Signaling pathways for modulation of mouse sperm motility by adenosine and catecholamine agonists

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line.

Previous studies of nucleoside transport in mammalian cells have identified two types of activities: the equilibrative nucleoside transporters and concentrative, Na+-nucleoside cotransporters. Characterization of the concentrative nucleoside transporters has been hampered by the presence in most cells and tissues of multiple transporters with overlapping permeant specificities. With the recent cloning of cDNAs encoding rat and human members of the concentrative nucleoside transporter (CNT) family, it is now possible to study the concentrative transporters in isolation by use of functional expression systems. We report here the isolation of a nucleoside transport-deficient subline of L1210 mouse leukemia (L1210/DNC3) that is a suitable recipient for stable expression of cloned nucleoside transporter cDNAs. We have used L1210/DNC3 as the recipient in gene transfer studies to develop a stable cell line (L1210/DU5) that produces the recombinant concentrative nucleoside transporter with selectivity for pyrimidine nucleosides (CNT1) that was initially identified in rat intestine (Q.Q. Huang, S.Y. Yao, M.W. Ritzel, A.R.P. Paterson, C.E. Cass, and J.D. Young. 1994. J. Biol. Chem. 269: 17,757-17,760). L1210/DU5 was used to examine the permeant selectivity of recombinant rat CNT1 by comparing a series of nucleoside analogs with respect to (i) inhibition of inward fluxes of [3H]thymidine, (ii) initial rates of transport of 3H-analog, and (iii) cytotoxicity to L1210/DU5 versus the parental transport-deficient cell line. By all three criteria, recombinant CNT1 transported 5-fluoro-2'-deoxyuridine and 5-fluorouridine well and cytosine arabinoside poorly. Although some purine nucleosides (2'-deoxyadenosinedeoxyadeno-2'-deoxyadenosine, 7-deazaadenosine) were potent inhibitors of CNT1, they were poor permeants when uptake was measured directly by analysis of isotopic fluxes or indirectly by comparison of cytotoxicity ratios. We conclude that comparison of analog cytotoxicity to L1210/DU5 versus L1210/DNC3 is a reliable indirect predictor of transportability, suggesting that cytotoxicity assays with a panel of such cell lines, each with a different recombinant nucleoside transporter, would be a valuable tool in the development of antiviral and antitumor nucleoside analogs.     

Nucleoside transport in human colonic epithelial cell lines: evidence for two Na+-independent transport systems in T84 and Caco-2 cells.

RT-PCR of RNA isolated from monolayers of the human colonic epithelial cell lines T84 and Caco-2 demonstrated the presence of mRNA for the two cloned Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2, but not for the cloned Na+-dependent concentrative nucleoside transporters, CNT1 and CNT2. Uptake of [3H]uridine by cell monolayers in balanced Na+-containing and Na+-free media confirmed the presence of only Na+-independent nucleoside transport mechanisms. This uptake was decreased by 70-75% in the presence of 1 microM nitrobenzylthioinosine, a concentration that completely inhibits ENT1, and was completely blocked by the addition of 10 microM dipyridamole, a concentration that inhibits both ENT1 and ENT2. These findings indicate the presence in T84 and Caco-2 cells of two functional Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2.     

Recent advances in studies on biochemical and structural properties of equilibrative and concentrative nucleoside transporters

Nucleoside transporters (NT) facilitate the movement of nucleosides and nucleobases across cell membranes. NT-mediated transport is vital for the synthesis of nucleic acids in cells that lack de novo purine synthesis. Some nucleosides display biological activity and act as signalling molecules. For example, adenosine exerts a potent action on many physiological processes including vasodilatation, hormone and neurotransmitter release, platelet aggregation, and lipolysis. Therefore, carrier-mediated transport of this nucleoside plays an important role in modulating cell function, because the efficiency of the transport processes determines adenosine availability to its receptors or to metabolizing enzymes. Nucleoside transporters are also key elements in anticancer and antiviral therapy with the use of nucleoside analogues. Mammalian cells possess two major nucleoside transporter families: equilibrative (ENT) and concentrative (CNT) Na(+)-dependent ones. This review characterizes gene loci, substrate specificity, tissue distribution, membrane topology and structure of ENT and CNT proteins. Regulation of nucleoside transporters by various factors is also presented.     

Molecular biology and regulation of nucleoside and nucleobase transporter proteins in eukaryotes and prokaryotes.

The molecular cloning of cDNAs encoding nucleoside transporter proteins has greatly advanced understanding of how nucleoside permeants are translocated across cell membranes. The nucleoside transporter proteins identified thus far have been categorized into five distinct superfamilies. Two of these superfamilies, the equilibrative and concentrative nucleoside transporters, have human members and these will be examined in depth in this review. The human equilibrative nucleoside transporters translocate nucleosides and nucleobases bidirectionally down their concentration gradients and are important in the uptake of anticancer and antiviral nucleoside drugs. The human concentrative nucleoside transporters cotranslocate nucleosides and sodium unidirectionally against the nucleoside concentration gradients and play a vital role in certain tissues. The regulation of nucleoside and nucleobase transporters is being studied more intensely now that more tools are available. This review provides an overview of recent advances in the molecular biology and regulation of the nucleoside and nucleobase transporters.     

Equilibrative nucleoside transporter family members from Leishmania donovani are electrogenic proton symporters

Leishmania donovani express two members of the equilibrative nucleoside transporter family; LdNT1 encoded by two closely related and linked genes, LdNT1.1 and LdNT1.2, that transport adenosine and pyrimidine nucleosides and LdNT2 that transports inosine and guanosine exclusively. LdNT1.1, LdNT1.2, and LdNT2 have been expressed in Xenopus laevis oocytes and found to be electrogenic in the presence of nucleoside ligands for which they mediate transport. Further analysis revealed that ligand uptake and transport currents through LdNT1-type transporters are proton-dependent. In addition to the flux of protons that is coupled to the transport reaction, LdNT1 transporters mediate a variable constitutive proton conductance that is blocked by substrates and dipyridamole. Surprisingly, LdNT1.1 and LdNT1.2 exhibit different electrogenic properties, despite their close sequence homology. This electrophysiological study provides the first demonstration that members of the equilibrative nucleoside transporter family can be electrogenic and establishes that these three permeases, unlike their mammalian counterparts, are probably concentrative rather than facilitative transporters.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.     

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.     

Macrophages require different nucleoside transport systems for proliferation and activation.

To evaluate the mechanisms involved in macrophage proliferation and activation, we studied the regulation of the nucleoside transport systems. In murine bone marrow-derived macrophages, the nucleosides required for DNA and RNA synthesis are recruited from the extracellular medium. M-CSF induced macrophage proliferation and DNA and RNA synthesis, whereas interferon gamma (IFN-gamma) led to activation, blocked proliferation, and induced only RNA synthesis. Macrophages express at least the concentrative systems N1 and N2 (CNT2 and CNT1 genes, respectively) and the equilibrative systems es and ei (ENT1 and ENT2 genes, respectively). Incubation with M-CSF only up-regulated the equilibrative system es. Inhibition of this transport system blocked M-CSF-dependent proliferation. Treatment with IFN-gamma only induced the concentrative N1 and N2 systems. IFN-gamma also down-regulated the increased expression of the es equilibrative system induced by M-CSF. Thus, macrophage proliferation and activation require selective regulation of nucleoside transporters and may respond to specific requirements for DNA and RNA synthesis. This report also shows that the nucleoside transporters are critical for macrophage proliferation and activation.     

Hypoxanthine effect on equilibrative and concentrative adenosine transport in human lymphocytes: implications in the phatogenesis of Lesch-Nyhan syndrome

We postulated that increased levels of hypoxanthine, a main characteristic of hypoxanthine phosphoribosyltransferase (HPRT) deficiency, may influence adenosine function which could be related to some of the neurological features of the Lesch-Nyhan syndrome. We have examined the effect of hypoxanthine on different adenosine transporters in peripheral blood lymphocytes from control subjects. Increased hypoxanthine concentrations (25 microM) significantly decreased adenosine transport. The equilibrative adenosine transporters (79.6% of the adenosine transport), both NBTI sensitive and NBTI insensitive, were affected significantly. In contrast, the concentrative adenosine transporters were not influenced by hypoxanthine. These results supports the hypothesis that increased hypoxanthine levels influence equilibrative (predominantly NBTI-insensitive type) adenosine transporters.     

Hypoxanthine Effect on Equilibrative and Concentrative Adenosine Transport in Human Lymphocytes. Implications in the Phatogenesis of Lesch-Nyhan Syndrome

We postulated that increased levels of hypoxanthine, a main characteristic of hypoxanthine phosphoribosyltransferase (HPRT) deficiency, may influence adenosine function which could be related to some of the neurological features of the Lesch-Nyhan syndrome. We have examined the effect of hypoxanthine on different adenosine transporters in peripheral blood lymphocytes from control subjects. Increased hypoxanthine concentrations (25 μM) significantly decreased adenosine transport. The equilibrative adenosine transporters (79.6% of the adenosine transport), both NBTI sensitive and NBTI insensitive, were affected significantly. In contrast, the concentrative adenosine transporters were not influenced by hypoxanthine. These results supports the hypothesis that increased hypoxanthine levels influence equilibrative (predominantly NBTI-insensitive type) adenosine transporters.     

Substrate specificity, kinetics, and stoichiometry of sodium-dependent adenosine transport in L1210/AM mouse leukemia cells

Two equilibrative (facilitated diffusion) nucleoside transport processes and a concentrative Na(+)-dependent co-transport process contribute to zero-trans inward fluxes of nucleosides in L1210 mouse leukemia cells. Na(+)-linked inward adenosine fluxes in L1210/AM cells (a clone deficient in adenosine, deoxyadenosine, and deoxycytidine kinase activities) were measured as initial rates of [3H]adenosine influx in medium containing Na+ salts and 10 microM dipyridamole. The Na(+)-linked transporter distinguished between the D- and L-enantiomers of adenosine, the latter being a virtual nonpermeant in the initial-rate assay. Adenine arabinoside, inosine, 2'-deoxyadenosine and 2'-deoxyadenosine derivatives with halogen atoms at the purine C-2 position were recognized as substrates of the Na(+)-linked system because of their inhibition of adenosine (10 microM) fluxes under the condition of Na(+)-dependence with IC50 values ranging between 25 and 183 microM; uridine, deoxycytidine, and cytosine arabinoside (each at 400 microM) inhibited adenosine fluxes by 10-40%. Inward Na(+)-linked adenosine fluxes were saturable with respect to extracellular adenosine and Na+ concentrations [( Na+]o); Km and Vmax values for adenosine influx were 9.4 +/- 2.6 microM and 1.67 +/- 0.2 pmol/microliter cell water/s when [Na+]o was 100 mM. The stoichiometry of Na+:adenosine co-transport, determined by Hill analysis of the dependence of adenosine fluxes on [Na+]o, was 1:1. The thiol-reactive agents, N-ethylmaleimide (NEM), showdomycin and p-chloromercuriphenylsulphonate (pCMPS), inhibited Na(+)-linked adenosine fluxes with IC50 values of 40, 10, and 2 microM, respectively. This inhibition was partially reversed by the presence of adenosine in incubation media containing pCMPS, but not NEM. Thiol groups accessible to pCMPS may be involved in substrate recognition by the transporter and in the permeation step.     

Sodium-dependent, concentrative nucleoside transport in cultured intestinal epithelial cells

In a simple salts medium, monolayers of IEC-6 intestinal cells achieved concentrations of unmetabolized formycin B (an analog of inosine) about 6-fold higher than in the medium. Rates of formycin B influx were a saturable function of Na+ concentrations in the medium. Although IEC-6 cells possess sites with high affinity for nitrobenzylthioinosine, a potent inhibitor of equilibrative (facilitated diffusion) nucleoside transport systems in certain cell types, the inhibitor had only minor effects on formycin B uptake in IEC-6 cells, but reduced efflux of the analog from these cells. These findings indicate the joint presence in IEC-6 cells of nucleoside transporters of two types, one that is concentrative and Na+-dependent, and another that is sensitive to nitrobenzylthioinosine and apparently equilibrative.     

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.     

Long term endocrine regulation of nucleoside transporters in rat intestinal epithelial cells

We studied the regulation of nucleoside transporters in intestinal epithelial cells upon exposure to either differentiating or proliferative agents. Rat intestinal epithelial cells (line IEC-6) were incubated in the presence of differentiating (glucocorticoids) or proliferative (EGF and TGF-alpha) agents. Nucleoside uptake rates and nucleoside transporter protein and mRNA levels were assessed. The signal transduction pathways used by the proliferative stimuli were analyzed. We found that glucocorticoids induce an increase in sodium-dependent, concentrative nucleoside transport rates and in protein and mRNA levels of both rCNT2 and rCNT1, with negligible effects on the equilibrative transporters. EGF and TGF-alpha induce an increase in the equilibrative transport rate, mostly accounted for by an increase in rENT1 activity and mRNA levels, rENT2 mRNA levels remaining unaltered. This effect is mimicked by another proliferative stimulus that functions as an in vitro model of epithelial wounding. Here, rENT1 activity and mRNA levels are also increased, although the signal transduction pathways used by the two stimuli are different. We concluded that differentiation of rat intestinal epithelial cells is accompanied by increased mature enterocyte features, such as concentrative nucleoside transport (located at the brush border membrane of the enterocyte), thus preparing the cell for its ultimate absorptive function. A proliferative stimulus induces the equilibrative nucleoside activities (mostly through ENT1) known to be located at the basolateral membrane, allowing the uptake of nucleosides from the bloodstream for the increased demands of the proliferating cell.     

Functional redundancy of two nucleoside transporters of the ENT family (CeENT1, CeENT2) required for development of Caenorhabditis elegans

The genome of Caenorhabditis elegans encodes multiple homologues of the two major families of mammalian equilibrative and concentrative nucleoside transporters. As part of a programme aimed at understanding the biological rationale underlying the multiplicity of eukaryote nucleoside transporters, we have now demonstrated that the nematode genes ZK809.4 (ent-1) and K09A9.3 (ent-2) encode equilibrative transporters, which we designate CeENT1 and CeENT2 respectively. These transporters resemble their human counterparts hENT1 and hENT2 in exhibiting similar broad permeant specificities for nucleosides, while differing in their permeant selectivities for nucleobases. They are insensitive to the classic inhibitors of mammalian nucleoside transport, nitrobenzylthioinosine, dilazep and draflazine, but are inhibited by the vasoactive drug dipyridamole. Use of green fluorescent protein reporter constructs indicated that the transporters are present in a limited number of locations in the adult, including intestine and pharynx. Their potential roles in these tissues were explored by using RNA interference to disrupt gene expression. Although disruption of ent-1 or ent-2 expression alone had no effect, simultaneous disruption of both genes yielded pronounced developmental defects involving the intestine and vulva.     

Synthesis and biological evaluation of phloridzin analogs as human concentrative nucleoside transporter 3 (hCNT3) inhibitors

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective and neuroprotective agents. Although quite a few potent inhibitors of the equilibrative nucleoside transporters are known, largely missing are the concentrative nucleoside transporter inhibitors. Phloridzin (3, K_i = 16.00 μM) is a known moderate inhibitor of the concentrative nucleoside transporters. We have synthesized and evaluated analogs of phloridzin at the hCNT3 nucleoside transporter. Within the series of synthesized analogs compound 16 (K_i = 2.88 μM), possessing a ribofuranose sugar unit instead of a glucopyranose as present in phloridzin, exhibited the highest binding affinity at the hCNT3 transporter. Phloridzin and compound 16 have also been shown to be selective for the hCNT3 transporter as compared with the hENT1 transporter. Compound 16 can serve as a new lead which after further modifications could yield selective and potent hCNT3 inhibitors.