-ATPase immunostaining towards the apical surface of IC, and metabolic alkalosis was accompanied by H(+)-ATPase redistribution towards the basal surface of IC. Therefore, acute metabolic acidosis produced changes consistent with increased activity of Type A IC and decreased activity of Type B IC, whereas acute metabolic alkalosis produced changes corresponding to increased activity of Type B IC and decreased activity of Type A IC. These data demonstrate that acute systemic acidosis and alkalosis modulate the cellular distribution of two key transporters involved in proton secretion in the distal nephron.

Polarized expression of membrane proteins in renal epithelial cells: involvement of specialized transport vesicles and intracellular pathways.

The polarized insertion of membrane proteins in epithelial cells is a highly regulated process that involves the coordinated interaction of many subsets of intracellular proteins. Thus, like cog-wheels in a complex machine, the different parts of the trafficking pathway must work together to ensure the correct functioning of an individual cell and of the entire epithelium in which it resides. The individual portions of the trafficking pathway include (1) sorting of membrane proteins into the correct apically or basolaterally targeted vesicles at the level of the trans-Golgi network; (2) delivery of these vesicles to their correct membrane destination by the microtubular (and probably the actin) cytoskeleton; (3) fusion of the vesicles with the appropriate membrane domain via a complex set of fusion proteins. This review has briefly outlined some of these processes and has used examples from the kidney to illustrate their importance to normal renal function.

Renal type II Na/Pi-cotransporter is strongly impaired whereas the Na/sulphate-cotransporter and aquaporin 1 are unchanged in cadmium-treated rats.

The cellular mechanisms of cadmium (Cd) nephrotoxicity are poorly understood. In this study we investigated the cellular causes of the Cd-induced phosphaturia in the rat. Compared to controls, Cd-treated rats (2 mg Cd/kg body weight, s.c. for 14 days) showed a marked polyuria, proteinuria and phosphaturia. As studied by the rapid filtration technique in isolated cortical brush-border membrane vesicles (BBMV), Na+-gradient-driven uptake of phosphate ([32Pi]) and of [3H] glucose were markedly decreased in Cd-treated rats, whereas uptake of sulphate ([35S]) remained unchanged. By Western blotting of BBMV proteins and by indirect immunocytochemistry in 4-micron thick frozen fixed kidney sections, using an antibody against the type II Na/Pi-cotransporter (NaPi-2), we found a diminished expression of this protein in the brush-border membrane from Cd-treated rats. How ever, the expression of the water channel aquaporin 1, estimated from the specific antibody staining in brush-border membranes, remained unchanged by Cd. Northern blot analysis showed a strong reduction of 2.7 kb NaPi-2-related mRNA in Cd-affected kidneys. Our data indicate that: (1) Cd may reduce reabsorption of Pi in proximal tubules by affecting the expression of the functional Na/Pi-cotransporters in the luminal membrane, and (2) Cd effects on brush-border transporters are selective.

Cellular distribution of the aquaporins: a family of water channel proteins.

A group of transmembrane proteins that are related to the major intrinsic protein of lens fibers (MIP26) have been named "aquaporins" to reflect their role as water channels. These proteins are located at strategic membrane sites in a variety of epithelia, most of which have well-defined physiological functions in fluid absorption or secretion. However, some aquaporins have been localized in cell types where their role is at present unknown. Most of the aquaporins are delivered to the plasma membrane in a non-regulated (constitutive) fashion, but AQP2 enters the regulated exocytotic pathway and its membrane expression is controlled by the action of the antidiuretic hormone, vasopressin. These pathways of constitutive versus regulated delivery to the plasma membrane have been reconstituted in transfected LLC-PK1 epithelial cells, indicating that the information encoded within the protein sequence is sufficient to allow sorting of newly synthesized protein into distinct intracellular vesicles. Finally, different members of the aquaporin family can be targeted to apical, basolateral or both apical and basolateral plasma membrane domains of polarized epithelial cells. This implies that signals for the polarized targeting of these proteins also is located in non-homologous regions of these similar proteins. Thus, future investigations on the aquaporin family of proteins will provide important information not only on the physiology of membrane transport processes in many cell types, but also on the targeting and trafficking signals that allow proteins to enter distinct intracellular vesicular pathways in epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ecto-adenosinetriphosphatase in rat small intestinal brush-border membranes.

Antibodies against the holo ecto-adenosinetriphosphatase (ATPase) of rat liver and antibodies against COOH-terminal peptides of the long isoform of this enzyme reacted in Western blots with a 105-kDa band from small intestinal brush-border membranes. Indirect immunofluorescence revealed reactive proteins predominantly at the apical surface of enterocytes with some staining of basolateral membranes and of vascular endothelium. Similar results were obtained with monoclonal antibodies against HA4, a protein from rat liver closely related to the ecto-ATPase. Since these results suggested the presence of an ecto-ATPase, ATP hydrolysis was studied in intact, right-side-out brush-border membrane vesicles. Nearly half of ATP hydrolysis was caused by alkaline phosphatase (AP). Besides purine and pyrimidine trinucleotides, AP also hydrolyzed ADP, AMP, pyrophosphate, and 4-nitrophenylphosphate. Inactivation of AP by cleavage of its membrane anchor and by removal of the Zn2+ necessary for its function left the ecto-ATPase that was activated by Ca2+ and Mg2+ and hydrolyzed purine and pyrimidine trinucleotides and dinucleotides, but not AMP, pyrophosphate, and 4-nitrophenylphosphate. These features are characteristic of an ATP diphosphohydrolase (EC 3.6.1.5, also called apyrase). The physiological role of the small intestinal ecto-ATPase may be the degradation of nutrient nucleotides.