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Biomedical subjects

S L Alper

Publications and source records attributed to S L Alper.

At least 91 records · Page 5Linked to original sources
PubMed1996

Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton.

The red blood cell (RBC) membrane protein AE1 provides high affinity binding sites for the membrane skeleton, a structure critical to RBC integrity. AE1 biosynthesis is postulated to be required for terminal erythropoiesis and membrane skeleton assembly. We used targeted mutagenesis to assess AE1 function in vivo. RBCs lacking AE1 spontaneously shed membrane vesicles and tubules, leading to severe spherocytosis and hemolysis, but the levels of the major skeleton components, the synthesis of spectrin in mutant erythroblasts, and skeletal architecture are normal or nearly normal. The results indicate that AE1 does not regulate RBC membrane skeleton assembly in vivo but is essential for membrane stability. We postulate that stabilization is achieved through AE1-lipid interactions and that loss of these interactions is a key pathogenic event in hereditary spherocytosis.

Animals↗
PubMed1996

Proteolytic cleavage sites of native AE2 anion exchanger in gastric mucosal membranes.

The AE2 anion exchanger in pig and rabbit gastric mucosal membranes was subjected to limited proteolysis with trypsin, chymotrypsin, and papain, and to enzymatic N-deglycosylation. A monoclonal antibody to the AE2 C-terminal peptide was raised, characterized, and used to purify pig AE2 and its C-terminal cleavage products. Five distinct proteolytic cleavage sites within the AE2 transmembrane domain were defined by amino acid sequencing. The amino acid sequence of pig AE2 in the region encompassing the N-glycosylated Z-loop was also determined by RT-PCR. Tryptic cleavage of pig AE2 in the Z-loop produced C-terminal glycopeptides and was unaffected by deglycosylation, whereas the smaller rabbit AE2 C-terminal tryptic peptide lacked oligosaccharide, consistent with the respective amino acid sequences. The third consensus N-glycosylation site in pig Z-loop was heterogeneously glycosylated. Rapid papain cleavage in the Z-loop and slower cleavage in loop 7-8 produced C-terminal peptide products which were not N-glycosylated. Chymotryptic cleavage of the rabbit AE2 Z-loop required prior deglycosylation. Chymotryptic cleavage in the pig AE2 Z-loop produced C-terminal glycopeptides. Prior deglycosylation of pig AE2 unmasked novel, ionic strength-sensitive chymotryptic cleavage sites in the adjacent exofacial loop 7-8. These results provide experimental confirmation for some aspects of AE2 topography previously predicted from primary structure alone.

Amino Acid Sequence↗
PubMed1996

The cytoplasmic and transmembrane domains of AE2 both contribute to regulation of anion exchange by pH.

We have compared regulation by pH of AE1 (band 3)- and AE2-mediated 36Cl- uptake into Xenopus oocytes. 36Cl- influx was assayed at varying extracellular pH (pHo) values between 9.0 and 5.0 under conditions in which corresponding intracellular pH (pHi) values were at or near steady-state. Wild type (WT) AE1 displayed a broad convex pH versus activity curve, with peak activity at pHo 7.0 and 63% of maximal activity at pHo 5.0. In contrast, WT AE2 displayed a steep pH versus activity curve, with peak activity at pHo9.0 and full suppression at pHo 5.0. The structural basis of these differing pH sensitivities was examined by expression of cRNAs encoding chimeric and truncated proteins. Mutant polypeptides were expressed in oocytes and detected at the cell surface. The AE2cyto/AE1memb polypeptide displayed a broad pH versus activity curve similar to that of WT AE1. In contrast, the AE1cyto/AE2memb polypeptide displayed a steep pH versus activity curve, which was shifted toward acid pH values from that of WT AE2 by 0.69 +/- 0.04 pHo units. Moreover, whereas the pH versus activity curves of AE2 Delta99 and WT AE2 were indistinguishable, AE2 Delta510 exhibited a pH versus activity curve acid-shifted from that of WT AE2 by 0.66 +/- 0.13 pHo units (indistinguishable from that of AE1cyto/AE2memb). The data suggest that a pH sensor resides within the transmembrane region of AE2. The affinity for protons of this pH sensor is influenced by a modifier site located between residues 99 and 510 of the N-terminal cytoplasmic domain of AE2. Acidification of oocytes with acetate suggested that pHi accounted for some but not all of the measured pH dependence of AE2.

Acetates↗
PubMed1996

Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease.

Pathologic water loss from sickle erythrocytes concentrates the abnormal hemoglobin and promotes sickling. The Ca2+-activated K+ channel (Gardos channel) contributes to this deleterious dehydration in vitro, and blockade of K+ and water loss via this channel could be a potential therapy in vivo. We treated five subjects who have sickle cell anemia with oral clotrimazole, a specific Gardos channel inhibitor. Patients were started on a dose of 10 mg clotrimazole/kg/d for one week. Protocol design allowed the daily dose to be escalated by 10 mg/kg each week until significant changes in erythrocyte density and K+ transport were achieved. Blood was sampled three times a week for hematological and chemical assays, erythrocyte density, cation content, and K+ transport. At dosages of 20 mg clotrimazole/kg/d, all subjects showed Gardos channel inhibition, reduced erythrocyte dehydration, increased cell K+ content, and somewhat increased hemoglobin levels. Adverse effects were limited to mild/moderate dysuria in all subjects, and a reversible increase in plasma alanine transaminase and aspartic transaminase levels in two subjects treated with 30 mg clotrimazole/kg/d. This is the first in vivo evidence that the Gardos channel causes dehydration of sickle erythrocytes, and that its pharmacologic inhibition provides a realistic antisickling strategy.

Administration, Oral↗
PubMed1996

Combination therapy of erythropoietin, hydroxyurea, and clotrimazole in a beta thalassemic mouse: a model for human therapy.

beta thalassemia (beta thal) in DBA/2J mice is a consequence of the spontaneous and complete deletion of the beta major globin gene. Homozygous beta thal mice have clinical and biological features similar to those observed in human beta thal intermedia. Erythrocytes in human beta thal are characterized by a relative cell dehydration and reduced K+ content. The role of this erythrocyte dehydration in the reduced erythrocyte survival, which typifies the disease, has not previously been evaluated. We examined for 1 month the effects on the anemia and the erythrocyte characteristics of beta thal mice of daily treatment with either clotrimazole (CLT), an inhibitor of red blood cell (RBC) dehydration via the Gardos channel, or human recombinant erythropoietin (r-HuEPO), or hydroxyurea (HU). The use of either r-HuEPO or HU induced a significant increase in hemoglobin (Hb), hematocrit (Hct), erythrocyte K+ and a decrease in percent reticulocytes, suggesting improved erythrocyte survival. CLT alone decreased only mean corpuscular hemoglobin concentration (MCHC) and cell density and increased cell K+. Thus, though the Gardos channel plays a major role in cell dehydration of murine beta thal erythrocyte survival. Combination therapy with r-HuEPO plus HU produced no incremental benefit beyond those of single drug therapy. However, addition of CLT to r-HuEPO, to HU, or to combined r-HuEPO plus HU led to statistically significant increase in Hb, Hct, and erythrocyte K+ compared with any of the regimens without CLT. These results suggest that CLT not only inhibits erythrocyte dehydration, but also potentiates the erythropoietic and cellular survival responses to r-HuEPO and HU.

Animals↗
PubMed1996

Clotrimazole and efaroxan inhibit red cell Gardos channel independently of imidazoline I1 and I2 binding sites.

In the present report, we investigated the potential involvement of imidazoline I1 and I2 binding sites in the inhibition of the Ca(2+)-activated K+ channel (Gardos channel) by clotrimazole in human red cells. Ca(2+)-activated 86Rb influx was inhibited by clotrimazole and efaroxan but not by the imidazoline binding site ligands clonidine, moxonidine, cirazoline and idazoxan (100 microM). Binding studies with [3H]idazoxan and [3H]p-aminoclonidine did not reveal the expression of I1 and I2 binding sites in erythrocytes. These data indicate that the effects of clotrimazole and efaroxan on the erythrocyte Ca(2+)-activated K+ channel may be mediated by a 'non-I1/non-I2' binding site.

Adrenergic alpha-Antagonists↗
PubMed1996

Resistance to osmotic lysis in BXD-31 mouse erythrocytes: association with upregulated K-Cl cotransport.

The decreased osmotic fragility and reduced K+ content of BXD-31 mouse erythrocytes arise from variation at a single genetic locus. We compared ion transport in erythrocytes from BXD-31 mice and the parental strain, DBA/2J. The strains had similar rates for Na-K pump, Na/H exchange, Na-K-2Cl cotransport, Ca2+ activated K+ channel, or AE1-mediated SO4 transport. In contrast, K-Cl cotransport was twice as active in BXD-31 as in DBA/2J cells. Cl- dependent K+ efflux from BXD-31 cells displayed steep activation by acid pH (with maximal transport occurring at pH 6.75), whereas DBA/2J erythrocytes displayed a far less dramatic response to pH. Both strains displayed regulatory volume decrease in response to cell swelling. However, a 62% greater loss of cell K+ via K-Cl cotransport was observed in the BXD-31 strain. Furthermore the decreased osmotic fragility of BXD-31 red blood cells was normalized by treatment with nystatin to achieve normal cell K+ and water content. Thus upregulated K-Cl cotransport induces cell dehydration and K+ deficit in BXD-31 erythrocytes and causes their characteristic resistance to osmotic lysis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

HCO3(-)-dependent conformational change in gastric parietal cell AE2, a glycoprotein naturally lacking sialic acid.

Although the AE1 chloride/bicarbonate exchanger of the red blood cell is among the most thoroughly investigated of membrane transport proteins, less is known about the related AE2 polypeptide of parietal cells. We have studied enzymatic deglycosylation of native AE2 polypeptide in gastric mucosal membranes from pig and rabbit. Deglycosylation of AE2 was maximal at low ionic strength. Deglycosylation of AE2 in membranes was preferentially inhibited by bicarbonate compared with other anions. This inhibition was maximal at alkaline pH and was not evident after detergent solubilization of AE2. Deglycosylation of AE2 increased its susceptibility to proteolytic degradation, but the presence of bicarbonate protected against this degradation. Bicarbonate failed to inhibit deglycosylation of the membrane glycoproteins AE1 and gastric H(+)-K(+)-adenosinetriphosphatase beta-subunit or deglycosylation of the soluble glycoproteins fetuin and ribonuclease B. These data suggest that bicarbonate induces a conformational change in AE2 that can protect the polypeptide from deglycosylation and proteolysis. Pig AE2 was purified in sodium dodecyl sulfate, and its monosaccharide composition was determined after blotting onto polyvinylidene fluoride membrane. AE2 was found to be devoid of sialic acid, with a composition suggestive of the presence of lactosamine-type chains.

Animals↗
PubMed1995

Expression and function of bicarbonate/chloride exchangers in the preimplantation mouse embryo.

Bicarbonate/chloride (HCO3-/Cl-) exchangers regulate intracellular pH in the alkaline range. Previously, it has been shown that mouse embryos at the two-cell stage exhibit this activity, but that the otherwise ubiquitous mechanisms for regulating intracellular pH in the acid-to-neutral range are undetectable. We have examined mouse embryos during preimplantation development (one-cell zygote through blastocyst) to determine whether HCO3-/Cl- exchange activity exists at all stages, whether it is necessary for preimplantation development, and whether messenger RNAs from the known HCO3-/Cl- exchanger genes are expressed. We have found that all stages of preimplantation embryo have detectable HCO3-/Cl- exchange activity. In addition, inhibition of this activity with the stilbene anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) disrupts intracellular pH homeostasis and markedly inhibits embryo development from the two-cell stage to blastocysts in culture under conditions of moderately high external pH. Finally, mRNA encoding two members of the band 3-related AE anion exchanger gene family are expressed in preimplantation embryos.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis.

To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3sMEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHIS in the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine subsitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors.

Alleles↗
PubMed1995

Overexpression of AE1 Prague, but not of AE1 SAO, inhibits wild-type AE1 trafficking in Xenopus oocytes.

Mutations in the AE1 (band 3) anion exchanger of human erythrocytes have been associated with altered red cell shape and heritable disease. The Southeast Asian Ovalocytosis (SAO) AE1 mutation, a 27 nt deletion producing the delta 400-408 form of AE1, and the AE1 Prague mutation, a 10 nt insertion producing a frameshift after AE1 aa 821 leading to premature termination, are found only in the heterozygous state. We therefore examined accumulation and function of wt AE1 polypeptide in Xenopus oocytes when coexpressed with AE1 SAO and with AE1 Prague. Our SAO construct lacked the K56E (AE1 Memphis) polymorphism present in the endogenous AE1 SAO protein. Neither mutant AE1 mediated Cl- uptake into cRNA-injected Xenopus oocytes. Coinjection of mutant and wt cRNAs led to dose-dependent inhibition of wt function by AE1 Prague, but not by SAO. Though in vitro translation of the two mutants revealed little difference in their insertion into microsomal membranes, AE1 Prague accumulated in Xenopus oocytes to lower levels than did AE1 SAO or wt. Unlike AE1 SAO polypeptide, AE1 Prague polypeptide was not detectable at the oocyte surface. Moreover, overexpression of AE1 Prague, in contrast to AE1 SAO, reduced the accumulation of wt AE1, at the oocyte surface. This inhibition occurred in the absence of detectable heteromer formation between the AE1 Prague and wt AE1 polypeptides.

Animals↗
PubMed1995

Ca(2+)-activated K+ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins.

Despite recent progress in the molecular characterization of high-conductance Ca(2+)-activated K+ (maxi-K) channels, the molecular identities of intermediate conductance Ca(2+)-activated K+ channels, including that of mature erythrocytes, remains unknown. We have used various peptide toxins to characterize the intermediate conductance Ca(2+)-activated K+ channels (Gardos pathway) of human and rabbit red cells. With studies on K+ transport and on binding of 125I-charybdotoxin (ChTX) and 125I-kaliotoxin (KTX) binding in red cells, we provide evidence for the distinct nature of the red cell Gardos channel among described Ca(2+)-activated K+ channels based on (i) the characteristic inhibition and binding patterns produced by ChTX analogues, iberiotoxin (IbTX) and IbTX-like ChTX mutants, and KTX (1-37 and 1-38 variants); (ii) the presence of some properties heretofore attributed only to voltage-gated channels, including inhibition of K transport by margatoxin (MgTX) and by stichodactyla toxin (StK); (iii) and the ability of scyllatoxin (ScyTX) and apamin to displace bound 125I-charybdotoxin, a novel property for K+ channels. These unusual pharmacological characteristics suggest a unique structure for the red cell Gardos channel.

Animals↗
PubMed1995

Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalinization.

Xenopus oocytes express endogenous Na+/H+ exchange activity but lack significant endogenous Cl-/HCO3- exchange activity. Coupled operation of Na+/H+ exchange and Cl-/HCO3- exchange contributes in many cell types to the cellular response to hypertonic stress. We therefore examined in Xenopus oocytes the osmotic regulation of chloride transport mediated by recombinant anion exchanger proteins AE2 and AE1. Hypotonicity was without effect on either anion transporter. Hypertonicity activated AE2-associated 36Cl- influx and efflux in a time- and osmolarity-dependent manner, whether incremental osmoles were charged or uncharged, but had no measurable effect on AE1 function. Hypertonic stimulation of AE2 was completely inhibited by Na+ removal or by addition of amiloride. In contrast, neither maneuver altered isotonic activity of AE2. Hypertonicity also induced amiloride-sensitive elevation of oocyte intracellular pH (pHi), and shifted the sigmoidal relationship of extracellular pH vs. AE2 activity > or = 0.5 units to the acid. Injection of pH 7.4 buffer into oocytes attenuated both hypertonic alkalinization and activation of AE2-associated 36Cl- influx, without inhibition of isotonic AE2 function. These data demonstrate that recombinant AE2 expressed in Xenopus oocytes is activated by increased pHi and that hypertonic activation of AE2 is secondary to hypertonic activation of Na+/H+ exchange.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Cation transport in mouse erythrocytes: role of K(+)-Cl- cotransport in regulatory volume decrease.

We investigated cation transport and cell volume regulation in erythrocytes of CD1 and C57/B6 mice. Swelling of cells from either strain stimulated K+ efflux that was insensitive to ouabain, bumetanide, and clotrimazole. Seventy-five percent of swelling-induced K+ efflux was Cl- dependent (inhibited by sulfamate or methanesulfonate, partially by NO3-, but not by SCN-) and was inhibited by okadaic acid (OA; 50% inhibitory concentration = 18 +/- 6 nM in CD1 and 10 +/- 4 nM in C57/B6). In both strains, K+ efflux into isotonic medium was stimulated by staurosporine or by N-ethylmaleimide, and the latter was partially blocked by pretreatment of cells with OA. When cells of either strain were incubated in hypotonic medium or preswollen isosmotically with nystatin, OA-sensitive regulatory volume decrease (RVD) and K+ loss were observed. RVD produced by hypotonic swelling was prevented by Cl- replacement with sulfamate or methanesulfonate. These properties suggest the presence in outbred and inbred mouse erythrocytes of RVD mediated by K(+)-Cl- cotransport.

Animals↗
PubMed1995

Regional and segmental localization of AE2 anion exchanger mRNA and protein in rat kidney.

Chloride/base exchange activity has been detected in every mammalian nephron segment in which it has been sought. However, in contrast to the Cl-/HCO3- exchanger AE1 in type A intercalated cells, localization of AE2 within the kidney has not been reported. We therefore studied AE2 expression in rat kidney. AE2 mRNA was present in cortex, outer medulla, and inner medulla. Semiquantitative polymerase chain reaction of cDNA from microdissected tubules revealed AE2 cDNA levels as follows [copies of cDNA derived per mm tubule (+/- SE)]: proximal convoluted tubule, 688 +/- 161; proximal straight tubule, 652 +/- 189; medullary thick ascending limb, 1,378 +/- 226; cortical thick ascending limb, 741 +/- 24; cortical collecting duct, 909 +/- 71; and outer medullary collecting duct, 579 +/- 132. AE2 cDNA was also amplified in thin limbs and in inner medullary collecting duct. AE2 polypeptide was detected in all kidney regions. AE2 mRNA and protein were also detected in several renal cell lines. The data are compatible with the postulated roles of AE2 in maintenance of intracellular pH and chloride concentration and with its possible participation in transepithelial transport.

Animals↗
PubMed1995

Depletion of intercalated cells from collecting ducts of carbonic anhydrase II-deficient (CAR2 null) mice.

The kidneys of mice (CAR2-null mice) that are genetically devoid of carbonic anhydrase type II (CAII) were screened by immunocytochemistry with antibodies that distinguish intercalated and principal cells. Immunofluorescent localization of the anion exchanger AE1 and of the 56-kDa subunit of the vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) was used to identify intercalated cells, while the AQP2 water channel was used as a specific marker for principal cells of the collecting duct. The CAII deficiency of the CAR2-null mice was first confirmed by the absence of immunofluorescent staining of kidney sections exposed to an anti-CAII antibody. Cells positive for AE1 and H(+)-ATPase were common in all collecting duct regions in normal mice but were virtually absent from the inner stripe of the outer medulla and the inner medulla of CAR2-null mice. The number of positive cells was also reduced threefold in the cortical collecting duct of CAR2-null animals compared with normal mice. In parallel, the percentage of AQP2-positive cells was correspondingly increased in the collecting tubules of CAII-deficient mice, whereas the total number of cells per tubule remained unchanged. These results suggest that intercalated cells are severely depleted and are replaced by principal cells in CAII-deficient mice. Quantitative analysis and double staining showed that, in the cortex, both type A and type B intercalated cells are equally affected. Elucidation of the mechanism(s) responsible for this phenotype will be of importance in understanding the origin and development of intercalated cells in the kidney.

Animals↗