NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

In chloride-secretory epithelia, the basolateral Na-K2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl ؊ and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K ؉ that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1 ؊/؊ ) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl ؊ channel, were reduced in jejunum, cecum, and trachea of Nkcc1 ؊/؊ mice, indicating that NKCC1 contributes to cAMP-induced Cl ؊ secretion. In contrast, secretion of gastric acid in adult Nkcc1 ؊/؊ stomachs and enterotoxin-stimulated fluid secretion in the intestine of suckling Nkcc1 ؊/؊ mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K ؉ movements involved in generation of the K ؉ -rich endolymph and the endocochlear potential.

The relative importance of plasma membrane Ca 2؉ -ATPase (PMCA) 1 and PMCA4 was assessed in mice carrying null mutations in their genes (Atp2b1 and Atp2b4). Loss of both copies of the gene encoding PMCA1 caused embryolethality, whereas heterozygous mutants had no overt disease phenotype. Despite widespread and abundant expression of PMCA4, PMCA4 null (Pmca4 ؊/؊ ) mutants exhibited no embryolethality and appeared outwardly normal. Loss of PMCA4 impaired phasic contractions and caused apoptosis in portal vein smooth muscle in vitro; however, this phenotype was dependent on the mouse strain being employed. Pmca4 ؊/؊ mice on a Black Swiss background did not exhibit the phenotype unless they also carried a null mutation in one copy of the Pmca1 gene. Pmca4 ؊/؊ male mice were infertile but had normal spermatogenesis and mating behavior. Pmca4؊/؊ sperm that had not undergone capacitation exhibited normal motility but could not achieve hyperactivated motility needed to traverse the female genital tract. Ultrastructure of the motility apparatus in Pmca4 ؊/؊ sperm tails was normal, but an increased incidence of mitochondrial condensation indicated Ca 2؉ overload. Immunoblotting and immuno-histochemistry showed that PMCA4 is the most abundant isoform in testis and sperm and that it is localized to the principle piece of the sperm tail, which is also the location of the major Ca 2؉ channel (CatSper) required for sperm motility. These results are consistent with an essential housekeeping or developmental function for PMCA1, but not PMCA4, and show that PMCA4 expression in the principle piece of the sperm tail is essential for hyperactivated motility and male fertility.

Mutations in the gene encoding the thiazide-sensitive Na ؉ -Cl ؊ cotransporter (NCC) of the distal convoluted tubule cause Gitelman's syndrome, an inherited hypokalemic alkalosis with hypomagnesemia and hypocalciuria. These metabolic abnormalities are secondary to the deficit in NaCl reabsorption, but the underlying mechanisms are unclear. To gain a better understanding of the role of NCC in sodium and fluid volume homeostasis and in the pathogenesis of Gitelman's syndrome, we used gene targeting to prepare an NCC-deficient mouse. Null mutant (Ncc ؊/؊ ) mice appear healthy and are normal with respect to acid-base balance, plasma electrolyte concentrations, serum aldosterone levels, and blood pressure. Ncc ؊/؊ mice retain Na ؉ as well as wild-type mice when fed a Na ؉ -depleted diet; however, after 2 weeks of Na ؉ depletion the mean arterial blood pressure of Ncc ؊/؊ mice was significantly lower than that of wild-type mice. In addition, Ncc ؊/؊ mice exhibited increased renin mRNA levels in kidney, hypomagnesemia and hypocalciuria, and morphological changes in the distal convoluted tubule. These data indicate that the loss of NCC activity in the mouse causes only subtle perturbations of sodium and fluid volume homeostasis, but renal handling of Mg 2؉ and Ca 2؉ are altered, as observed in Gitelman's syndrome.

The cytochrome P450 (CYP1A1) enzyme metabolically activates many polycyclic aromatic hydrocarbons, including benzo [a]pyrene (BaP), to DNA-and protein-binding intermediates that are associated with toxicity, mutagenesis, and carcinogenesis. As a result, it is widely accepted that CYP1A1 potentiates the toxicity of this class of chemicals. In distinct contrast, we show here that CYP1A1 inducibility is essential in the detoxication of oral BaP. We compared Cyp1a1(Ϫ/Ϫ) knockout mice, having the genetic absence of the CYP1A1 enzyme, with Cyp1a1(ϩ/ϩ) wild-type mice. At an oral BaP dose of 125 mg/ kg/day, Cyp1a1(Ϫ/Ϫ) mice died within 30 days whereas Cyp1a1(ϩ/ϩ) mice displayed no outward signs of toxicity. The rate of BaP clearance was 4-fold slower in Cyp1a1(Ϫ/Ϫ) than Cyp1a1(ϩ/ϩ) mice. The cause of death in Cyp1a1(Ϫ/Ϫ) mice receiving oral BaP seemed to be immunotoxicity, including toxic chemical depression of the bone marrow; some toxic effects in Cyp1a1(Ϫ/Ϫ) mice were noted at a BaP dose as low as 1.25 mg/kg/day. DNA post-labeling studies demonstrated dramatically higher BaP-DNA adduct levels in all Cyp1a1(Ϫ/Ϫ) tissues assayed, with the exception of the small intestine, which is probably a major site of BaP metabolism in Cyp1a1(ϩ/ϩ) mice. Different BaP-DNA adduct patterns were also observed between the two genotypes receiving oral BaP. Despite previous studies in vitro and in cell culture that have shown a participatory role for CYP1A1 in BaP toxicity, the present data indicate that, in the intact animal, inducible CYP1A1 is extremely important in detoxication and protection against oral BaP toxicity.

Multiple isoforms of the Na+/H+ exchanger (NHE) are expressed at high levels in gastric epithelium, but the physiological role of individual isoforms is unclear. To study the function of NHE2, which is expressed in mucous, zymogenic, and parietal cells, we prepared mice with a null mutation in the NHE2 gene. Homozygous null mutants exhibit no overt disease phenotype, but the cellular composition of the oxyntic mucosa of the gastric corpus is altered, with parietal and zymogenic cells reduced markedly in number. Net acid secretion in null mutants is reduced slightly relative to wild-type levels just before weaning and is abolished in adult animals. Although mature parietal cells are observed, and appear morphologically to be engaged in active acid secretion, many of the parietal cells are in various stages of degeneration. These results indicate that NHE2 is not required for acid secretion by the parietal cell, but is essential for its long-term viability. This suggests that the unique sensitivity of NHE2 to inhibition by extracellular H+, which would allow upregulation of its activity by the increased interstitial alkalinity that accompanies acid secretion, might enable this isoform to play a specialized role in maintaining the long-term viability of the parietal cell.

Plasma membrane Ca 2؉-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.Calmodulin-dependent plasma membrane Ca 2ϩ -ATPases (PMCAs) 1 are highly regulated enzymes that maintain the appropriate concentrations of intracellular free Ca 2ϩ by extruding Ca 2ϩ from the cell (1, 2). There are four mammalian PMCA isoforms (PMCA1-4), each encoded by a distinct gene (3-8), and additional diversity is generated by alternative splicing of exons encoding the regulatory domains (7, 9 -11). Variants of PMCA1 and PMCA4 are expressed in many different tissues and cell types, whereas variants of PMCA2 and PMCA3 exhibit a highly restricted distribution (5,8,12). This suggests that specific isoforms and splice variants serve different physiological functions. However, despite extensive information about PMCA structural diversity, expression patterns, and biochemical and regulatory characteristics, little is known about the functions of individual isoforms in vivo.Its unique biochemical characteristics (13, 14) and tissue specificity (5, 10 -12) suggest that PMCA2 might serve specialized physiological functions. In situ hybridization studies revealed that expression of PMCA2 is particularly high in Purkinje neurons of the cerebellum (15) and in the spiral ganglion nerves of the inner ear and outer hair cells of the cochlea (16). The observation that PMCA2 is the predominant isoform in outer hair cells (16) suggests that it might be the isoform that is expressed at high levels in stereocilia (17), which comprise hair bundles, the sensory organelles that mediate mechanoelectrical transduction by hair cells of both the vestibular and auditory systems (18,19). A recent study demonstrated that PMCA activity in the stereocilia of vestibular hair cells regulates hair bundle Ca 2ϩ concentrations and indicated that it ...

Testicular necrosis is a sensitive endpoint for cadmium (Cd 2؉ , Cd) toxicity across all species tested. Resistance to Cd-induced testicular damage is a recessive trait assigned to the Cdm locus on mouse chromosome 3. We first narrowed the Cdm-gene-containing region to 880 kb. SNP analysis of this region from two sensitive and two resistant inbred strains demonstrated a 400-kb haplotype block consistent with the Cd-induced toxicity phenotype; in this region is the Slc39a8 gene encoding a member of the solute-carrier superfamily. Slc39a8 encodes SLC39A8 (ZIP8), whose homologs in plant and yeast are putative zinc transporters. We show here that ZRT-, IRT-like protein (ZIP)8 expression in cultured mouse fetal fibroblasts leads to a >10-fold increase in the rate of intracellular Cd influx and accumulation and 30-fold increase in sensitivity to Cd-induced cell death. The complete ZIP8 mRNA and intron-exon splice junctions have no nucleotide differences between two sensitive and two resistant strains of mice; by using situ hybridization, we found that ZIP8 mRNA is prominent in the vascular endothelial cells of the testis of the sensitive strains of mice but absent in these cells of resistant strains. Slc39a8 is therefore the Cdm gene, defining sensitivity to Cd toxicity specifically in vascular endothelial cells of the testis. metal influx ͉ vascular endothelial cells ͉ solute carrier gene superfamily ͉ in situ hybridization C d is a toxic and carcinogenic nonessential metal (1), which can enter the body through the intestine, skin, and lung and accumulates in the kidney (1-3). The level of Cd in the environment has risen with advances in industrialization, and the role of Cd in human disease is of increasing concern. The mechanisms of Cd toxicity are poorly understood, although it is known that Cd exerts its effects intracellularly, and there are polypeptides such as metallothionein (4) and reduced glutathione (5) that bind Cd and afford protection. The subcellular events by which Cd is taken up by cells or removed from cells remain obscure, although such knowledge could provide potential therapeutic targets for protection or intervention against Cd toxicity. Several proteins transport Cd into bacteria, yeast, plants, and mammalian cells in culture (6-11), but their specific roles in causing toxicity are unclear; these studies underscore the difficulties in extrapolating from observations in cell culture to the intact animal.Nature has provided a fascinating genetic system as a foothold into identifying a gene involved in Cd toxicity. It is known that Cd-induced testicular necrosis is common across all animal species having testes: rodents, opossum, armadillos, frogs, pigeons, roosters, and fish (12-17). Cellular events that precede Cd-induced testicular toxicity indicate that vascular endothelial cell injury is the earliest and, perhaps, the causative event (16,(18)(19)(20)(21)(22)(23)(24).Some inbred mouse strains are resistant to Cd-induced testicular toxicity (25). The resistance phenotype segregates largely as a...