Quercetin relaxes guinea pig gallbladder strips.

Quercetin, a phytoestrogen and flavonoid, relaxes intestinal and vascular smooth muscle. The purpose of this study was to determine if quercetin had an effect on gallbladder smooth muscle. An in vitro technique was used to determine the effects of quercetin on gallbladder strips and which system(s) mediated the relaxation. Paired t tests were used; differences between means of P < .05 were considered significant. Adding quercetin before cholecystokinin or KCl produced a significant (P < .001) decrease in the amount of tension (0.80 ± 0.04 vs 0.48 ± 0.04 g cholecystokinin octapeptide and 0.8 ± 0.06 vs 0.54 ± 0.05 g KCl, respectively). When the protein kinase C (PKC) inhibitors bisindolymaleimide IV and chelerythrine Cl- were simultaneously, a significant (P < .001) reduction in the quercetin-induced relaxation (45.7% ± 4.3% vs 27.6% ± 3.4%) was observed. To determine if protein kinase A (PKA) mediated the quercetin-induced relaxation, PKA inhibitor 14-22 amide myristolated was used. It significantly (P < .05) decreased the amount (40.4% ± 3.7% vs 34.5% ± 3.3%) of quercetin-induced relaxation. The use of 2-APB also significantly (P < .001) reduced the amount of quercetin-induced relaxation (51.2% ± 3.5% vs 14.8% ± 3.6%). l-NG-methyl-l-arginine acetate salt, a nitric oxide synthase inhibitor, significantly (P < 001) decreased the quercetin-induced relaxation (45.7% ± 4.2% vs 35.2% ± 3.6%). KT5823, a PKC inhibitor, had no effect on the quercetin-induced relaxation. Quercetin blocked extracellular Ca2+ entry which affected downstream events such as activation of PKC, PKA, intracellular Ca2+ release, and activation of nitric oxide synthase. Quercetin relaxed cholecystokinin octapeptide and KCl-induced tension in a concentration dependent manner. Thus quercetin-induced relaxation was mediated by multiple signaling pathways.

Gallbladder motility and the sex of the guinea pig.

Progesterone (P), 17ß-estradiol (E2), and dihydrotestosterone (DHT) affect gallbladder motility. When gallbladders were taken from women and men, women had more estrogen and P receptors than men. Both P and E2 had an inhibitory effect upon gallbladder contractility in men and premenopausal and postmenopausal women. Similar findings have been reported in gallbladder strips from male and female guinea pigs. In the present study, there was no significant difference in the amount of E2-, P-, or DHT-induced relaxation of CCK-induced tension when the responses in gallbladder strips from male and female guinea pigs were compared. Three metabolites of P were used: 17-hydroxyprogesterone (17-P), 20α-hydroxyprogesterone (20-P), and 21-hydroxyprogesterone (21-P). There was no significant difference in the responses from strips from male and female guinea pigs. In order to determine if the effects of E2 and P were additive, strips from male animals were exposed to either E2 or P and the amount of relaxation recorded. After recovery, the strips were exposed to E2 or P in reverse order to ensure the order of treatment had no effect. Then, the strips were treated with both E2 and P simultaneously and the relaxation recorded. This procedure was repeated with strips from female guinea pigs. The effect of E2 and P was found to be additive; however, the response of the strips from each sex were not significantly different. It is concluded that the sex of the guinea pig has no significant effect on the response to the sex hormones used.

Curcumin Relaxes Precontracted Guinea Pig Gallbladder Strips via Multiple Signaling Pathways.

BACKGROUND: Curcumin (diferuloymethane) is the active ingredient of the dietary spice turmeric. Curcumin modulates various signalling molecules, including inflammatory agents, transcription factors, protein kinases and cell cycle regulatory proteins. The purpose of this study was to determine if curcumin had an effect on gallbladder motility. METHODS: A pharmacologic in vitro technique was used. Since curcumin relaxed both cholecystokinin octapeptide- (CCK) and KCl-induced tension of guinea pig gallbladder strips in a concentration dependent manner, an in vitro technique was used to determine which second messenger system(s) mediated the observed relaxation. Paired t-tests, t-tests and analysis of variance were used for statistical analysis. Differences between mean values of P < 0.05 were considered significant. RESULTS: To determine if protein kinase A (PKA) mediated the curcumin-induced relaxation, PKA inhibitor 14-22 amide myristolated (PKA-IM) was used. PKA-IM had no significant effect on the amount of curcumin-induced relaxation. When the protein kinase C (PKC) inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together, a significant (P < 0.01) reduction in the curcumin-induced relaxation was observed. The use of tetraethylammonium chloride (TEA) caused a significant (P < 0.01) decrease in the amount of curcumin-induced relaxation. Adding curcumin prior to the KCl caused a significant (P < 0.001) decrease in the amount of KCl-induced tension. CONCLUSIONS: The results suggested that the curcumin-induced relaxation is mediated by multiple signaling pathways including the PKC second messenger system, inhibiting extracellular Ca2+ entry and K+ channels.

The Resveratrol-induced Relaxation of Cholecystokinin Octapeptide- or KCl-induced Tension in Male Guinea Pig Gallbladder Strips Is Mediated Through L-type Ca2+Channels.

BACKGROUND/AIMS: Resveratrol (3,5,4'-trihydroxystilbene) is a polyphenolic compound (stilbene) and a phytoalexin. The purpose of this study was to determine the mechanism which mediated the resveratrol-induced relaxation of cholecystokinin octapeptide- or KCl-induced tension in male guinea pig gallbladder strips. METHODS: Gallbladder strips were prepared and suspended in in vitro chambers filled with Krebs-Henseleit solution. The strips were attached to force displacement transducers, and the changes in tension were recorded on a polygraph. All reagents were added directly into the chambers. RESULTS: To determine if intracellular Ca(2+) release mediated the resveratrol-induced relaxation of cholecystokinin octapeptide-induced tension, 2-aminoethoxydiphenylborane (2-APB) was used. 2-APB significantly (P < 0.01) decreased the amount of RSVL-induced relaxation. To determine if protein kinase A (PKA) mediated the resveratrol-induced relaxation, PKA inhibitor 14-22 amide myristolated (PKA-IM) was used. PKA-IM had no effect on resveratrol-induced relaxation. Neither KT5823, N(G)-methyl-L-arginine acetate salt, a nitric oxide synthase inhibitor, nor fulvestrant had a significant effect on the amount of resveratrol-induced relaxation. Genistein, a protein tyrosine kinase inhibitor, significantly (P < 0.01) increased the RSVL-induced relaxation. To determine if protein kinase C mediated the RSVL-induced relaxation, the protein kinase C inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together, and a significant (P < 0.05) increase in resveratrol-induced relaxation was observed. The pretreatment of the strips with resveratrol significantly (P < 0.001) decreased the amount of KCl- and cholecystokinin octapeptide- induced tension. CONCLUSIONS: Resveratrol-induced relaxation is mediated by its effects on L-type Ca(2+) channels and intracellular Ca(2+) release.

The flavonoid chrysin, an endocrine disrupter, relaxes cholecystokinin- and KCl-induced tension in male guinea pig gallbladder strips through multiple signaling pathways.

The bioflavonoids have effects on vascular smooth muscle and gastrointestinal smooth muscle. The flavone and phytoestrogen, chrysin, has been shown to have a vasorelaxant effect on resistance blood vessels. This effect was mediated by nitric oxide (NO). Chrysin inhibited aromatase/estrogen biosynthesis in postmenopausal women. The purpose of this study was to determine if chrysin had an effect on cholecystokinin- or KCl-induced tension in male guinea pig gallbladder strips. In addition, the second messenger(s) system(s) that mediated the effect were to be determined. A pharmacologic approach was used. Male guinea pig gallbladder strips were placed in in vitro chambers filled with Krebs solution, maintained at 37 °C, and gassed with 95% O2-5% CO2. Changes in tension were recorded using a polygraph. It was shown that the PKA/cAMP second messenger system mediated part of the observed chrysin-induced relaxation of cholecystokinin-induced tension, the PKC system also mediated part of the relaxation, and the inhibition of both extracellular Ca(2+) entry and intracellular Ca(2+) release also mediated the chrysin-induced relaxation. This is the first report of chrysin having an effect on gallbladder smooth muscle contraction.

Growth hormone and retinal ganglion cell function: QNR/D cells as an experimental model.

Retinal ganglion cells (RGCs) have been shown to be sites of growth hormone (GH) production and GH action in the embryonic (embryo day 7, ED7) chick neural retina. Primary RGC cell cultures were previously used to determine autocrine or paracrine actions of GH in the retina, but the antibody used in their immunopanning (anti-Thy-1) is no longer available. We have therefore characterized an immortalized neural retina (QNR/D) cell line derived from ED7 embryonic quail as a replacement experimental model. These cells express the GH gene and have GH receptor (GHR)-immunoreactivity. They are also immunoreactive for RGC markers (islet-1, calretinin, RA4) and neural fibers (neurofilament, GAP 43, vimentin) and they express the genes for Thy-1, neurotrophin 3 (NTF3), neuritin 1 (NRN1) and brn3 (POU4F). These cells are also electrically active and therefore resemble the RGCs in the neural retina. They are also similarly responsive to exogenous GH, which induces overexpression of the neurotrophin 3 and insulin-like growth factor (IGF) 1 genes and stimulates cell survival, as in the chick embryo neural retina. QNR/D cells are therefore a useful experimental model to assess the actions of GH in retinal function.

A comparison of the effects of various sex steroids on cholecystokinin- and KCl-induced tension in female guinea pig gallbladder strips.

Estrogen (E) has been shown to have an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. During pregnancy E and progesterone (P) levels are elevated. A biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of 17ß-estradiol (E2), dihydrotestosterone (DHT), P, 17-hydroxyprogesterone (17-P), and a P metabolite, 20α-hydroxyprogesterone (20-P) on contraction in female guinea pig gallbladder strips. DHT, P, 17-P, 20-P, and E2 each induced a concentration-dependent relaxation of cholecystokinin octapeptide (CCK) induced tension. DHT, E2, and P also induced a concentration-dependent relaxation of KCl-induced tension. When the response to E2 was compared to strips from young female guinea pigs with those taken from guinea pigs in late pregnancy, there was no significant difference in the response to either 50 or 100 µM E2; however, 10 µM E2 caused a significant increase (p<0.05) in the amount of relaxation in strips from pregnant guinea pigs. Treatment of the strips from young guinea pigs with PKA inhibitor 14-22 amide myristolated had no significant effect on the E2-induced relaxation. Treatment of the strips with 2-APB, an inhibitor of IP3 induced Ca(2+) release, produced a significant (p<0.001) increase in the amount of E2-induced relaxation when either CCK or KCl were used. Neither KT5823, a PKG inhibitor, nor L-NMMA, a nitric oxide (NO) synthase inhibitor, had a significant effect on the E2-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl(-), PKC blockers, were used in combination with no significant effect on the amount of CCK-induced tension, but significantly (p<0.01) increased the amount of E2-induced relaxation. When either E2 or P were added to the chambers 3 min prior to either CCK or KCl, a significant decrease (p<0.001) in the amount of tension generated was observed. The inhibition of extracellular Ca(2+) entry mediates both P-induced and E2-induced relaxation of CCK- and KCl-induced tension in female guinea pig gallbladder strips.

Transport of A1 adenosine receptor agonist tecadenoson by human and mouse nucleoside transporters: evidence for blood-brain barrier transport by murine equilibrative nucleoside transporter 1 mENT1.

The high density of A1 adenosine receptors in the brain results in significant potential for central nervous system (CNS)-related adverse effects with A1 agonists. Tecadenoson is a selective A1 adenosine receptor agonist with close similarity to adenosine. We studied the binding and transmembrane transport of tecadenoson by recombinant human equilibrative nucleoside transporters (hENTs) hENT1 and hENT2, and human concentrative nucleoside transporters (hCNTs) hCNT1, hCNT2, and hCNT3 in vitro and by mouse mENT1 in vivo. Binding affinities of the five recombinant human nucleoside transporters for tecadenoson differed (hENT1 > hCNT1 > hCNT3 > hENT2 > hCNT2), and tecadenoson was transported largely by hENT1. Pretreatment of mice with a phosphorylated prodrug of nitrobenzylmercaptopurine riboside, an inhibitor of mENT1, significantly decreased brain exposure to tecadenoson compared with that of the untreated (control) group, suggesting involvement of mENT1 in transport of tecadenoson across the blood-brain barrier (BBB). In summary, ENT1 was shown to mediate the transport of tecadenoson in vitro with recombinant and native human protein and in vivo with mice. The micromolar apparent Km value of tecadenoson for transport by native hENT1 in cultured cells suggests that hENT1 will not be saturated at clinically relevant (i.e., nanomolar) concentrations of tecadenoson, and that hENT1-mediated passage across the BBB may contribute to the adverse CNS effects observed in clinical trials. In contrast, in cases in which a CNS effect is desired, the present results illustrate that synthetic A1 agonists that are transported by hENT1 could be used to target CNS disorders because of enhanced delivery to the brain.

Human SLC2A9a and SLC2A9b isoforms mediate electrogenic transport of urate with different characteristics in the presence of hexoses.

Human SLC2A9 (GLUT9) is a novel high-capacity urate transporter belonging to the facilitated glucose transporter family. In the present study, heterologous expression in Xenopus oocytes has allowed us to undertake an in-depth radiotracer flux and electrophysiological study of urate transport mediated by both isoforms of SLC2A9 (a and b). Addition of urate to SLC2A9-producing oocytes generated outward currents, indicating electrogenic transport. Urate transport by SLC2A9 was voltage dependent and independent of the Na(+) transmembrane gradient. Urate-induced outward currents were affected by the extracellular concentration of Cl(-), but there was no evidence for exchange of the two anions. [(14)C]urate flux studies under non-voltage-clamped conditions demonstrated symmetry of influx and efflux, suggesting that SLC2A9 functions in urate efflux driven primarily by the electrochemical gradient of the cell. Urate uptake in the presence of intracellular hexoses showed marked differences between the two isoforms, suggesting functional differences between the two splice variants. Finally, the permeant selectivity of SLC2A9 was examined by testing the ability to transport a panel of radiolabeled purine and pyrimidine nucleobases. SLC2A9 mediated the uptake of adenine in addition to urate, but did not function as a generalized nucleobase transporter. The differential expression pattern of the two isoforms of SLC2A9 in the human kidney's proximal convoluted tubule and its electrogenic transport of urate suggest that these transporters play key roles in the regulation of plasma urate levels and are therefore potentially important participants in hyperuricemia and hypouricemia.

17ß-Estradiol relaxes cholecystokinin- and KCl-induced tension in male guinea pig gallbladder strips.

Estrogen has been shown to have an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. Since estrogen and progesterone levels are elevated during pregnancy, a biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of 17ß-estradiol (E2) on contraction in male guinea pig gallbladder strips. E2 induced a concentration-dependent relaxation of either CCK-induced tension or KCl-induced tension. Pretreatment of the strips with PKA inhibitor 14-22 amide myristolated had no significant effect on the E2-induced relaxation. Pretreatment of strips with 2-APB, and inhibitor of IP(3) induced Ca(2+) release, produced a significant (p<0.001) increase in the amount of E2-induced relaxation when either CCK or KCl were used to induce tension. KT5823, an inhibitor of PKG, also significantly (p<0.001) increased the amount of E2-induced relaxation. Genistein, an inhibitor of protein tyrosine kinase, had no significant effect on the E2-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl- when used in combination had no significant effect on the amount of CCK-induced tension, but significantly (p<0.001) increased the amount of E2-induced relaxation. When E2 was added to the chambers prior to either CCK or KCl, a significant decrease (p<0.001) in the amount of tension generated was observed. The inhibition of extracellular Ca(2+) entry mediates the E2-induced relaxation of CCK- and KCl-induced tension in male guinea pig gallbladder strips.

Interaction of fused-pyrimidine nucleoside analogs with human concentrative nucleoside transporters: High-affinity inhibitors of human concentrative nucleoside transporter 1.

Human concentrative nucleoside transporters (hCNTs) mediate electrogenic secondary active transport of physiological nucleosides and nucleoside drugs into cells. Six fused-pyrimidine ribonucleosides and one 2'-deoxynucleoside were assessed for their abilities to inhibit [(3)H]uridine transport in the yeast Saccharomyces cerevisiae producing recombinant hCNT1, hCNT2 or hCNT3. Six of the analogs inhibited hCNT1 with K(i) values<1µM whereas only two analogs inhibited hCNT3 with K(i) values<1µM and none inhibited hCNT2. To assess if the inhibitory analogs were also permeants, currents evoked were measured in oocytes of Xenopus laevis producing recombinant hCNT1, hCNT2 or hCNT3. Significant inward currents, indicating permeant activity, were generated with (i) three of the analogs in hCNT1-producing oocytes, (ii) none of the analogs in hCNT2-producing oocytes and (iii) all of the analogs in hCNT3-producing oocytes. Four were not, or were only very weakly, transported by hCNT1. The thienopyrimidine 2'-deoxynucleoside (dMeThPmR, 3) and ribonucleoside (MeThPmR, 4) were the most active inhibitors of uridine transport in hCNT1-producing oocytes and were an order of magnitude more effective than adenosine, a known low-capacity transport inhibitor of hCNT1. Neither was toxic to cultured human leukemic CEM cells, and both protected CEM cell lines with hCNT1 but not with hENT1 against gemcitabine cytotoxicity. In summary, dMeThPmR (3) and MeThPmR (4) were potent inhibitors of hCNT1 with negligible transportability and little apparent cytotoxicity, suggesting that pending further evaluation for toxicity against normal cells, they may have utility in protecting normal hCNT1-producing tissues from toxicities resulting from anti-cancer nucleoside drugs that enter via hCNT1.

Conserved glutamate residues Glu-343 and Glu-519 provide mechanistic insights into cation/nucleoside cotransport by human concentrative nucleoside transporter hCNT3.

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na(+) and H(+) to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na(+)/nucleoside and H(+)/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H(+) dependence (all mutants), shift in Na(+):nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na(+)-specific hCNT1, uncoupled Na(+) currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.

Growth hormone production and action in N1E-115 neuroblastoma cells.

Neuroblastoma cells are undifferentiated cells derived from the neural crest and are commonly used as models for studying neural function. Mouse N1E-115 neuroblastoma cells are derived from cancerous tissue and provide a model for studying the oncogenesis of neural cells. As growth hormone (GH) has been implicated as an autocrine or paracrine involved in neural regulation and in the induction or progression of cancer, the possibility that N1E-115 cells are sites of GH production and GH action was assessed. Using RT-PCR, cultured N1E-115 cells were found to express the mouse GH and GH receptor (GHR) genes. Immunocytochemistry demonstrated that both of the translated proteins (GH and its receptor) were abundantly present in the cytoplasm of these cells and their co-localization was established by confocal cytochemistry. GH action in these cells was determined in cells cultured for 72 h in the presence or absence of 10(-6) M or 10(-9) M mouse GH, which induced neurite sprouting and increased axon growth. In summary, the expression of GH and its receptor in GH responsive tumor-derived N1E-115 neuroblastoma cells suggests they provide a useful experimental model to assess GH actions in neural function or neural oncogenesis.

Testosterone and dihydrotestosterone inhibit gallbladder motility through multiple signalling pathways.

Testosterone (T) has been shown to cause vasodilation in rabbit coronary arteries through a nongenomic pathway. Part of this T-induced relaxation was shown to be mediated by opening voltage dependent K(+) channels. T infusion also reduces peripheral resistance in human males with heart failure. The effects of T or its active metabolite 5-alpha dihydrotestosterone (DHT) are not well studied. This study investigates the effect of T and DHT on contraction in guinea pig gallbladder strips. T or DHT induced a concentration-dependent relaxation of cholecystokinin octapeptide (CCK)-induced tension. Pretreatment of the strips with PKA inhibitor 14-22 amide myristolated had no significant effect on the relaxation induced by either T or DHT. Pretreatment of strips with 2-APB, an inhibitor of IP(3) induced Ca(2+) release, produced a significant (p<0.001) reduction in the T- or DHT-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl(-) when used in combination had no significant effect on the amount of CCK-induced tension, but significantly (p<0.01) decreased the amount of T- or DHT-induced relaxation. The flavone chrysin, an aromatase inhibitor, and genistein, an isoflavone, each produced a significant (p<0.01) reduction in CCK-induced tension. Chrysin significantly (p<0.05) increased T-induced relaxation; however, genistein had no effect on T-induced relaxation. It is concluded that T and DHT inhibits gallbladder motility rapidly by nongenomic actions of the hormones. Multiple pathways that include inhibition of intracellular Ca(2+) release, inhibition of extracellular Ca(2+) entry, and the actions of PKC may mediate this effect.

A conformationally mobile cysteine residue (Cys-561) modulates Na+ and H+ activation of human CNT3.

In humans, the SLC28 concentrative nucleoside transporter (CNT) protein family is represented by three Na+-coupled members; human CNT1 (hCNT1) and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, whereas hCNT3 transports both purine and pyrimidine nucleosides and nucleoside drugs. Belonging to a phylogenetic CNT subfamily distinct from hCNT1/2, hCNT3 also mediates H+/nucleoside cotransport. Using heterologous expression in Xenopus oocytes, we have characterized a cysteineless version of hCNT3 (hCNT3C-). Processed normally to the cell surface, hCNT3C- exhibited hCNT3-like transport properties, but displayed a decrease in apparent affinity specific for Na+ and not H+. Site-directed mutagenesis experiments in wild-type and hCNT3C- backgrounds identified intramembranous Cys-561 as the residue responsible for this altered Na+-binding phenotype. Alanine at this position restored Na+ binding affinity, whereas substitution with larger neutral amino acids (threonine, valine, and isoleucine) abolished hCNT3 H+-dependent nucleoside transport activity. Independent of these findings, we have established that Cys-561 is located in a mobile region of the hCNT3 translocation pore adjacent to the nucleoside binding pocket and that access of p-chloromercuribenzene sulfonate to this residue reports a specific H+-induced conformational state of the protein ( Slugoski, M. D., Ng, A. M. L., Yao, S. Y. M., Smith, K. M., Lin, C. C., Zhang, J., Karpinski, E., Cass, C. E., Baldwin, S. A., and Young, J. D. (2008) J. Biol. Chem. 283, 8496-8507 ). The present investigation validates hCNT3C- as a template for substituted cysteine accessibility method studies of CNTs and reveals a pivotal functional role for Cys-561 in Na+- as well as H+-coupled modes of hCNT3 nucleoside transport.

A proton-mediated conformational shift identifies a mobile pore-lining cysteine residue (Cys-561) in human concentrative nucleoside transporter 3.

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 mediates transport of a broad range of purine and pyrimidine nucleosides and nucleoside drugs, whereas hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively. All three hCNTs are Na(+)-coupled. Unlike hCNT1/2, however, hCNT3 is also capable of H(+)-mediated nucleoside cotransport. Using site-directed mutagenesis in combination with heterologous expression in Xenopus oocytes, we have identified a C-terminal intramembranous cysteine residue of hCNT3 (Cys-561) that reversibly binds the hydrophilic thiol-reactive reagent p-chloromercuribenzene sulfonate (PCMBS). Access of this membrane-impermeant probe to Cys-561, as determined by inhibition of hCNT3 transport activity, required H(+), but not Na(+), and was blocked by extracellular uridine. Although this cysteine residue is also present in hCNT1 and hCNT2, neither transporter was affected by PCMBS. We conclude that Cys-561 is located in the translocation pore in a mobile region within or closely adjacent to the nucleoside binding pocket and that access of PCMBS to this residue reports a specific H(+)-induced conformational state of the protein.

Inhibition of TRPP3 channel by amiloride and analogs.

TRPP3, a member of the transient receptor potential (TRP) superfamily of cation channels, is a Ca2+-activated channel permeable to Ca2+, Na+, and K+. TRPP3 has been implicated in sour tasting in bipolar cells of tongue and in regulation of pH-sensitive action potential in spinal cord neurons. TRPP3 is also present in excitable and nonexcitable cells of other tissues, including retina, brain, heart, testis, and kidney, with unknown functions. In this study, we examined the functional modulation of TRPP3 channel by amiloride and its analogs, known to inhibit several ion channels and transporters and respond to all taste stimuli, using Xenopus laevis oocyte expression, electrophysiology, and radiotracer measurements. We found that amiloride and its analogs inhibit TRPP3 channel activities with different affinities. Radiolabeled (45)Ca2+ uptake showed that TRPP3-mediated Ca2+ transport was inhibited by amiloride, phenamil, benzamil, and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Two-microelectrode voltage clamp experiments revealed that TRPP3-mediated Ca2+-activated currents are substantially inhibited by amiloride analogs, in an order of potency of phenamil > benzamil > EIPA > amiloride, with IC50 values of 0.14, 1.1, 10.5, and 143 microM, respectively. The inhibition potency positively correlated with the size of inhibitors. Using cell-attached patch clamping, we showed that the amiloride analogs decrease the open probability and mean open time but have no effect on single-channel conductance. Study of inhibition by phenamil in the presence of previously reported inhibitor tetrapentylammonium indicates that amiloride and organic cation inhibitors compete for binding the same site on TRPP3. TRPP3 may contribute to previously reported in vivo amiloride-sensitive cation transport.

Conserved glutamate residues are critically involved in Na+/nucleoside cotransport by human concentrative nucleoside transporter 1 (hCNT1).

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50(Na+) and/or Km(uridine) values and diminished Vmax(Na+) and Vmax(uridine) values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.

Cation coupling properties of human concentrative nucleoside transporters hCNT1, hCNT2 and hCNT3.

The SLC28 family of concentrative nucleoside transporter (CNT) proteins in mammalian cells contains members of two distinct phylogenic subfamilies. In humans, hCNT1 and hCNT2 belong to one subfamily, and hCNT3 to the other. All three CNTs mediate inwardly-directed Na(+)/nucleoside cotransport, and are either pyrimidine nucleoside-selective (hCNT1), purine nucleoside-selective (hCNT2), or broadly selective for both pyrimidine and purine nucleosides (hCNT3). While previous studies have characterized cation interactions with both hCNT1 and hCNT3, little is known about the corresponding properties of hCNT2. In the present study, heterologous expression in Xenopus oocytes in combination with radioisotope flux and electrophysiological techniques has allowed us to undertake a side-by-side comparison of hCNT2 with other hCNT family members. Apparent K (50) values for Na(+) activation were voltage-dependent, and similar in magnitude for all three transporters. Only hCNT3 was also able to couple transport of uridine to uptake of H(+). The Na(+)/nucleoside stoichiometry of hCNT2, as determined from both Hill coefficients and direct charge/flux measurements, was 1:1. This result was the same as for hCNT1, but different from that of hCNT3 (2:1). The charge-to-(22)Na(+) uptake stoichiometry was 1:1 for all three hCNTs. In parallel with their division into two separate CNT subfamilies, hCNT2 shares common cation specificity and coupling characteristics with hCNT1, which differ markedly from those of hCNT3.

Specific mutations in transmembrane helix 8 of human concentrative Na+/nucleoside cotransporter hCNT1 affect permeant selectivity and cation coupling.

The Na+/nucleoside cotransporters hCNT1 (650 residues) and hCNT2 (658 residues) are 72% identical in amino acid sequence and contain 13 putative transmembrane helices (TMs). Both transport uridine and adenosine but are otherwise selective for pyrimidine (system cit) and purine (system cif) nucleosides, respectively. Previously, we used site-directed mutagenesis and functional expression in Xenopus oocytes to identify two pairs of adjacent residues in TMs 7 and 8 of hCNT1 (Ser319-Gln320 and Ser353-Leu354) that, when converted to the corresponding residues in hCNT2 (Gly-Met and Thr-Val, respectively), changed the permeant selectivity of the transporter from cit to cif. We now report an investigation of the effects of corresponding mutations in TM 8 alone and demonstrate unique S353T- and L354V-induced changes in nucleoside specificity and cation coupling, respectively. hCNT1 mutation S353T produced a profound decrease in cytidine transport efficiency (Vmax/Km ratio) and, in combination with L354V (S353T/L354V), resulted in a novel uridine-preferring transport phenotype. In addition, the L354V mutation markedly increased the apparent affinity of hCNT1 for Na+ and Li+. Both hCNT1 TM 8 residues exhibited uridine-protectable inhibition by p-chloromercuribenzene sulfonate when converted to Cys, suggesting that they occupy positions within or closely adjacent to a common cation/nucleoside translocation pore.