We describe an analysis of the NCX1 gene and show that various tissues express different alternat... more We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.
We report the labeling (internalization) of skeletal myoblasts (SMs) with a novel class of oxygen... more We report the labeling (internalization) of skeletal myoblasts (SMs) with a novel class of oxygen-sensing paramagnetic spin probe for noninvasive tracking and in situ monitoring of oxygenation in stem cell therapy using electron paramagnetic resonance (EPR) spectroscopy. SM cells were isolated from thigh muscle biopsies of mice and propagated in culture. Labeling of SM cells with the probe was achieved by coincubating the cells with submicron-sized (270 +/- 120 nm) particulates of the probe in culture for 48 h. The labeling had no significant effect on the viability or proliferation of the cells. The SM cells labeled with the probe were transplanted in the infarcted region of mouse hearts. The engraftment of the transplanted cells in the infarct region was verified by using MY-32 staining for skeletal myocytes. The in situ Po(2) in the heart was determined noninvasively and repeatedly for 4 wk after transplantation. The results showed significant enhancement of myocardial oxygenation at the site of cell transplant compared with untreated control. In conclusion, labeling of SM cells with the oxygen-sensing spin probe offers a unique opportunity for the noninvasive monitoring of transplanted cells as well as in situ tissue Po(2) in infarcted mouse hearts.
In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H a... more In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H and Cl/HCO(3) exchange on the villus cell brush border membrane (BBM). Although constitutive nitric oxide (cNO) has been demonstrated to alter gastrointestinal tract functions, how cNO may specifically alter these two transporters to regulate coupled NaCl absorption is unknown. In villus cells, inhibition of cNO synthase (cNOS) with l-N(G)-nitroarginine methylester (l-NAME) stimulated Na/H exchange whereas Cl/HCO(3) exchange was unaffected. In villus cell BBM vesicles (BBMV) prepared from rabbits treated with l-NAME, Na/H exchange was also stimulated. d-NAME, an inactive analog of l-NAME, and N(6)-(1-imonoethyl)-l-lysine dihydrochloride, a more selective inhibitor of inducible NO synthase, did not affect Na/H exchange. Kinetic studies demonstrated that the mechanism of stimulation is secondary to an increase in the maximal rate of uptake of Na, without an alteration in the affinity of the transporter for Na. Northern blot studies demonstrated an increase in the message for the BBM Na/H exchanger NHE3, and Western blot studies showed that the immunoreactive protein levels of NHE3 was increased when cNOS was inhibited. Thus these results indicate that cNO under nominal physiological states most likely maintains an inhibitory tone on small intestinal coupled NaCl absorption by specifically inhibiting BBM Na/H expression.
Neutral Na-amino acid cotransport by system ATB(0) [e.g., Na-alanine cotransport (NAcT)] is an im... more Neutral Na-amino acid cotransport by system ATB(0) [e.g., Na-alanine cotransport (NAcT)] is an important means of assimilation of amino acids in the intestine. NAcT is inhibited during chronic intestinal inflammation by an alteration in the affinity for the amino acid. How glucocorticoids, a standard of treatment for diseases characterized by chronic intestinal inflammation, may affect NAcT during chronic enteritis is not known. Thus we first demonstrated that methylprednisolone (MP) stimulated NAcT in the normal intestine. The mechanism of stimulation was secondary to an increase in cotransporter numbers without an alteration in the affinity for the amino acid. Treatment with MP reversed the reduction in NAcT in villus cells from the chronically inflamed intestine. MP also alleviated the decrease in Na-K-ATPase activity in villus cells during chronic enteritis. However, MP treatment reversed the NAcT inhibition in villus cell brush border membrane vesicles from the inflamed intestine, which suggested an effect of MP at the level of the cotransporter itself. Kinetic studies demonstrated that the reversal of NAcT inhibition by MP was secondary to restoration in the affinity for the amino acid without a change in the V(max). Unaltered steady-state mRNA and immunoreactive protein levels of NAcT also indicated that the number of cotransporters was unchanged after MP treatment in the chronically inflamed intestine. These results indicated that MP reversed NAcT inhibition in the chronically inflamed intestine by restoring the affinity of the transporter for the amino acid while it stimulated NAcT in the normal intestine by increasing the cotransporter numbers. Therefore, MP differentially regulates NAcT in the normal and chronically inflamed intestine.
We describe an analysis of the NCX1 gene and show that various tissues express different alternat... more We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.
We report the labeling (internalization) of skeletal myoblasts (SMs) with a novel class of oxygen... more We report the labeling (internalization) of skeletal myoblasts (SMs) with a novel class of oxygen-sensing paramagnetic spin probe for noninvasive tracking and in situ monitoring of oxygenation in stem cell therapy using electron paramagnetic resonance (EPR) spectroscopy. SM cells were isolated from thigh muscle biopsies of mice and propagated in culture. Labeling of SM cells with the probe was achieved by coincubating the cells with submicron-sized (270 +/- 120 nm) particulates of the probe in culture for 48 h. The labeling had no significant effect on the viability or proliferation of the cells. The SM cells labeled with the probe were transplanted in the infarcted region of mouse hearts. The engraftment of the transplanted cells in the infarct region was verified by using MY-32 staining for skeletal myocytes. The in situ Po(2) in the heart was determined noninvasively and repeatedly for 4 wk after transplantation. The results showed significant enhancement of myocardial oxygenation at the site of cell transplant compared with untreated control. In conclusion, labeling of SM cells with the oxygen-sensing spin probe offers a unique opportunity for the noninvasive monitoring of transplanted cells as well as in situ tissue Po(2) in infarcted mouse hearts.
In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H a... more In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H and Cl/HCO(3) exchange on the villus cell brush border membrane (BBM). Although constitutive nitric oxide (cNO) has been demonstrated to alter gastrointestinal tract functions, how cNO may specifically alter these two transporters to regulate coupled NaCl absorption is unknown. In villus cells, inhibition of cNO synthase (cNOS) with l-N(G)-nitroarginine methylester (l-NAME) stimulated Na/H exchange whereas Cl/HCO(3) exchange was unaffected. In villus cell BBM vesicles (BBMV) prepared from rabbits treated with l-NAME, Na/H exchange was also stimulated. d-NAME, an inactive analog of l-NAME, and N(6)-(1-imonoethyl)-l-lysine dihydrochloride, a more selective inhibitor of inducible NO synthase, did not affect Na/H exchange. Kinetic studies demonstrated that the mechanism of stimulation is secondary to an increase in the maximal rate of uptake of Na, without an alteration in the affinity of the transporter for Na. Northern blot studies demonstrated an increase in the message for the BBM Na/H exchanger NHE3, and Western blot studies showed that the immunoreactive protein levels of NHE3 was increased when cNOS was inhibited. Thus these results indicate that cNO under nominal physiological states most likely maintains an inhibitory tone on small intestinal coupled NaCl absorption by specifically inhibiting BBM Na/H expression.
Neutral Na-amino acid cotransport by system ATB(0) [e.g., Na-alanine cotransport (NAcT)] is an im... more Neutral Na-amino acid cotransport by system ATB(0) [e.g., Na-alanine cotransport (NAcT)] is an important means of assimilation of amino acids in the intestine. NAcT is inhibited during chronic intestinal inflammation by an alteration in the affinity for the amino acid. How glucocorticoids, a standard of treatment for diseases characterized by chronic intestinal inflammation, may affect NAcT during chronic enteritis is not known. Thus we first demonstrated that methylprednisolone (MP) stimulated NAcT in the normal intestine. The mechanism of stimulation was secondary to an increase in cotransporter numbers without an alteration in the affinity for the amino acid. Treatment with MP reversed the reduction in NAcT in villus cells from the chronically inflamed intestine. MP also alleviated the decrease in Na-K-ATPase activity in villus cells during chronic enteritis. However, MP treatment reversed the NAcT inhibition in villus cell brush border membrane vesicles from the inflamed intestine, which suggested an effect of MP at the level of the cotransporter itself. Kinetic studies demonstrated that the reversal of NAcT inhibition by MP was secondary to restoration in the affinity for the amino acid without a change in the V(max). Unaltered steady-state mRNA and immunoreactive protein levels of NAcT also indicated that the number of cotransporters was unchanged after MP treatment in the chronically inflamed intestine. These results indicated that MP reversed NAcT inhibition in the chronically inflamed intestine by restoring the affinity of the transporter for the amino acid while it stimulated NAcT in the normal intestine by increasing the cotransporter numbers. Therefore, MP differentially regulates NAcT in the normal and chronically inflamed intestine.
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Papers by S. Wisel