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M Donowitz

Publications and source records attributed to M Donowitz.

At least 73 records · Page 4Linked to original sources
PubMed1995

Cloning, tissue distribution, and functional analysis of the human Na+/N+ exchanger isoform, NHE3.

We previously isolated a 1.4-kb partial cDNA from a human kidney cortex library. Using both library screening and reverse transcription-polymerase chain reaction of human kidney RNA, we obtained the entire coding region of the human NHE3 cDNA. The human NHE3 cDNA encoded a protein of 834 amino acids with a calculated relative molecular weight of 92,906. It exhibited 89 and 88% amino acid identity with rat and rabbit NHE3, respectively. The stable transfection of a composite human NHE3 cDNA into Na+/H+ exchanger-deficient PS120 cells established Na+/H+ exchange. Functionally, human NHE3 was similar to the rabbit and rat NHE3 homologues, being relatively resistant to inhibition by amiloride, half-maximal inhibition (IC50) = 49.0 microM, and ethylisopropylamiloride, IC50 = 6.6 microM, and being stimulated by fibroblast growth factor but inhibited by phorbol 12-myristate 13-acetate. However, unlike the rabbit or rat NHE3, human NHE3 message was not restricted to kidney, intestine, stomach, and brain. Northern analysis of multiple human tissues detected NHE3 message, in descending order, as follows: kidney >> small intestine >> testes > ovary > colon = prostate > thymus > peripheral leukocyte = brain > spleen > placenta. Message in the kidney, small intestine, and colon was primarily of 6.7 kb, whereas both 6.7- and 8.9-kb bands were expressed nearly equivalently in the other tissues. No NHE3 message was detected in the human heart, lung, liver, skeletal muscle, or pancreas.

Amino Acid Sequence↗
PubMed1995

Mammalian Na+/H+ exchanger gene family: structure and function studies.

Na+/H+ exchangers are integral plasma membrane proteins that exchange extracellular Na+ for intracellular H+ with a stoichiometry of one for one. They are inhibitable by the diuretic amiloride and have multiple cellular functions, including intracellular pH homeostasis, cell volume control, and electroneutral NaCl absorption in epithelia. The presence of multiple forms of the exchangers was demonstrated by the recent cloning of four mammalian Na+/H+ exchangers, NHE1, NHE2, NHE3, and NHE4. All of these cloned Na+/H+ exchangers have 10-12 putative transmembrane helixes and a long cytoplasmic carboxyl domain. Despite the structural similarity, these Na+/H+ exchanger isoforms differ in their tissue distribution, kinetic characteristics, and response to external stimuli. The present review deals with the recent developments in the molecular identification of the Na+/H+ exchanger gene family, the functional characteristics, and the short-term regulation of Na+/H+ exchange at molecular and cellular levels.

Amino Acid Sequence↗
PubMed1995

Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers.

The involvement of tyrosine phosphorylation in the regulation of epithelial cell Cl- secretion is unknown. Therefore, the purpose of these studies was to determine if tyrosine kinase activation was involved in the regulation of Cl- secretion, using the tyrosine kinase inhibitors, genistein and tyrphostin 47, and human intestinal epithelial cells (T84 cells) as an intestinal Cl- secretory model. Genistein rapidly but reversibly stimulated sustained apical Cl- secretion in monolayers of T84 cells without increasing intracellular cyclic nucleotides or Ca2+ levels. Tyrphostin 47 also stimulated Cl- secretion in T84 monolayers, although it was short-lived. Transfection experiments in 3T3 fibroblasts and IEC-6 intestinal cells utilizing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) showed that genistein and tyrphostin 47 stimulated 125I efflux only in CFTR-transfected cells and not in CFTR-negative cells. Thus genistein- and tyrphostin 47-stimulated Cl- secretion involved CFTR. Genistein also acted synergistically with the Ca(2+)- and protein kinase C-dependent acetylcholine analogue, carbachol, to stimulate Cl- secretion in T84 monolayers. However, the Cl- secretory response to saturating concentrations of the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin, or the guanosine 3',5'-cyclic monophosphate (cGMP) agonist, Escherichia coli heat-stable enterotoxin, was not further enhanced by genistein. Although the mechanism of activation of Cl- secretion is unclear, these data suggest that tyrosine kinase activity limits basal Cl- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical membrane Cl- secretion, most likely through activation of the CFTR-Cl- channel. Moreover, genistein does not itself act through cAMP or cGMP elevation but appears to share a common Cl- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory responses to a Ca(2+)- and protein kinase C-dependent agonist.

Calcium↗
PubMed1994

Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein.

A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-alpha-D-galactosaminide (Bz alpha GalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the Bz alpha GalNAc treatment of PS120/NHE2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylgalactosamine↗
PubMed1994

Effects of galanin on short circuit current and electrolyte transport in rabbit ileum.

Galanin decreased short circuit current (Isc) and increased active Na+ and Cl- absorption in rabbit ileum. In the absence of calcium, the galanin-induced decrease in Isc was inhibited by approximately 60%. Tetrodotoxin significantly reduced the effect of galanin on Isc, and tetrodotoxin and EGTA totally blocked the effect, indicating that the nonneuronal mediator of the effect is Ca2+ dependent. Galanin binding to basolateral membranes prepared from ileal epithelial cells was specific and of high affinity. These results suggest the involvement of this peptide in the regulation of intestinal epithelial cell function.

Animals↗
PubMed1994

Premarin-induced ischemic colitis.

There are numerous literature reports of oral contraceptive-induced (estrogen and progesterone) ischemic bowel disease. However, Premarin-induced (equine conjugated estrogen) intestinal ischemia has rarely been reported. We describe three cases of Premarin-induced ischemic colitis. In contrast to oral contraceptive-induced ischemic colitis, Premarin-induced ischemia is restricted to the colon, has not required surgical therapy, can have a chronic or chronic intermittent course, may be reversible despite continued usage of Premarin, and may present with nonspecific abdominal and colonic symptoms.

Adult↗
PubMed1994

Brush-border tyrosine phosphorylation stimulates ileal neutral NaCl absorption and brush-border Na(+)-H+ exchange.

The drug genistein, a tyrosine (Tyr) kinase inhibitor, was used to define a role for Tyr phosphorylation in regulation of basal and stimulated neutral NaCl absorption in rabbit ileum. Brush-border vesicles contain Tyr-phosphorylated peptides. Genistein freeze-thawed into the vesicles caused a concentration-dependent inhibition of at least three peptides with M(r) 111,000, 83,000, and 80,000. Studied with the Ussing chamber-voltage clamp technique, genistein added to the ileal mucosal surface inhibited neutral NaCl absorption. Direct addition of genistein to brush-border vesicles made from ileal villus cells inhibited brush-border Na(+)-H+ exchange but not D-glucose-stimulated Na+ uptake. These effects were not duplicated by genistin, a drug with similar structure to genistein but lacking Tyr kinase inhibiting properties. Serosal but not mucosal epidermal growth factor (EGF) stimulated NaCl absorption. Mucosal genistein but not genistin also altered second-messenger regulation of neutral NaCl absorption, inhibiting the effect of Ca2+ ionophore A-23187 and of serosal EGF but not affecting the transport changes caused by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). In contrast, the Cl secretory effects indicated by the increase in short-circuit current for all three agents, A-23187, EGF, and 8-BrcAMP, were inhibited by mucosal genistein. These results suggest that 1) a Tyr kinase is involved in basally stimulating ileal neutral NaCl absorption and brush-border Na(+)-H+ exchange; 2) EGF stimulates NaCl absorption by an effect exerted from the serosal surface, but the effect also involves a brush-border Tyr kinase; 3) brush-border Tyr kinase is involved in the ability of Ca2+ ionophore A-23187 to inhibit neutral NaCl absorption but is not involved in the transport effects of cAMP. This study suggests that Tyr kinase(s) acting over short time periods is involved in stimulation of neutral NaCl absorption and brush-border Na(+)-H+ exchange and also in Ca(2+)-induced inhibition of NaCl absorption. These studies represent the first example of a brush-border Tyr kinase being involved in short-term signal transduction in intestinal epithelial cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Na+/H+ exchange mediates meal-stimulated ileal absorption.

BACKGROUND: The ingestion of a meal increases water and electrolyte absorption from the jejunoileum. Recent observations have implicated Na(+)-glucose cotransport as a primary mediator of meal-stimulated jejunal absorption. The current experiments tested the hypothesis that Na+/H+ exchange is a major mediator of basal and meal-stimulated ileal absorption. METHODS: Absorption studies (n = 36) were performed on dogs with 25 cm ileal Thiry-Vella fistulas. Six groups were studied for 4 hours. Luminal perfusion with 14C-polyethylene glycol was used to calculate absorption of water, sodium, chloride, and glucose. A 480 kcal canine meal was ingested in groups 2, 3, 5, and 6. Luminal amiloride (1 mmol/L) was used to inhibit Na+/H+ exchange in groups 1, 3, 4, and 6. To assess Na(+)-glucose cotransport the luminal perfusate contained either 10 mmol/L glucose (groups 1 through 3) or 10 mmol/L mannitol, a nontransported hexose (groups 4 through 6). RESULTS: Ileal absorption increased significantly in response to a meal in the presence of both glucose (group 2) and mannitol (group 5). Amiloride caused a significant reduction in basal and postmeal water and electrolyte absorption, largely independent of the glucose-mannitol content of the luminal perfusate. Mannitol significantly reduced basal absorption of water and electrolytes. CONCLUSIONS: These data indicate that both Na+/H+ exchange and Na(+)-glucose cotransport contribute to basal ileal water and electrolyte absorption, whereas Na+/H+ exchange appears to be the primary mediator of meal-stimulated ileal absorption. Na+/H+ exchange appears to play a major role in the physiologic regulation of postprandial ileal absorption.

Amiloride↗
PubMed1993

Kinetics and regulation of three cloned mammalian Na+/H+ exchangers stably expressed in a fibroblast cell line.

The kinetics and second messenger regulation of three cloned mammalian intestinal Na+/H+ exchangers were studied using fluorometric techniques. These exchangers, NHE1, NHE2, and NHE3, were stably expressed in PS120 fibroblasts, which lack an endogenous Na+/H+ exchanger. H+ kinetic data indicated cooperativity by internal protons, with Hill coefficients of approximately 2 for all three isoforms. In contrast, Na+ kinetic data fit Michaelis-Menten kinetics, with Km (Na+) 15-18 mM and a Hill coefficient of approximately 1. The exchangers were all activated by growth factors and thrombin; in NHE1 these agonists increased the apparent affinity for intracellular H+, but did not change Vmax, while for NHE2 and NHE3 the effect was on Vmax alone. Phorbol ester stimulated NHE1 and NHE2, but inhibited NHE3 with a decrease in Vmax. ATP-depletion decreased Vmax and the apparent affinity for H+ for all three isoforms, and reduced the Hill coefficient to approximately 1, suggesting that a basal level of phosphorylation was required for the cooperativity. The differences in kinetics and second messenger regulation suggest that the NHE isoforms may serve different cellular functions. The up- and down-regulation of NHE3 by kinases indicates that this isoform may be involved in a specialized function such as Na+ absorption.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Functional characteristics of a cloned epithelial Na+/H+ exchanger (NHE3): resistance to amiloride and inhibition by protein kinase C.

We previously cloned an isoform Na+/H+ exchanger (NHE3), which was expressed only in intestine, kidney, and stomach. We show here the functional characteristics of NHE3 as a Na+/H+ exchanger by stably transfecting NHE3 cDNA into PS120 cells, a fibroblast cell line that lacks endogenous Na+/H+ exchangers. NHE3 was 39- and 160-fold more resistant to inhibition by amiloride and ethylisopropyl amiloride, respectively, than NHE1, the housekeeping Na+/H+ exchanger isoform. Although both exchangers were stimulated by serum, NHE3 was inhibited by phorbol 12-myristate 13-acetate (PMA), which stimulated NHE1. Mechanistically, serum and PMA stimulated NHE1 by an increase in the apparent affinity of the exchanger for intracellular H+. In contrast, serum stimulated and PMA inhibited NHE3 by a Vmax change. When NHE3 was stably expressed in Caco-2 cells, an intestinal epithelial cell line, NHE3 was functionally expressed in the apical membrane. Thus, NHE3 is a good candidate to be an epithelial brush border Na+/H+ exchanger. Furthermore, Na+/H+ exchangers can be rapidly regulated by mechanisms that change either the Vmax or the affinity for intracellular H+, depending on the Na+/H+ exchanger subtype.

Amiloride↗
PubMed1993

Leu143 in the putative fourth membrane spanning domain is critical for amiloride inhibition of an epithelial Na+/H+ exchanger isoform (NHE-2).

A family of Na+/H+ exchanger isoforms (called NHE1, NHE2, and NHE3) which exhibits a wide range of amiloride sensitivity has recently been cloned and characterized. A part of the domain, which determines amiloride sensitivity in the epithelial Na+/H+ exchanger isoform, NHE2, was identified by site-directed mutagenesis and functional studies using cDNAs stably expressed in a fibroblast cell line. It has previously been reported that AR300, an amiloride resistant mutant of the ubiquitous Na+/H+ exchanger isoform, NHE1, is 30-fold more resistant to methylpropyl amiloride (MPA) compared to NHE1 and contains a single amino acid substitution of L167F in the fourth putative transmembrane helix, which corresponds to L143 in NHE2. Therefore, in the present study point mutational substitutions were introduced into the equivalent of this fourth transmembrane helix of rabbit NHE2 (including Y144F; L143F; L143F and Y144F) to mimic the corresponding amino acids in NHE1, NHE3 (another epithelial isoform) and AR300, respectively. NHE2/L143F (mimicking NHE3) increased the IC50 for amiloride by 5-fold and for ethylisopropyl amiloride (EIPA) by 20-fold. Similarly, NHE2/L143F and Y144F (mimicking AR300) increased the resistance to both amiloride and EIPA by 10-fold. On the other hand, NHE2/Y144F (mimicking NHE1) did not affect the sensitivity to amiloride or EIPA, and this mutant, like wild type NHE2, is partially resistant to EIPA. Thus, amino acid 143 of NHE2 is critical for, but is not the only amino acid responsible for, amiloride and EIPA inhibition of Na+/H+ exchange. That none of the mutations studied altered the Na+ affinity of these Na+/H+ exchangers further suggests that amiloride binding and Na+ transport sites are not identical.

Amiloride↗
PubMed1993

Cloning and expression of a rabbit cDNA encoding a serum-activated ethylisopropylamiloride-resistant epithelial Na+/H+ exchanger isoform (NHE-2).

A unique Na+/H+ exchanger isoform, NHE-2, was cloned and characterized. NHE-2 is a protein of 809 amino acids with a calculated size of 90,787. It exhibits overall amino acid identity of 50, 44, and 60% with other cloned mammalian Na+/H+ exchangers NHE-1, NHE-3, and NHE-4, respectively. Northern blot analysis of poly(A+) RNA isolated from rabbit ileum, kidney cortex, and kidney medulla using NHE-2 cDNA as a probe revealed messages of 5.2, 4.2, and 3.2 kilobases with relative abundance (in descending order) kidney medulla > kidney cortex > ileum. More detailed tissue distribution of message was performed by ribonuclease protection assay. NHE-2 was predominantly expressed in kidney, intestine, and adrenal gland with a small amount in skeletal muscle and trachea. Stable expression of NHE-2 in PS120 fibroblasts confirmed that NHE-2 is a functional Na+/H+ exchanger which is defined by amiloride-sensitive Na+-dependent alkalinization of acid-loaded cells. NHE-2 has the same Ki for amiloride inhibition as NHE-1 (1 microM) but is 25-fold more resistant to ethylisopropylamiloride inhibition than is NHE-1 (500 versus 20 nM). Like NHE-1, NHE-2 can be activated by serum. Expression of NHE-2 in a polarized human intestinal epithelial cell line, Caco-2 cells, results in functional expression of NHE-2 in the apical membrane. Thus, we conclude that NHE-2 is a candidate to be an apical membrane Na+/H+ exchanger in intestinal and renal epithelial cells.

Adenocarcinoma↗
PubMed1993

Glucocorticoid stimulation of ileal Na+ absorptive cell brush border Na+/H+ exchange and association with an increase in message for NHE-3, an epithelial Na+/H+ exchanger isoform.

Methylprednisolone stimulates rabbit ileal neutral NaCl absorption; and aminoglutethimide, which decreases glucocorticoid levels, decreases NaCl absorption. Studies were carried out to determine the mechanism of these effects and to determine which members of the gene family of mammalian Na+/H+ exchangers were involved. Rabbits were treated subcutaneously with methylprednisolone (40 mg daily for 24 or 72 h), aminoglutethimide (100 mg twice daily for 72 h), or saline as a control. Ileal brush border membranes were prepared by magnesium precipitation, and brush border Na+/H+ exchange was determined by 22Na+ uptake over 3-8 s. The 22Na+ uptake experiments were performed in the presence of a voltage clamp using either valinomycin/potassium or tetramethylammonium/nitrate to eliminate potential contributions by other electrogenic transport processes. Methylprednisolone treatment approximately doubled ileal brush border Na+/H+ exchange, whereas aminoglutethimide led to a 50% decrease in Na+/H+ exchange. These effects were specifically on Na+ uptake with an acid inside pH gradient, whereas diffusive Na+ uptake (no pH gradient), glucose-dependent Na+ uptake, and glucose and Na+ equilibrium volumes were not affected. To determine if the increase in Na+/H+ exchange was associated with an increase in message expression, mRNA levels were measured by ribonuclease protection assay. Methylprednisolone stimulated the NHE-3 mRNA level by 4-6-fold at 24 h, which remained increased at 72 h. In contrast, messages for NHE-1 and NHE-2 were not affected by methylprednisolone. In summary, 1) methylprednisolone stimulation of rabbit ileal Na+ absorption is due to stimulation of ileal villus cell brush border Na+/H+ exchange; 2) basal ileal brush border Na+/H+ exchange is dependent on glucocorticoid levels; and 3) an increase in NHE-3 message, but not in NHE-1 or NHE-2 message, correlates with the stimulation of ileal brush border Na+/H+ exchange. It is likely that NHE-3 is an Na+/H+ exchanger that is involved in ileal Na+ absorption.

Aminoglutethimide↗
PubMed1993

Physical and genetic mapping of a human apical epithelial Na+/H+ exchanger (NHE3) isoform to chromosome 5p15.3.

A gene family of Na+/H+ exchanger isoforms has been identified. Characterization of rabbit NHE3 suggests that it is the apical epithelial Na+/H+ exchanger isoform responsible for transepithelial, electroneutral Na+ absorption in intestinal and renal epithelial cells. We have previously isolated from a human kidney cortex library a partial human NHE3 cDNA, clone HKC-3. Using HKC-3 to probe human/rodent somatic cell hybrid mapping panels, the human NHE3 gene was physically mapped to the distal portion of chromosome 5p15.3. Southern analysis of EcoRI-digested human genomic DNAs of CEPH pedigrees probed with HKC-3 detected three polymorphic sites containing a total of nine alleles that segregate in a Mendelian fashion. The observed heterozygosity for the NHE3 locus in unrelated individuals was 71%. Linkage analysis between NHE3 and other markers known to map at 5p15 confirmed the localization of NHE3 to chromosome 5p15.3, making NHE3 the most telomeric gene identified on the short arm of chromosome 5.

Animals↗
PubMed1993

Identification of PTH-responsive Na/H-exchanger isoforms in a rabbit proximal tubule cell line (RKPC-2).

Renal epithelial cells may express apical and basolateral Na/H exchangers which are different in their physiological regulation and different in their sensitivities to the inhibitor amiloride. In the present study RKPC-2 cells [a Simian virus 40 (SV-40) transformed cell line of rabbit S2 proximal tubular origin] were examined for localization (apical vs basolateral) and regulation of Na/H-exchange activity(ies) by parathyroid hormone (PTH). In addition, using specific cDNA probes we determined the expression of multiple isoforms of Na/H exchangers in RKPC-2 cells. By the use of BCECF [2',7',bis(2-carboxyethyl)-5,6-carboxyfluorescein intracellular pH (pHi) indicator] and single cell fluorescence microscopy, Na/H-exchange activities (defined as initial rate of Na-dependent pHi recovery) were found on the apical and basolateral membrane of RKPC-2 cells; apical and basolateral transport activities differed in sensitivity to dimethylamiloride, the basolateral being more sensitive. Northern blot analysis demonstrated the presence of a 5.2-kb transcript, related to Na/H-exchanger activity NHE-1, and a 3.2-kb transcript, related to Na/H-exchanger activity NHE-2. PTH (10(-8) M) inhibited apically and basolaterally located Na/H-exchanger activities. The inhibitory effect of PTH was mimicked by 8-bromo-adenosine 3'5'-cyclic monophosphate (cAMP); it was blunted in the presence of H-89 (inhibitor of protein kinase A) and was unaffected by calphostin C (inhibitor of protein kinase C). In contrast to 8-bromo-cAMP (and PTH), exposure of RKPC-2 cells to phorbol 12-myristate 13-acetate (TPA) caused a significant stimulation of both Na/H-exchange activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗