P2Y receptor regulation of sodium transport in human mammary epithelial cells

Primary human mammary epithelial (HME) cells were immortalized by stable, constitutive expression of the catalytic subunit of human telomerase. Purinergic receptors were identified by RT-PCR and quantitative RT-PCR from mRNA isolated from primary and immortalized cells grown to confluence on membrane filters. Several subtypes of P2Y receptor mRNA were identified including P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors. RT-PCR experiments also revealed expression of A(2b) adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (I(sc)) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na(+) channel (ENaC) alpha-, beta-, and gamma-subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive Na(+) transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation with UTP, UDP, or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) produced increases in intracellular calcium concentration that were significantly reduced following pretreatment with the calcium-chelating agent BAPTA-AM. Concentration-response relationships indicated that the rank order of potency for these agonists was UTP > UDP > ATPgammaS. Basolateral stimulation with UTP produced a rapid but transient increase in I(sc) that was significantly reduced if cells were pretreated with BAPTA-AM or benzamil. Moreover, basolateral treatment with either charybdotoxin or clotrimazole significantly inhibited the initial UTP-dependent increase in I(sc) and eliminated the sustained current response. These results indicate that human mammary epithelial cells express multiple P2 receptor subtypes and that Ca(2+) mobilization evoked by P2Y receptor agonists stimulates Na(+) absorption by increasing the activity of Ca(2+)-activated K(+) channels located in the basolateral membrane.     

Role of Ca2+-activated K+ channels in duodenal mucosal ion transport and bicarbonate secretion

Stimulation of muscarinic receptors in the duodenal mucosa raises cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), thereby regulating duodenal epithelial ion transport. However, little is known about the downstream molecular targets that account for this Ca(2+)-mediated biological action. Ca(2+)-activated K(+) (K(Ca)) channels are candidates, but the expression and function of duodenal K(Ca) channels are poorly understood. Therefore, we determined whether K(Ca) channels are expressed in the duodenal mucosa and investigated their involvement in Ca(2+)-mediated duodenal epithelial ion transport. Two selective blockers of intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channels, clotrimazole (30 muM) and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; 10 muM), significantly inhibited carbachol (CCh)-induced duodenal short-circuit current (I(sc)) and duodenal mucosal bicarbonate secretion (DMBS) in mice but did not affect responses to forskolin and heat-stable enterotoxin of Escherichia coli. Tetraethylammonium, 4-aminopyridine, and BaCl(2) failed to inhibit CCh-induced I(sc) and DMBS. A-23187 (10 muM), a Ca(2+) ionophore, and 1-ethyl-2-benzimidazolinone (1-EBIO; 1 mM), a selective opener of K(Ca) channels, increased both I(sc) and DMBS. The effect of 1-EBIO was more pronounced with serosal than mucosal addition. Again, both clotrimazole and TRAM-34 significantly reduced A23187- or 1-EBIO-induced I(sc) and DMBS. Moreover, clotrimazole (20 mg/kg ip) significantly attenuated acid-stimulated DMBS of mice in vivo. Finally, the molecular identity of IK(Ca) channels was verified as KCNN4 (SK4) in freshly isolated murine duodenal mucosae by RT-PCR and Western blotting. Together, our results suggest that the IK(Ca) channel is one of the downstream molecular targets for [Ca(2+)](cyt) to mediate duodenal epithelial ion transport.     

Adenosine triphosphate induces inhibition of Na(+) absorption in mouse endometrial epithelium: a Ca(2+)-dependent mechanism

The present study investigated the inhibitory effect of extracellular ATP on Na(+) absorption and the possible underlying mechanism in cultured mouse endometrial epithelium using the short-circuit current (I(SC)) technique. The cultured epithelia exhibited a Na(+)-dependent basal current that could be predominately blocked by the epithelial Na(+) channel (ENaC) blocker, amiloride (10 microM). Apical addition of ATP (10 microM) induced a reduction in basal I(SC). However, in the presence of amiloride or when apical Na(+) was removed, the ATP-induced reduction was abolished and an increase in the I(SC) was observed with kinetic characteristics similar to those reported previously for the ATP-induced Cl(-) secretion, indicating that ATP could induce both Cl(-) secretion and inhibition of Na(+) absorption. Further reduction in I(SC) after ATP challenge could be obtained with forskolin (10 microM), which indicates that different inhibitory mechanisms are involved. The ATP-induced inhibition of Na(+) absorption, but not that induced by forskolin, could be abolished by the P(2) receptor antagonist, reactive blue (100 microM), indicating the involvement of a P(2) receptor in mediating the ATP response. ATP and uridine 5'-diphosphate (UDP; 100 microM), a relatively selective agonist for the pyrimidinoceptor, induced separate I(SC) reduction, and distinct I(SC) increases in the presence of amiloride, regardless of the order of drug administration, indicating the involvement of two receptor populations. The ATP-induced inhibition of Na(+) absorption was mimicked by the Ca(2+) ionophore, ionomycin (1 microM), whereas the Ca(2+) chelators, EGTA and BAPTA-AM, abolished the ATP-induced, but not the forskolin-induced, inhibition of Na(+) absorption, suggesting the involvement of a Ca(2+)-dependent pathway. In the presence of the Cl(-) channel blocker, DIDS (100 microM), both inhibitory and stimulatory responses to ATP were abolished, suggesting the involvement of a Ca(2+)-activated Cl(-) channels (CaCCs) in mediating both ATP responses. The ATP-induced as well as the forskolin-induced reduction in I(SC) was not observed when Cl(-) was removed from the bathing solution, indicating that Cl(-) permeation is important for the inhibition of Na(+) absorption. The results suggest the presence of a Ca(2+)-dependent ENaC-inhibiting mechanism involving CaCC in mouse endometrial epithelial cells. Thus, extracellular nucleotides may play an important role in the fine-tuning of the uterine fluid microenvironment by regulating both Cl(-) secretion and Na(+) absorption across the endometrium.     

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na(+) conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K(+) permeability > Cs(+) permeability > Na(+) permeability. Conductance increased and rectification decreased when either Mg(2+) or both Ca(2+) and Mg(2+) were removed from the internal or external solution or both. The conductance could be blocked by Ba(2+), but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (I(sc)) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both I(sc) and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in I(sc) was followed by a subsequent increase. The cAMP-mediated increase in I(sc) was reduced by low serosal Na(+) and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na(+) reduced (28)Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na(+)/Mg(2+) exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.     

Modulation of Extracellular γ-Aminobutyric Acid in the Ventral Pallidum Using In Vivo Microdialysis

Intracranial microdialysis was used to investigate the origin of extracellular gamma-aminobutyric acid (GABA) in the ventral pallidum. Changes in basal GABA levels in response to membrane depolarizers, ion-channel blockers, and receptor agonists were determined. Antagonism of Ca2+ fluxes with high Mg2+ in a Ca(2+)-free perfusion buffer decreased GABA levels by up to 30%. Inhibition of voltage-dependent Na+ channels by the addition of tetrodotoxin also significantly decreased basal extracellular GABA concentrations by up to 45%, and blockade of Ca2+ and Na+ channels with verapamil reduced extracellular GABA by as much as 30%. The addition of either the GABAA agonist, muscimol, or the GABAB agonist, baclofen, produced a 40% reduction in extracellular GABA. GABA release was stimulated by high K+ and the addition of veratridine to increase Na+ influx. High K(+)-induced release was predominantly Ca(2+)-dependent, whereas the effect of veratridine was potentiated in the absence of extracellular Ca2+. Both high K(+)- and veratridine-induced elevations in extracellular GABA were inhibited by baclofen, whereas only veratridine-induced release was antagonized by muscimol. These results demonstrate that at least 50% of basal extracellular GABA in the ventral pallidum is derived from Ca(2+)- or Na(+)-dependent mechanisms. They also suggest that Na(+)-dependent release of GABA via reversal of the uptake carrier can be shown in vivo.     

Nucleotides and Mg2+ ions differentially regulate K+ channels and non-selective cation channels present in cells forming the stomatal complex

Voltage-dependent inward-rectifying (K(in)) and outward-rectifying (K(out)) K(+) channels are capable of mediating K(+) fluxes across the plasma membrane. Previous studies on guard cells or heterologously expressed K(+) channels provided evidence for the requirement of ATP to maintain K(+) channel activity. Here, the nucleotide and Mg(2+) dependencies of time-dependent K(in) and K(out) channels from maize subsidiary cells were examined, showing that MgATP as well as MgADP function as channel activators. In addition to K(out) channels, these studies revealed the presence of another outward-rectifying channel type (MgC) in the plasma membrane that however gates in a nucleotide-independent manner. MgC represents a new channel type distinguished from K(out) channels by fast activation kinetics, inhibition by elevated intracellular Mg(2+) concentration, permeability for K(+) as well as for Na(+) and insensitivity towards TEA(+). Similar observations made for guard cells from Zea mays and Vicia faba suggest a conserved regulation of channel-mediated K(+) and Na(+) transport in both cell types and species.     

Tamapin,a venom peptide from the Indian red scorpion (Mesobuthus tamulus) that targets small conductance Ca2+-activated K+ channels and afterhyperpolarization currents in central neurons

The biophysical properties of small conductance Ca(2+)-activated K(+) (SK) channels are well suited to underlie afterhyperpolarizations (AHPs) shaping the firing patterns of a conspicuous number of central and peripheral neurons. We have identified a new scorpion toxin (tamapin) that binds to SK channels with high affinity and inhibits SK channel-mediated currents in pyramidal neurons of the hippocampus as well as in cell lines expressing distinct SK channel subunits. This toxin distinguished between the SK channels underlying the apamin-sensitive I(AHP) and the Ca(2+)-activated K(+) channels mediating the slow I(AHP) (sI(AHP)) in hippocampal neurons. Compared with related scorpion toxins, tamapin displayed a unique, remarkable selectivity for SK2 versus SK1 ( approximately 1750-fold) and SK3 ( approximately 70-fold) channels and is the most potent SK2 channel blocker characterized so far (IC(50) for SK2 channels = 24 pm). Tamapin will facilitate the characterization of the subunit composition of native SK channels and help determine their involvement in electrical and biochemical signaling.     

Modulation of endothelial SK3 channel activity by Ca²⁺-dependent caveolar trafficking

Small- and intermediate-conductance Ca(2+)-activated K(+) channels (SK3/Kcnn3 and IK1/Kcnn4) are expressed in vascular endothelium. Their activities play important roles in regulating vascular tone through their modulation of intracellular concentration ([Ca(2+)](i)) required for the production of endothelium-derived vasoactive agents. Activation of endothelial IK1 or SK3 channels hyperpolarizes endothelial cell membrane potential, increases Ca(2+) influx, and leads to the release of vasoactive factors, thereby impacting blood pressure. To examine the distinct roles of IK1 and SK3 channels, we used electrophysiological recordings to investigate IK1 and SK3 channel trafficking in acutely dissociated endothelial cells from mouse aorta. The results show that SK3 channels undergo Ca(2+)-dependent cycling between the plasma membrane and intracellular organelles; disrupting Ca(2+)-dependent endothelial caveolae cycling abolishes SK3 channel trafficking. Moreover, transmitter-induced changes in SK3 channel activity and surface expression modulate endothelial membrane potential. In contrast, IK1 channels do not undergo rapid trafficking and their activity remains unchanged when either exo- or endocytosis is block. Thus modulation of SK3 surface expression may play an important role in regulating endothelial membrane potential in a Ca(2+)-dependent manner.     

Suppression of ATP-induced Cl(-)secretion by enhanced expression of epithelial Na(+)channels in mouse endometrial epithelium

We have studied the effect of enhanced expression of epithelial Na(+)channels (ENaC) on the ATP-induced Cl(-)secretion in the mouse epithelium using short-circuit current (I(SC)) and RT-PCR techniques. The amiloride sensitivity of basal current (I(b)) across the cultured endometrial epithelia was found to vary with the magnitude of the I(b), the higher the I(b)the greater its sensitivity to amiloride, indicating possible elevation of ENaC. However, the magnitude of ATP-induced I(SC), previously demonstrated to be mediated by Ca(2+)-activated chloride channel (CaCC), decreased as the amiloride sensitivity of the I(b)increased, suggesting a possible inhibitory effect of elevated expression of ENaC on ATP-mediated chloride secretion. The Matrigel treatment for culturing the endometrial epithelia affected the amiloride sensitivity of the I(b)as well as the ATP-induced I(SC)reversedly. Competitive RT-PCR demonstrated that the expression of both ENaC gamma subunits and CaCC was enhanced in Matrigel-treated cultures. However, the observed reduction in the ATP-induced or CaCC-mediated I(SC)could not be explained by the CaCC expression pattern. These data suggest that inhibition of CaCC function is due to enhanced ENaC expression. Therefore, in addition to interacting with CFTR, ENaC also appears to interact with CaCC in the mouse endometrial epithelium. Physiologically the present findings indicate that enhanced expression of ENaC leads to suppression of other Cl(-)channels, such as CFTR and CaCC, thereby preconditioning the endometrium in favour of overall salt and water absorption as observed during embryo implantation.     

Defective cholinergic Cl(-) secretion and detection of K(+) secretion in rectal biopsies from cystic fibrosis patients

Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl(-) secretion and an inverse response of the short-circuit current (I(sc)) toward stimulation with carbachol (CCh). Alternative Cl(-) channels are found in airway epithelia and have been attributed to residual Cl(-) secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversed I(sc) upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca(2+)-dependent Cl(-) conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage and I(sc) were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive I(sc) in CF rectal biopsies but caused a negative I(sc) in non-CF subjects. The CCh-induced negative I(sc) in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positive I(sc) was significantly enhanced in CF and was caused by activation of a luminal K(+) conductance, as shown by the use of the K(+) channel blockers Ba(2+) and tetraethylammonium. Moreover, a cAMP-dependent luminal K(+) conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl(-) channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl(-) conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca(2+)- and cAMP-dependent K(+) secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.     

A Ba2+-resistant, acid-sensitive K+ conductance in Na+-absorbing H441 human airway epithelial cells

By analysis of whole cell membrane currents in Na(+)-absorbing H441 human airway epithelial cells, we have identified a K(+) conductance (G(K)) resistant to Ba(2+) but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K(+) current (I(Bl)) whereas Ba(2+) has only a weak inhibitory effect. I(Bl) was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in I(Bl), acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K(+) channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current (I(SC)), and basolateral bupivacaine, lidocaine, quinidine, and Ba(2+) inhibited I(SC) at concentrations similar to those needed to inhibit I(Bl), suggesting that the K(+) channels underlying I(Bl) are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K(+) (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie G(K) in unstimulated cells and so maintain the driving force for Na(+) absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology.     

The Rice Monovalent Cation Transporter OsHKT2;4: Revisited Ionic Selectivity

The family of plant membrane transporters named HKT (for high-affinity K(+) transporters) can be subdivided into subfamilies 1 and 2, which, respectively, comprise Na(+)-selective transporters and transporters able to function as Na(+)-K(+) symporters, at least when expressed in yeast (Saccharomyces cerevisiae) or Xenopus oocytes. Surprisingly, a subfamily 2 member from rice (Oryza sativa), OsHKT2;4, has been proposed to form cation/K(+) channels or transporters permeable to Ca(2+) when expressed in Xenopus oocytes. Here, OsHKT2;4 functional properties were reassessed in Xenopus oocytes. A Ca(2+) permeability through OsHKT2;4 was not detected, even at very low external K(+) concentration, as shown by highly negative OsHKT2;4 zero-current potential in high Ca(2+) conditions and lack of sensitivity of OsHKT2;4 zero-current potential and conductance to external Ca(2+). The Ca(2+) permeability previously attributed to OsHKT2;4 probably resulted from activation of an endogenous oocyte conductance. OsHKT2;4 displayed a high permeability to K(+) compared with that to Na(+) (permeability sequence: K(+) > Rb(+) ≈ Cs(+) > Na(+) ≈ Li(+) ≈ NH(4)(+)). Examination of OsHKT2;4 current sensitivity to external pH suggested that H(+) is not significantly permeant through OsHKT2;4 in most physiological ionic conditions. Further analyses in media containing both Na(+) and K(+) indicated that OsHKT2;4 functions as K(+)-selective transporter at low external Na(+), but transports also Na(+) at high (>10 mm) Na(+) concentrations. These data identify OsHKT2;4 as a new functional type in the K(+) and Na(+)-permeable HKT transporter subfamily. Furthermore, the high permeability to K(+) in OsHKT2;4 supports the hypothesis that this system is dedicated to K(+) transport in the plant.     

Control of K(Ca) channels by calcium nano/microdomains

Transient elevations in cytoplasmic Ca(2+) trigger a multitude of Ca(2+)-dependent processes in CNS neurons and many other cell types. The specificity, speed, and reliability of these processes is achieved and ensured by tightly restricting Ca(2+) signals to very local spatiotemporal domains, "Ca(2+) nano- and microdomains," that are centered around Ca(2+)-permeable channels. This arrangement requires that the Ca(2+)-dependent effectors reside within these spatial boundaries where the properties of the Ca(2+) domain and the Ca(2+) sensor of the effector determine the channel-effector activity. We use Ca(2+)-activated K(+) channels (K(Ca)) with either micromolar (BK(Ca) channels) or submicromolar (SK(Ca) channels) affinity for Ca(2+) ions to provide distance constraints for Ca(2+)-effector coupling in local Ca(2+) domains and review their significance for the cell physiology of K(Ca) channels in the CNS. The results may serve as a model for other processes operated by local Ca(2+) domains.     

Role of potassium channels in catecholamine secretion in the rat adrenal gland

We elucidated the functional contribution of K(+) channels to cholinergic control of catecholamine secretion in the perfused rat adrenal gland. The small-conductance Ca(2+)-activated K(+) (SK(Ca))-channel blocker apamin (10-100 nM) enhanced the transmural electrical stimulation (ES; 1-10 Hz)- and 1, 1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 microM)-induced increases in norepinephrine (NE) output, whereas it did not affect the epinephrine (Epi) responses. Apamin enhanced the catecholamine responses induced by acetylcholine (6-200 microM) and methacholine (10-300 microM). The putative large-conductance Ca(2+)-activated K(+) channel blocker charybdotoxin (10-100 nM) enhanced the catecholamine responses induced by ES, but not the responses induced by cholinergic agonists. Neither the K(A) channel blocker mast cell degranulating peptide (100-1000 nM) nor the K(V) channel blocker margatoxin (10-100 nM) affected the catecholamine responses. These results suggest that SK(Ca) channels play an inhibitory role in adrenal catecholamine secretion mediated by muscarinic receptors and also in the nicotinic receptor-mediated secretion of NE, but not of Epi. Charybdotoxin-sensitive Ca(2+)-activated K(+) channels may control the secretion at the presynaptic site.     

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

Effects of carbon monoxide on ion transport across rat distal colon

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current (I(sc)). A maximal response was achieved at a concentration of 2.5·10(-4) mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl(-) and HCO(3)(-) underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the I(sc) induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na(+)-K(+)-2Cl(-) cotransporter, combined with 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid sodium salt, an inhibitor of the basolateral Cl(-)/HCO(3)(-) exchanger, inhibited the CORM-2-induced I(sc). Membrane permeabilization experiments indicated an activation of basolateral K(+) and apical Cl(-) channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca(2+) concentration. This increase depended on the influx of extracellular Ca(2+), but not on the release of Ca(2+) from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.     

I(Ca(TTX)) channels are distinct from those generating the classical cardiac Na(+) current

The Na(+) current component I(Ca(TTX)) is functionally distinct from the main body of Na(+) current, I(Na). It was proposed that I(Ca(TTX)) channels are I(Na) channels that were altered by bathing media containing Ca(2+), but no, or very little, Na(+). It is known that Na(+)-free conditions are not required to demonstrate I(Ca(TTX).) We show here that Ca(2+) is also not required. Whole-cell, tetrodotoxin-blockable currents from fresh adult rat ventricular cells in 65 mm Cs(+) and no Ca(2+) were compared to those in 3 mM Ca(2+) and no Cs(+) (i.e., I(Ca(TTX))). I(Ca(TTX)) parameters were shifted to more positive voltages than those for Cs(+). The Cs(+) conductance-voltage curve slope factor (mean, -4.68 mV; range, -3.63 to -5.72 mV, eight cells) is indistinguishable from that reported for I(Ca(TTX)) (mean, -4.49 mV; range, -3.95 to -5.49 mV). Cs(+) current and I(Ca(TTX)) time courses were superimposable after accounting for the voltage shift. Inactivation time constants as functions of potential for the Cs(+) current and I(Ca(TTX)) also superimposed after voltage shifting, as did the inactivation curves. Neither of the proposed conditions for conversion of I(Na) into I(Ca(TTX)) channels is required to demonstrate I(Ca(TTX)). Moreover, we find that cardiac Na(+) (H1) channels expressed heterologously in HEK 293 cells are not converted to I(Ca(TTX)) channels by Na(+)-free, Ca(2+)-containing bathing media. The gating properties of the Na(+) current through H1 and those of Ca(2+) current through H1 are identical. All observations are consistent with two non-interconvertable Na(+) channel populations: a larger that expresses little Ca(2+) permeability and a smaller that is appreciably Ca(2+)-permeable.