Exposing cardiomyocytes to subclinical concentrations of doxorubicin rapidly reduces their creatine transport

Doxorubicin is commonly used to treat leukemia, lymphomas, and solid tumors, such as soft tissue sarcomas or breast cancer. A major side effect of doxorubicin therapy is dose-dependent cardiotoxicity. Doxorubicin's effects on cardiac energy metabolism are emerging as key elements mediating its toxicity. We evaluated the effect of doxorubicin on [(14)C]creatine uptake in rat neonatal cardiac myocytes and HL-1 murine cardiac cells expressing the human creatine transporter protein. A significant and irreversible decrease in creatine transport was detected after an incubation with 50-100 nmol/l doxorubicin. These concentrations are well below peak plasma levels (5 μmol/l) and within the ranges (25-250 nmol/l) for steady-state plasma concentrations reported after the administration of 15-90 mg/m(2) doxorubicin for chemotherapy. The decrease in creatine transport was not solely because of increased cell death due to doxorubicin's cytotoxic effects. Kinetic analysis showed that doxorubicin decreased V(max), K(m), and creatine transporter protein content. Cell surface biotinylation experiments confirmed that the amount of creatine transporter protein present at the cell surface was reduced. Cardiomyocytes rely on uptake by a dedicated creatine transporter to meet their intracellular creatine needs. Our findings show that the cardiomyocellular transport capacity for creatine is substantially decreased by doxorubicin administration and suggest that this effect may be an important early event in the pathogenesis of doxorubicin-mediated cardiotoxicity.     

Creatine transporter activity and content in the rat heart supplemented by and depleted of creatine

The intracellular creatine concentration is an important bioenergetic parameter in cardiac muscle. Although creatine uptake is known to be via a NaCl-dependent creatine transporter (CrT), its localization and regulation are poorly understood. We investigated CrT kinetics in isolated perfused hearts and, by using cardiomyocytes, measured CrT content at the plasma membrane or in total lysates. Rats were fed control diet or diet supplemented with creatine or the creatine analog beta-guanidinopropionic acid (beta-GPA). Creatine transport in control hearts followed saturation kinetics with a K(m) of 70 +/- 13 mM and a V(max) of 3.7 +/- 0.07 nmol x min(-1) x g wet wt(-1). Creatine supplementation significantly decreased the V(max) of the CrT (2.7 +/- 0.17 nmol x min(-1) x g wet wt(-1)). This was matched by an approximately 35% decrease in the plasma membrane CrT; the total CrT pool was unchanged. Rats fed beta-GPA exhibited a >80% decrease in tissue creatine and increase in beta-GPA(total). The V(max) of the CrT was increased (6.0 +/- 0.25 nmol x min(-1) x g wet wt(-1)) and the K(m) decreased (39.8 +/- 3.0 mM). The plasma membrane CrT increased about fivefold, whereas the total CrT pool remained unchanged. We conclude that, in heart, creatine transport is determined by the content of a plasma membrane isoform of the CrT but not by the total cellular CrT pool.     

Stimulation of the creatine transporter SLC6A8 by the protein kinase mTOR

Cellular accumulation of creatine is accomplished by the Na(+), Cl(-), and creatine transporter CreaT (SLC6A8). The mammalian target of rapamycin (mTOR) is a kinase stimulating cellular nutrient uptake. The present experiments explored whether SLC6A8 is regulated by mTOR. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes, creatine-induced a current which was significantly enhanced by coexpression of mTOR. Kinetic analysis revealed that mTOR enhanced maximal current without significantly altering affinity. Preincubation of the oocytes for 32 h with rapamycin (50 nM) decreased the creatine-induced current and abrogated its stimulation by mTOR. The effect of mTOR on CreaT was blunted by additional coexpression of the inactive mutant of the serum and glucocorticoid-inducible kinase (K119N)SGK1 and mimicked by coexpression of wild type SGK1. In conclusion, mTOR stimulates the creatine transporter SLC6A8 through mechanisms at least partially shared by the serum and glucocorticoid-inducible kinase SGK1.     

Stimulation of the creatine transporter SLC6A8 by the protein kinases SGK1 and SGK3

Creatine binds phosphate thus serving energy storage. Cellular creatine uptake is accomplished by the Na+,Cl-, creatine transporter CreaT (SLC6A8). The present study explored the regulation of SLC6A8 by the serum and glucocorticoid inducible kinase SGK1, a kinase upregulated during ischemia. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes creatine induced a current which was significantly enhanced by coexpression of wild type SGK1 and constitutively active (S422D)SGK1, but not inactive (K127N)SGK1. Kinetic analysis revealed that (S422D)SGK1 enhanced maximal current without significantly altering affinity. The effect of SGK1 was mimicked by the constitutively active isoform (S419D)SGK3 but not by inactive (K119N)SGK3, wild type isoform SGK2 or constitutively active related kinase (T308D,S473D)PKB. In conclusion, the kinases SGK1 and SGK3 increase SLC6A8 activity by increasing the maximal transport rate of the carrier. Deranged SGK1 and/or SGK3 dependent regulation of SLC6A8 may affect energy storage particularly in skeletal muscle, heart, and neurons.     

Electrolysis stimulates creatine transport and transporter cell surface expression in incubated mouse skeletal muscle: potential role of ROS

Electrical field stimulation of isolated, incubated rodent skeletal muscles is a frequently used model to study the effects of contractions on muscle metabolism. In this study, this model was used to investigate the effects of electrically stimulated contractions on creatine transport. Soleus and extensor digitorum longus muscles of male NMRI mice (35-50 g) were incubated in an oxygenated Krebs buffer between platinum electrodes. Muscles were exposed to [(14)C]creatine for 30 min after either 12 min of repeated tetanic isometric contractions (contractions) or electrical stimulation of only the buffer before incubation of the muscle (electrolysis). Electrolysis was also investigated in the presence of the reactive oxygen species (ROS) scavenging enzymes superoxide dismutase (SOD) and catalase. Both contractions and (to a lesser degree) electrolysis stimulated creatine transport severalfold over basal. The amount of electrolysis, but not contractile activity, induced (determined) creatine transport stimulation. Incubation with SOD and catalase at 100 and 200 U/ml decreased electrolysis-induced creatine transport by approximately 50 and approximately 100%, respectively. The electrolysis effects on creatine uptake were completely inhibited by beta-guanidino propionic acid, a competitive inhibitor of (creatine for) the creatine transporter (CRT), and were accompanied by increased cell surface expression of CRT. Muscle glucose transport was not affected by electrolysis. The present results indicate that electrical field stimulation of incubated mouse muscles, independently of contractions per se, stimulates creatine transport by a mechanism that depends on electrolysis-induced formation of ROS in the incubation buffer. The increased creatine uptake is paralleled by an increased cell surface expression of the creatine transporter.     

Acute activation of glucose uptake by glucose deprivation in L929 fibroblast cells

Glucose is a very important energy source for a wide variety of cells, and the ability of cells to respond to changes in glucose availability or other cell stresses is of critical importance. Many mammalian cells respond to acute stress by increasing the V(max) of transport through GLUT1; the most ubiquitously expressed glucose transporter isoform. This study investigated the acute response of glucose uptake to glucose deprivation in L929 fibroblast cells--a cell line that expresses only the GLUT1 transporter. Results indicated that glucose deprivation of only a minute activated glucose uptake 10-fold and reached a maximum of 20-fold within 10 min. The activation was dose dependent and only partially muted by addition of up to 20mM pyruvate as an alternate energy source. In contrast to the kinetics of acute metabolic stress, glucose deprivation decreased the K(m) of transport, but did not alter the V(max). Maximal activation of glucose transport by glucose deprivation was completely additive to activation of transport by methylene blue--a stimulant that increased the V(max) of transport without a change in the K(m). Glucose-deprived activation of glucose transport was not inhibited by wortmannin or herbimycin A, but was completely inhibited by phenylarsine oxide. Altogether, the data indicate that L929 fibroblast cells respond quickly and robustly to the cell stress of glucose deprivation and methylene blue treatment by two distinct activation pathways.     

AMPK and substrate availability regulate creatine transport in cultured cardiomyocytes

Profound alterations in myocellular creatine and phosphocreatine levels are observed during human heart failure. To maintain its intracellular creatine stores, cardiomyocytes depend upon a cell membrane creatine transporter whose regulation is not clearly understood. Creatine transport capacity in the intact heart is modulated by substrate availability, and it is reduced in the failing myocardium, likely adding to the energy imbalance that characterizes heart failure. AMPK, a key regulator of cellular energy homeostasis, acts by switching off energy-consuming pathways in favor of processes that generate energy. Our objective was to determine the effects of substrate availability and AMPK activation on creatine transport in cardiomyocytes. We studied creatine transport in rat neonatal cardiomyocytes and HL-1 cardiac cells expressing the human creatine transporter cultured in the presence of varying creatine concentrations and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Transport was enhanced in cardiomyocytes following incubation in creatine-depleted medium or AICAR. The changes in transport were due to alterations in V(max) that correlated with changes in total and cell surface creatine transporter protein content. Our results suggest a positive role for AMPK in creatine transport modulation for cardiomyocytes in culture.     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles.

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na(+)-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na(+)-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K(m)=18.7 microM; V(max)=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K(m)=11.5 microM and V(max)=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na(+)-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na(+)-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Multiple effects of mercuric chloride on hexose transport in Xenopus oocytes.

HgCl(2) had both stimulatory and inhibitory effects on [(3)H]2-deoxyglucose (DG) uptake in Xenopus laevis oocytes. The Hg dose response was complex, with 0.1-10 microM Hg increasing total DG uptake, 30-50 microM Hg inhibiting, and concentrations >100 microM increasing uptake. Analyses of the effects of Hg on DG transport kinetics and cell membrane permeability indicated that low concentrations of Hg stimulated mediated uptake, intermediate concentrations inhibited mediated uptake, but high Hg concentrations increased non-mediated uptake. 10 microM Hg increased the apparent V(max) for DG uptake, but caused little or no change in apparent K(m). Phenylarsine oxide prevented the increase in DG uptake by 10 microM Hg, suggesting that the increase was due to transporter recruitment. Microinjecting low doses of HgCl(2) into the cell increased mediated DG uptake. Higher intracellular doses of Hg increased both mediated and non-mediated DG uptake. Both insulin and Hg cause cell swelling in isotonic media and, for insulin, this swelling has been linked to the mechanism of hormone action. Osmotically swelling Xenopus oocytes stimulated DG transport 2-5-fold and this increase was due to an increased apparent V(max). Exposing cells to 10 microM Hg or 140 nM insulin both increased cellular water content by 18% and increased hexose transport 2-4-fold. These data indicate that low concentrations of Hg and insulin affect hexose transport in a similar manner and that for both an increase cellular water content could be an early event in signaling the increase in hexose transport.     

Creatine ethyl ester: a new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K(m) and V(max) values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.     

Selective transport of adenosine into porcine coronary smooth muscle

Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [(3)H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake (V(max)) of 67 +/- 7.0 pmol. mg protein(-1). min(-1) and a Michaelis constant (K(m)) of 10. 5 +/- 5.8 microM in isolated cells and a V(max) of 5.1 +/- 0.5 pmol. min(-1). mg wet wt(-1) and a K(m) of 17.6 +/- 2.6 microM in intact rings. NBTI inhibited transport into small arteries (IC(50) = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [(3)H]sucrose indicate the V(max) and K(m) for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.     

PIKfyve in the SGK1 mediated regulation of the creatine transporter SLC6A8.

The Na(+),Cl(-),creatine transporter CreaT (SLC6A8) mediates concentrative cellular uptake of creatine into a wide variety of cells. Previous observations disclosed that SLC6A8 transport activity is enhanced by mammalian target of rapamycin (mTOR) at least partially through the serum and glucocorticoid inducible kinase isoforms SGK1 and SGK3. As SLC6A8 does not contain a putative SGK consensus motif, the mechanism linking SGK1 with SLC6A8 activity remained elusive. A candidate kinase is the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3), which has previously been shown to regulate the glucose transporter GLUT4. The present experiments explored the possibility that SLC6A8 is regulated by PIKfyve. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes creatine induced a current which was significantly enhanced by coexpression of PIKfyve. The effect of PIKfyve on SLC6A8 was blunted by additional coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase (K127N)SGK1. The stimulating effect of PIKfyve was abrogated by replacement of the serine in the SGK consensus sequence by alanine ((S318A)PIKfyve). Moreover, coexpression of ( S318A)PIKfyve blunted the effect of SGK1 on SLC6A8 activity. The observations suggest that SGK1 regulates the creatine transporter SLC6A8 at least partially through phosphorylation and activation of PIKfyve and subsequent formation of PI(3,5)P(2).     

Studies on the mitochondrially bound form of rat brain creatine kinase

1. The development of the total rat brain creatine kinase was studied in brain homogenates. Until approx. 14-15 days after birth, the activity remains less than one-third that of the adult activity (207+/-6 units/g wet wt. s.d.; n=3). Over the next 10 days the activity increases markedly to the adult value and thereafter remains essentially constant. 2. In the adult brain, approx. 5% (11.9+/-2.2 units/g wet wt. s.d.; n=5) of the total creatine kinase is associated with the mitochondrial fraction. This creatine kinase could not be solubilized by sodium acetate solutions of up to 0.8m concentration, whereas 66% of the hexokinase associated with brain mitochondria was released under these conditions. 3. Rat brain mitochondria incubated in the presence of various concentrations of creatine (1, 5 and 10mm) and ADP (100mum) synthesized phosphocreatine at rates of approx. 4.5, 11 and 17.5nmol/min per mg of mitochondrial protein. Atractyloside (50mum) or oligomycin (1.5mug/mg of mitochondrial protein) completely inhibited the synthesis of phosphocreatine. 4. The apparent K(m) and V(max.) values of the mitochondrially bound rat brain creatine kinase were determined in both directions. The V(max.) in the direction of phosphocreatine synthesis is 237nmol/min per mg of mitochondrial protein, with an apparent K(m) for creatine of 1.67mm and for MgATP(2-) of 0.1mm, and in the reverse direction V(max.) is 489nmol/min per mg of mitochondrial protein, with an apparent K(m) for phosphocreatine of 0.4mm and for MgADP(-) of 27mum. 5. The results are discussed with reference to the role that the mitochondrially bound creatine kinase may play in the development of brain energy metabolism.     

Expression and possible role of creatine transporter in the brain and at the blood-cerebrospinal fluid barrier as a transporting protein of guanidinoacetate, an endogenous convulsant

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a K(m) value of 269 microM/412 microM which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that of d-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB.     

Uptake of creatine by cultured cells

We have examined the uptake of creatine by cultured monolayers of human IMR-90 flbroblasts, human uterine smooth muscle cells, calf aortic smooth muscle cells, and myoblasts and myotubes of the L₆E₉ rat skeletal muscle cell line. Creatine uptake is dependent on temperature and sensitive to the presence of Na⁺ in the extracellular medium. It is saturable, apparently concentrative, and inhibited by ouabain and structural analogs of creatine. In these respects, it resembles the process of creatine uptake by isolated preparations of skeletal muscle and brain tissues. Lineweaver-Burk plots of the data for variation in rate of uptake with concentration of creatine in the medium are nonlinear, suggesting that the process of uptake may be heterogeneous. Assuming the operation of two saturable processes of uptake, we calculated two values for apparent K_m and V for each cell line. Kinetic parameters of creatine uptake by the different cell types are similar. The lower values of K_m (0.02–0.04 mm) are in the physiological range of creatine concentration in mammalian plasma.     

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.     

Parkin increases dopamine uptake by enhancing the cell surface expression of dopamine transporter

Mutations of parkin, a protein-ubiquitin E3 ligase, are linked to Parkinson's disease (PD). Although a variety of parkin substrates have been identified, none of these is selectively expressed in dopaminergic neurons, whose degeneration plays a critical role in PD. Here we show that parkin significantly increased dopamine uptake in the human dopaminergic neuroblastoma cell line SH-SY5Y. This effect was accompanied by increased V(max) of dopamine uptake and unchanged K(m). Consistent with this, increased binding sites for dopamine transporter (DAT) ligand were observed in SH-SY5Y cells overexpressing parkin. The results were confirmed when parkin was transfected in HEK293 cells stably expressing DAT. In these cells, parkin enhanced the ubiquitination and degradation of DAT, increased its cell surface expression, and augmented dopamine uptake. The effects of parkin were significantly abrogated by its PD-causing mutations. Because the cell surface expression of functional DAT requires its oligomerization, misfolded DAT, induced either by the protein glycosylation inhibitor tunicamycin or by its C-terminal truncation, significantly attenuated cell surface expression of native DAT and reduced dopamine uptake. Expression of parkin, but not its T240R mutant, significantly alleviated these detrimental effects of misfolded DAT. Thus, our studies suggest that parkin increases dopamine uptake by enhancing the ubiquitination and degradation of misfolded DAT, so as to prevent it from interfering with the oligomerization and cell surface expression of native DAT. This function of parkin would enhance the precision of dopaminergic transmission, increase the efficiency of dopamine utilization, and reduce dopamine toxicity on neighboring cells.     

Regulation of human organic anion transporter 4 by progesterone and protein kinase C in human placental BeWo cells

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT4 is abundantly expressed in the placenta. In the current study, we examined the regulation of hOAT4 by pregnancy-specific hormones progesterone (P(4)) and 17beta-estradiol (E(2)) and by protein kinase C (PKC) in human placental BeWo cells. P(4) induced a time- and concentration-dependent downregulation of hOAT4 transport activity, whereas E(2) had no effect on hOAT4 function. The downregulation of hOAT4 activity by P(4) mainly resulted from a decreased cell surface expression without a change in total cell expression of the transporter, kinetically revealed as a decreased V(max) without significant change in K(m). Activation of PKC by phorbol 12,13-dibutyrate also resulted in an inhibition of hOAT4 activity through a decreased cell surface expression of the transporter. However, P(4)-induced downregulation of hOAT4 activity could not be prevented by treating hOAT4-expressing cells with the PKC inhibitor staurosporine. We concluded that both P(4) and activation of PKC inhibited hOAT4 activity through redistribution of the transporter from cell surface to the intracellular compartments. However, P(4) regulates hOAT4 activity by mechanisms independent of PKC pathway.     

Mechanism of glucocorticoid-mediated reversal of inhibition of Cl(-)/HCO(-)(3) exchange during chronic ileitis

In the normal ileum, coupled NaCl absorption occurs via the dual operation of Na(+)/H(+) and Cl(-)/HCO(-)(3) exchange on the brush-border membrane (BBM) of villus cells. In a rabbit model of chronic small intestinal inflammation we determined the cellular mechanism of inhibition of NaCl absorption and the effect of steroids on this inhibition. Cl(-)/HCO(-)(3) but not Na(+)/H(+) exchange was reduced in the BBM of villus cells during chronic ileitis. Cl(-)/HCO(-)(3) exchange was inhibited secondary to a decrease in the affinity for Cl(-) rather than an alteration in the maximal rate of uptake of Cl(-) (V(max)). Methylprednisolone (MP) stimulated Cl(-)/HCO(-)(3) exchange in the normal ileum by increasing the V(max) of Cl(-) uptake rather than altering affinity for Cl(-). MP reversed the inhibition of Cl(-)/HCO(-)(3) exchange in rabbits with chronic ileitis. However, MP alleviated the Cl(-)/HCO(-)(3) exchange inhibition by restoring the affinity for Cl(-) rather than altering the V(max) of Cl(-) uptake. These data suggest that glucocorticoids mediate the alleviation of Cl(-)/HCO(-)(3) exchange inhibition in chronically inflamed ileum by reversing the same mechanism that was responsible for inhibition of this transporter rather than exerting a direct effect on the transporter itself, as was the case in normal ileum.