Edward Karpinski

Recent molecular advances in studies of the concentrative Na+-dependent nucleoside transporter (CNT) family: identification and characterization of novel human and mouse proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib)

The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1 and hCNT2, found primarily in specialized epithelia, are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have orthologs in other mammalian species and belong to a gene family (CNT) that also includes members in lower vertebrates, insects, nematodes, pathogenic yeast and bacteria. The CNT transporter family also includes a newly identified human and mouse CNT3 transporter isoform. This paper reviews the studies of CNT transport proteins that led to the identification of hCNT3 and mCNT3, and gives an overview of the structural and functional properties of these latest CNT family members. hCNT3 and mCNT3 have primary structures that place them in a CNT subfamily separate from CNT1/2, transport a wide range of physiological pyrimidine and purine nucleosides and antineoplastic and antiviral nucleoside drugs (system cib), and exhibit a Na+:uridine coupling ratio of at least 2:1 (cf 1:1 for hCNT1/2). Cells and tissues containing hCNT3 transcripts include mammary gland, differentiated HL-60 cells, pancreas, bone marrow, trachea, liver, prostrate and regions of intestine, brain and heart. In HL-60 cells, hCNT3 is transcriptionally regulated by phorbol myristate (PMA). The hCNT3 gene, which contains an upstream PMA response element, mapped to 9q22.2 (cf chromosome 15 for hCNT1 and hCNT2).The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1 and hCNT2, found primarily in specialized epithelia, are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have orthologs in other mammalian species and belong to a gene family (CNT) that also includes members in lower vertebrates, insects, nematodes, pathogenic yeast and bacteria. The CNT transporter family also includes a newly identified human and mouse CNT3 transporter isoform. This paper reviews the studies of CNT transport proteins that led to the identification of hCNT3 and mCNT3, and gives an overview of the structural and functional properties of these latest CNT family members. hCNT3 and mCNT3 have primary structures that place them in a CNT subfamily separate from CNT1/2, transport a wide range of physiological pyrimidine and purine nucleosides and antineoplastic and antiviral nucleoside drugs (system cib), and exhibit a Na+:uridine coupling ratio of at least 2:1 (cf 1:1 for hCNT1/2). Cells and tissues containing hCNT3 transcripts include mammary gland, differentiated HL-60 cells, pancreas, bone marrow, trachea, liver, prostrate and regions of intestine, brain and heart. In HL-60 cells, hCNT3 is transcriptionally regulated by phorbol myristate (PMA). The hCNT3 gene, which contains an upstream PMA response element, mapped to 9q22.2 (cf chromosome 15 for hCNT1 and hCNT2).

Vascular Effects of Progesterone: Role of Cellular Calcium Regulation

Vascular actions of progesterone have been reported, independently of estrogen, affecting both blood pressure and other aspects of the cardiovascular system. To study possible mechanisms underlying these effects, we examined the effects of P in vivo in intact rats and in vitro in isolated artery and vascular smooth muscle cell preparations. In anesthetized Sprague-Dawley rats , bolus intravenous injections of P (100 &mgr;g/kg) significantly decreased pressor responses to norepinephrine (0.3 &mgr;g/kg). In vitro, progesterone (10−8 to 10−5 mmol/L) produced a significant, dose-dependent relaxation of isolated helical strips, both of rat tail artery precontracted with KCl (60 mmol/L) or arginine vasopressin (3 nmol/L), and of rat aorta precontracted with KCl (60 mmol/L) or norepinephrine (0.1 &mgr;mol/L). In isolated vascular smooth muscle cells, progesterone (5×10−7 mol/L) reversibly inhibited KCl (30 mmol/L) -induced elevation of cytosolic-free calcium by 64.1±5.5% (P <0.05), and in whole-cell patch-clamp experiments, progesterone (5×10−6 mol/L) reversibly and significantly blunted L-type calcium channel inward current, decreasing peak inward current to 65.7±4.3% of the control value (P <0.05). Our results provide evidence that progesterone is a vasoactive hormone, inhibiting agonist-induced vasoconstriction. The data further suggest that progesterone effects on vascular tissue may, at least in part, be mediated by modulation of the L-type calcium channel current activity and, consequently, of cytosolic-free calcium content.

Electrophysiological characterization of a recombinant human Na+-coupled nucleoside transporter (hCNT1) produced in Xenopus oocytes

Human concentrative nucleoside transporter 1 (hCNT1) mediates active transport of nucleosides and anticancer and antiviral nucleoside drugs across cell membranes by coupling influx to the movement of Na+ down its electrochemical gradient. The two‐microelectrode voltage‐clamp technique was used to measure steady‐state and presteady‐state currents of recombinant hCNT1 produced in Xenopus oocytes. Transport was electrogenic, phloridzin sensitive and specific for pyrimidine nucleosides and adenosine. Nucleoside analogues that induced inwardly directed Na+ currents included the anticancer drugs 5‐fluorouridine, 5‐fluoro‐2′‐deoxyuridine, cladribine and cytarabine, the antiviral drugs zidovudine and zalcitabine, and the novel thymidine mimics 1‐(2‐deoxy‐β‐d‐ribofuranosyl)‐2,4‐difluoro‐5‐methylbenzene and 1‐(2‐deoxy‐β‐d‐ribofuranosyl)‐2,4‐difluoro‐5‐iodobenzene. Apparent Km values for 5‐fluorouridine, 5‐fluoro‐2′‐deoxyuridine and zidovudine were 18, 15 and 450 μm, respectively. hCNT1 was Na+ specific, and the kinetics of steady‐state uridine‐evoked Na+ currents were consistent with an ordered simultaneous transport model in which Na+ binds first followed by uridine. Membrane potential influenced both ion binding and carrier translocation. The Na+–nucleoside coupling stoichiometry, determined directly by comparing the uridine‐induced inward charge movement to [14C]uridine uptake was 1: 1. hCNT1 presteady‐state currents were used to determine the fraction of the membrane field sensed by Na+ (61%), the valency of the movable charge (−0.81) and the average number of transporters present in the oocyte plasma membrane (6.8 × 1010 per cell). The hCNT1 turnover rate at −50 mV was 9.6 molecules of uridine transported per second.

The effects of parathyroid hormone on L-type voltage-dependent calcium channel currents in vascular smooth muscle cells and ventricular myocytes are mediated by a cyclic AMP dependent mechanism.

The present study demonstrated that L channel currents were decreased in smooth muscle cells, and increased in ventricular myocytes by both bovine parathyroid hormone, (bPTH‐(1–34)), and dibutyryl cyclic AMP (db‐cAMP), using the whole cell version of the patch clamp technique with Ba2+ as the charge carrier. The effects of bPTH‐(1–34) and db‐cAMP on L channel currents were additive but not synergistic. Furthermore, the effects of bPTH‐(1–34) on L channel currents in these 2 cell preparations were abolished in the presence of a cAMP antagonist. These results suggest that the effects of bPTH‐(1–34) on L channel currents in vascular smooth muscle cells and ventricular myocytes are mediated by a cAMP‐dependent mechanism.

α1D L-Type Ca2+-Channel Currents: Inhibition by a β-Adrenergic Agonist and Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Rat Pinealocytes

Abstract: In this study the subunits of the dihydropyridine‐sensitive L‐type Ca2+ channels (L‐channels) expressed in rat pinealocytes were characterized using reverse transcription (RT)‐PCR analysis, and the modulation of these channels by adrenergic agonists and by pituitary adenylate cyclase‐activating polypeptide (PACAP) was studied using the patch‐clamp technique. RT‐PCR analysis showed that rat pinealocytes expressed α1D, α2b, β2, and β4 Ca2+‐channel subunit mRNAs. Other α1 subunit transcripts were either not expressed or present at very low levels, indicating that the pinealocytes express predominantly α1D L‐channels. Electrophysiological studies confirmed that the pineal expressed a single population of L‐channels. The L‐channel currents were inhibited by two agonists that elevate cyclic AMP: the β‐adrenergic agonist isoproterenol and PACAP. Similar inhibition was observed with a cyclic AMP analogue, 8‐bromo‐cyclic AMP. The presence of a cyclic AMP antagonist, Rp‐adenosine 3′,5′‐cyclic monophosphorothioate, blocked the inhibition by isoproterenol and PACAP. Norepinephrine, a mixed α‐ and β‐adrenergic agonist, also inhibited the L‐channel currents, but the inhibition was smaller. The smaller inhibition by norepinephrine was secondary to the simultaneous activation of α‐ and β‐adrenergic receptors. These results indicate that (a) pinealocytes express predominantly α1D L‐channels, and (b) the β‐adrenergic agonist isoproterenol and PACAP inhibit the L‐channel currents through elevation of cyclic AMP. However, an α‐adrenergic‐mediated mechanism also appears to be involved in the effect of norepinephrine on the L‐channel currents.

Tetrandrine inhibits both T and L calcium channel currents in ventricular cells.

Summary: Tetrandrine, a putative Ca2+ channel blocker, is extracted from the Chinese medicinal herb, Radix stephania tetrandrae. In the present study, the whole-cell version of the patch clamp technique was used to investigate the effects of tetrandrine on both T and L calcium channel currents in primary cultured neonatal rat ventricular cells. We show that tetrandrine inhibits both T and L calcium channel currents in ventricular cells. This inhibition of inward Ca2+ currents is concentration dependent and reversible. Tetrandrine does not shift the I-V relationship of the calcium currents. These results clearly demonstrate that tetrandrine acts as a calcium channel antagonist in ventricular cells. Previous data show that tetrandrine may be regarded as a wide-spectrum calcium channel antagonist.

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 kidneys 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.

Parathyroid hormone selectively inhibits L-type calcium channels in single vascular smooth muscle cells of the rat.

1. The active synthetic N‐terminal fragment of bovine parathyroid hormone, bPTH‐(1‐34) at a concentration of 1 microM, decreased the peak amplitude of the long‐lasting (L‐type) calcium channel current by 37% (n = 14, P less than 0.01) in rat tail artery smooth muscle cells. By contrast, this fragment of parathyroid hormone (PTH) (1 microM) had no effect on the transient (T‐type) calcium channel current in the same cell preparation. 2. The inhibitory effect of bPTH‐(1‐34) on L‐channel currents was reversible and could be antagonized by the L‐channel agonist, Bay K 8644. In contrast, bPTH‐(1‐34) inhibited Bay K 8644‐induced amplification of L‐channel currents. 3. The inhibitory effect of bPTH‐(1‐34) on L‐Channel currents was dose dependent with a threshold concentration of less than 10(‐7), and voltage dependent with increased inhibition at more positive holding potentials. However, this effect of bPTH‐(1‐34) was not dependent on different pulse lengths or interpulse intervals. 4. The kinetics of deactivation of L‐channel currents were not changed although the instantaneous amplitude of the L‐channel tail current was reduced by bPTH‐(1‐34). 5. Application of bPTH‐(1‐34) antagonists (10(‐6) M‐bPTH‐(3‐34) and 10(‐5) M‐bPTH‐(7‐34] did not result in any significant change in the magnitude of L‐channel currents (n = 15 and n = 7, respectively). 6. Pre‐incubation of cells with bPTH‐(3‐34) for more than 15 min abolished the inhibitory effect of bPTH‐(1‐34) on L‐channel currents. 7. The present study provides direct evidence to demonstrate the PTH, an endogenous circulating hormone, is a selective inhibitor of L‐channel currents in vascular smooth muscle cells.

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 45Ca2+ 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 μM, 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.

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 K50 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-22Na+ 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.