Hyun Dju Kim

Regulation of intestinal Cl- and HCO3- secretion by uroguanylin

Uroguanylin is an intestinal peptide hormone that may regulate epithelial ion transport by activating a receptor guanylyl cyclase on the luminal surface of the intestine. In this study, we examined the action of uroguanylin on anion transport in different segments of freshly excised mouse intestine, using voltage-clamped Ussing chambers. Uroguanylin induced larger increases in short-circuit current ( I sc) in proximal duodenum and cecum compared with jejunum, ileum, and distal colon. The acidification of the lumen of the proximal duodenum (pH 5.0-5.5) enhanced the stimulatory action of uroguanylin. In physiological Ringer solution, a significant fraction of the I sc stimulated by uroguanylin was insensitive to bumetanide and dependent on[Formula: see text] in the bathing medium. Experiments using pH-stat titration revealed that uroguanylin stimulates serosal-to-luminal [Formula: see text]secretion ([Formula: see text]) together with a larger increase in I sc. Both[Formula: see text]and I sc were significantly augmented when luminal pH was reduced to pH 5.15. Uroguanylin also stimulated the[Formula: see text]and I sc across the cecum, but luminal acidity caused a generalized decrease in the bioelectric responsiveness to agonist stimulation. In cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice, the duodenal I sc response to uroguanylin was markedly reduced, but not eliminated, despite having a similar density of functional receptors. It was concluded that uroguanylin is most effective in acidic regions of the small intestine, where it stimulates both [Formula: see text] and Cl- secretion primarily via a CFTR-dependent mechanism.

CLONING, EXPRESSION, AND CHARACTERIZATION OF A NOVEL GUANYLATE-BINDING PROTEIN, GBP3 IN MURINE ERYTHROID PROGENITOR CELLS

We report the molecular cloning of a novel guanylate-binding protein (GBP), termed mouse GBP3 (mGBP3) in Friend virus-induced mouse erythroid progenitor (FVA) cells. The 71-kDa mGBP3 belongs to a family of known GBPs that contain the first two consensus motifs, GXXXXGK(S/T) and DXXG, but lack the third element, (N/T)KXD, found in typical GTP-binding proteins. Recombinant mGBP3 protein, expressed using a baculovirus expression system, binds to agarose-immobilized guanine nucleotides (GTP, GDP and GMP). Moreover, mGBP3 has been found to have an intrinsic GTPase activity with K(m) and Vmax values of 77 +/- 4 microM and 21 +/- 0.5 pmol min-1 microgram-1 of protein, respectively. The mGBP3 is distinct from the other GBPs, in that it does not have an isoprenylation/methylation motif CAAX at the carboxyl terminus. The mGBP3 appears to be localized in the cytosol based on immunofluorescence staining. Although the mGBP3 transcript is expressed to a varying degree in numerous mouse tissues, the message is most abundant in FVA cells. The mGBP3 transcript increases in FVA cells undergoing differentiation to a maximum within a few hours and then decreases to an undetectable level by 24 h. These results, taken together, suggest that mGBP3 is a novel member of a family of guanylate-binding proteins, which plays a role in the erythroid differentiation. The nucleotide sequence reported in this paper has been submitted to the GenBank with accession number U44731.

Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells.

A Na+/K+/Cl- cotransport pathway has been examined in the HT29 human colonic adenocarcinoma cell line using 86Rb as the K congener. Ouabain-resistant bumetanide-sensitive (OR-BS) K+ influx in attached HT29 cells was 17.9 +/- 0.9 nmol/min per mg protein at 25 degrees C. The identity of this pathway as a Na+/K+/Cl- cotransporter has been deduced from the following findings: (a) OR-BS K+ influx ceased if the external Cl- (Cl-o) was replaced by NO3- or the external Na+ (Na+o) by choline; (b) neither OR-BS 24Na+ nor 36Cl- influx was detectable in the absence of external K+ (K+o); and (c) concomitant measurements of 86Rb+, 22Na+, and 36Cl- influx indicated that the stoichiometry of the cotransport system approached a ratio of 1N+:1K+:2Cl-. In addition, OR-BS K+ influx was exquisitely sensitive to cellular ATP levels. Depletion of the normal ATP content of 35-40 nmol/mg protein to 10-15 nmol/mg protein, a concentration at which the ouabain-sensitive K+ influx was unaffected, completely abolished K+ cotransport. OR-BS K+ influx was slightly reduced by the divalent cations Ca2+, Ba2+, Mg2+ and Mn2+. Although changes in cell volume, whether shrinking or swelling, did not influence OR-BS K+ influx, ouabain-sensitive K+ influx was activated by cell swelling. As in T84 cells, we found that the OR-BS K+ influx in HT29 cells was stimulated by exogenous cyclic AMP analogues and by augmented cyclic AMP content in response to vasoactive intestinal peptide, forskolin, norepinephrine and forskolin or prostaglandin E1.

Cloning of ClC-2 chloride channel from murine duodenum and its presence in CFTR knockout mice.

We report the cloning of a murine ClC-2 chloride channel cDNA from duodenal epithelium by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers and by rapid amplification of cDNA ends (RACE)-PCR. Other than CFTR, this represents the first cloned chloride channel from intact intestine. The ClC-2 cDNA predicts encoding of a 908 amino acid polypeptide with a calculated M(r) of 99,373. The amino acid sequence of the murine ClC-2 chloride channel is over 94% identical to the ClC-2 chloride channel proteins of other species. Of interest is the finding that the ClC-2 mRNA is expressed about the same level in duodena from both CFTR knockout and wild-type mice. This is in keeping with the suggestion that ClC-2 might be a therapeutic target in cystic fibrosis.

The liver is an organ site for the release of inosine metabolized by non-glycolytic pig red cells.

The metabolic energy source used by the pig red cell, which is unable to metabolize blood-borne glucose, was examined. Potential physiological substrates include adenosine, inosine, ribose, deoxyribose, dihydroxyacetone and glyceraldehyde, of which inosine was previously implicated. A net ATP synthesis by red cells occurs during in situ perfusion through the adult miniature pig liver. HPLC analysis of the perfusate revealed the presence primarily of inosine and hypoxanthine. Inosine production by the liver was 0.015 mumol/g per min. Moreover, red cells maintain ATP when suspended in a balanced salt medium during a 6 h incubation at 38 degrees C, in which inosine is continuously infused to give an external concentration of no more than 3 mumol/l, mimicking its plasma level. Inosine consumption under these infusion conditions was 56 nmol/ml cell per h, which is two orders of magnitude lower than when inosine is present in millimolar concentration. The total red cell inosine consumption of 9.63 mumol/h is much less than the total liver inosine production of 212 mumol/h. These findings suggest that the liver is an organ site elaborating inosine, and that maintenance of a 3 mumol/l inosine in plasma is sufficient to meet the energy requirements of the pig red cells.

Volume-activated Na/H exchange activity in fetal and adult pig red cells: Inhibition by cyclic AMP

SummaryHyposmotic swelling of pig red cells leads to a selective increase in K permeability, whereas hyperosmotic cell shrinkage augments the Na permeability. In this regard, the ouabain-resistant (OR) Na flux of red cells of newborn and adult pigs is characterized in detail. A reduction in cell volume by approximately 18% leads to an increase in the OR Na efflux of fetal and adult cells by 15-and fourfold, respectively. The OR Na influx in both cell types is equally influenced by cell shrinkage. Depletion of cellular K does not influence the volume-activated OR Na efflux. Nor does OR Na influx require external K. Both OR Na efflux and influx activated by shrinkage are inhibited by the diuretics furosemide and amiloride. The rank order of decreasing anion sensitivity for diuretic-sensitive Na efflux was acetate > chloride > gluconate > nitrate. Cell shrinkage induced by the addition of hypertonic salts results in an acidification of the unbuffered and CO2-free media, provided that both Na and DIDS are present. The qcidification process can be reversed by either of the diuretic agents. These findings suggest that the shrinkageactivated OR Na flux is primarily mediated by a Na/H exchanger rather than by a Na/K/Cl cotransporter. Once loaded with either cAMP or cGMP, cell swelling can no longer activate the Na/H exchanger. The Na/H exchanger activity is detectable in the fetal cells of normal volume but quiescent in adult cells, indicating that the exchanger undergoes a developmental change during the transition from the fetal to adult stage.

Is adenosine a second metabolic substrate for human red blood cells

Adenosine is present in the micromolar range in human plasma. In this study, metabolism of adenosine, which was maintained between 0.62 +/- 0.03 and 2.92 +/- 0.43 microM by means of a continuous infusion using a Harvard infusion pump, was investigated in human red blood cells. It was found that lactate production increases linearly as the adenosine concentration was raised. Cells infused with an average adenosine concentration of 2 microM produced lactate comparable to that produced by 5 mM glucose. The extent to which ATP concentration is maintained by adenosine also depends on its concentration. After a 4 h infusion with an average adenosine concentration of 0.7 microM, ATP content amounts to 75% of the glucose control. Raising the adenosine infusion concentration to 1.5 microM results in a full maintenance of ATP levels and at concentrations higher than 1.5 microM, adenosine produces a net synthesis of ATP. A net synthesis of ATP also occurs with adenosine concentration below 1.5 microM, if supplemented with glucose. In contrast, inosine infusion provides only a partial support of ATP and fails to produce a net synthesis of ATP in the presence of glucose. In addition, the presence of purine nucleoside and glucose together influence the metabolism of each other, depending on inorganic phosphate content (Pi). At a Pi concentration of 1 mM, the glucose consumption rate is reduced by approx. 25% by purine nucleoside infusion and vice versa. In sharp contrast, glucose consumption at 16 mM Pi is potentiated by adenosine. These findings suggest that plasma adenosine contributes significantly to human red cell energetics, even though it is present at a concentration several orders of magnitude lower than glucose.

Forskolin inhibition of hexose transport in cardiomyocytes

The effects of insulin, forskolin, isoproterenol, and epinephrine on 3-O-methylglucose (hexose) transport and cell cyclic AMP levels were determined in adult rat cardiomyocytes. Insulin stimulated hexose transport in these cells an average of 2.5-fold. Initial hexose transport rates at 1 mM hexose were 3.75×10−2 nmol/mg cell protein/second in the absence of insulin, and 8.25×10−2 nmol/mg cell protein/second in the presence of 12.3 μM insulin. Forskolin at 5 μM nearly abolished hexose transport within 3 s of exposure, but did not increase cell cyclic AMP concentrations within 9 s. The apparentKi for hexose transport inhibition was about 0.3 μM forskolin. Epinephrine and isoproterenol at 50 μM increased cell cyclic AMP 4-fold during 9 s exposure, but did not affect hexose transport. Treatment of cells with these catecholamines of forskolin for up to 99 s increased cell cyclic AMP, but only forskolin inhibited hexose transport. We coclude from these results that forskolin acts on hexose transport independent of its action on adenyl cyclase, and that cyclic AMP does not inhibit or stimulate hexose transport.

Studies on avian erythrocyte metabolism: XVII. Kinetics and transport properties of myo-inositol in chicken reticulocytes

The uptake of myo-inositol was determined in a reticulocyte-enriched fraction prepared from chicken blood and compared with uptake in mature erythrocytes. While reticulocytes accumulated inositol at levels more than threefold that of the plasma concentration, erythrocyte levels were only slightly higher than that of the plasma concentration. The rate of uptake in reticulocytes was approximately 66 mumol/ml rbc/h compared to 5 mumol/ml rbc/h in mature erythrocytes when measured at an inositol medium concentration of 250 microM. The kinetic analysis of inositol influx by reticulocytes reveals a two component system: saturable and nonsaturable. The saturable component, which has a Km for inositol of approximately 222 microM, is Na-dependent. This Na-dependent saturable component, which presumably reflects active transport of inositol, accounts for 30-35% of the transport process. The saturable component is completely inhibited by amiloride but to a lesser extent by ouabain and bumetanide. Moreover, in the course of reticulocyte maturation, the saturable component is lost concomitantly with the completion of the synthesis of myo-inositol pentakisphosphate and the drastic decrease in the membrane permeability to inositol. In addition, phloretin and cytochalasin B, which bind to hexose carriers and inhibit hexose sugar transport, also inhibited inositol transport. The uptake of inositol was not affected by excesses of 3-O-methylglucose (100 mM) or by physiological concentrations of D-glucose. Thus, the transport mechanism of myo-inositol appears distinct from that of D-glucose.

Chapter 6 Ion Transport and Adenylyl Cyclase System in Red Blood Cells

Publisher Summary An impressive number of ion transport pathways, now known to exist in a variety of cells, were originally identified in red cell membranes— namely, Ca 2+ -activated K + channels, Cl − /HC0 3 − exchanger, ouabain inhibition of Na + /K + pump, Ca 2+ pump, and Na + /K + cotransport. Apart from the metabolic energy that energizes the Na + /K + pump, a different form of energy transduction, which can also fuel a solute movement against its chemical potential gradient, has been recognized as implicitly embodied in the pioneering discovery of Na + gradient-coupled glucose transport in intestinal epithelia. The transport of one ion influences the transport of the other in the same direction, termed “cotransport,” and in the opposite direction, termed “antiport,” involving a common element in the membrane. Cotransport system can mediate concomitant movement of more than two ions, as evidenced by the Na + /K + /Cl − cotransport pathway, as opposed to the Na + /H + antiport. The antiporter can operate, in conjunction with the Cl − /HC0 3 - exchanger, resulting in salt and water movement. There is growing evidence that hormones and neurotransmitters regulate ion transport. This chapter discusses the regulation of ion transport, by the adenylyl cyclase system, with particular emphasis on studies with red cells used as a model system. It describes the advances in the adenylyl cyclase system, followed by developmental changes in adenylyl cyclase in reticulocytes and in erythrocytes, the effects of cyclic adenosine monophosphate (AMP) in Na + I K + I Cl − cotransport, Na + /H + antiport, and K + /Cl − cotransport systems, and explains the observation, pertaining to the influence of adenosine receptor agonists, on KCl cotransport. The influence of cyclic AMP (cAMP) on ion transport in red cells is also explained in the chapter. Non-nucleated mammalian red cells are capable of responding to cAMP that are introduced exogenously.