Diphenylamine-2-carboxylate Research Articles

A maxi Cl- channel in cultured pavement cells from the gills of the freshwater rainbow trout Oncorhynchus mykiss.

Primary cultures of pavement cells from the gills of a freshwater fish, the rainbow trout Oncorhynchus mykiss, have been studied for the first time using the patch-clamp technique. Gigaohm seals were obtained in approximately 95% of cells studied, and channel activity was evident in a high proportion (>90%). A large-conductance Cl- channel was evident in 6 % of cell-attached and in 31% of inside-out patches. Single-channel conductance in inside-out patches was 372 pS, and current/voltage relationships were linear over the range -60 to +60 mV. The channel was activated by patch excision, and activation was often associated with polarization of the patch. The mean number of channels per patch was 1.9, and there were several subconductance states. The relationship between channel activity (NP(o)) and voltage was in the form of an inverted U, and channel activity was highest between 0 and +20 mV. Large-conductance Cl- channels showed a progressive time-dependent reduction in current in response to sustained polarization to voltages outside the range -20 mV to +20 mV. Permeability ratios (P) of Cl- to other anions were P(HCO3)/P(Cl)=0.81, P(SO4)/P(Cl)=0.31 and P(isethionate)/P(Cl)=0.53. The channel was blocked by Zn2+, SITS, DIDS and diphenylamine carboxylate. This is the first description of a large-conductance Cl- channel in gill cells from freshwater or marine species. Possible functions of the channel are discussed.

Janus kinase 2 (JAK2) regulates prolactin-mediated chloride transport in mouse mammary epithelial cells through tyrosine phosphorylation of Na+-K+-2Cl- cotransporter.

Epithelial chloride (Cl-) transport is achieved by the coordinated action of symporters such as the Na+-K+-2Cl- cotransporter (NKCC1) and chloride channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). As a secretory tissue, mammary epithelial cells are obvious candidates for such mechanisms, but Cl- transport and its hormonal regulation have been poorly delineated in mammary epithelial cells. We determined whether the mammary epithelial cell line, HC11, transports chloride and whether this was regulated by PRL, a hormone known to stimulate ion transport. HC11 cells express both CFTR and NKCC1. Exposure to PRL or PGE1 increased Cl- transport in HC11 cells. This was inhibited by the NKCC1 blocker, furosemide, and by the Cl- channel inhibitor, diphenylamine 2-carboxylate. Dose and time course of PRL action indicate that PRL had maximal effect on Cl- transport at 1 microg/ml and at 10 min of stimulation. Examination of the signaling pathways suggests that the PRL effect on Cl- transport does not involve an increase in [Ca2+]i or MAP kinase activity. RT-PCR analyses indicate that HC11 cells express mRNA for Janus kinase 1 (JAK1), JAK2, and signal transducer and activator of transcription 5 (STAT5) but not for JAK3. PRL treatment of HC11 cells increased phosphorylation of STAT5. The JAK2 inhibitor AG490 blocked phosphorylation of STAT5 and PRL-induced, but not PGE1-induced, Cl- transport. NKCC1, but not CFTR, is tyrosine phosphorylated in HC11 cells. PRL enhanced tyrosine phosphorylation of NKCC1, and this effect was attenuated by the JAK2 inhibitor AG490. These results are the first demonstrations of a role for tyrosine phosphorylation of NKCC1 and of the PRL-JAK2 cascade in the regulation of Cl- transport.

DIFFERENT PHYSIOLOGICAL MECHANISMS CONTROL ISOVOLUMETRIC REGULATION AND REGULATORY VOLUME DECREASE IN CHICK EMBRYO CARDIOMYOCYTES

Cultured chick embryo cardiac myocytes submitted to a 180mOsm/kg hyposmotic solution swell present a regulatory volume decrease (RVD). This RVD is mediated by a Ca2+influx followed by a 40% loss of total taurine content accompanied by the loss of lesser amounts of other osmolytes. Kidney cells respond to a gradual change in osmolality by maintaining their volume at the initial level. This is termed isovolumetric regulation (IVR), which may activate regulatory processes other than those observed with sudden changes in osmolality. When cardiac myocytes were exposed to a gradual change in osmolality, they show a partial IVR which is not dependent upon extracellular Ca2+. Potassium channel blockers, quinidine and Ba2+, and the chloride channel blocker, diphenylamine-2-carboxylate (DPC), compromise IVR in our model. Tritiated taurine loss and total intracellular K+contents were analyzed in cultured cardiomyocytes submitted to a gradual change in osmolality. The cultured cells lost approximately 10% of their taurine and 35% of their total K+. These findings suggest that different compensatory mechanisms are activated when cells are exposed to stepwise and gradual changes in osmolality. Inorganic osmolytes (through conductive pathways) are preferentially mobilized during the physiological and/or patho-physiological IVR situation, perhaps reflecting energetic conservation in response to a less traumatic event for the cardiac myocytes.

CAMP activates an ATP-permeable pathway in neonatal rat cardiac myocytes

The molecular mechanisms associated with intracellular ATP release by the heart are largely unknown. In this study the luciferin-luciferase assay and patch-clamp techniques were used to characterize the pathways responsible for ATP release in neonatal rat cardiac myocytes (NRCM). Spontaneous ATP release by NRCM was significantly increased after cAMP stimulation under physiological conditions. cAMP stimulation also induced an anion-selective electrodiffusional pathway that elicited linear, diphenylamine-2-carboxylate (DPC)-inhibitable Cl(-) currents in either symmetrical MgCl(2) or NaCl. ATP, adenosine 5'-O-(3-thiotriphosphate), and the ATP derivatives ADP and AMP, permeated this pathway; however, GTP did not. The cAMP-induced ATP currents were inhibited by DPC and glibenclamide and by a monoclonal antibody raised against the R domain of the cystic fibrosis transmembrane conductance regulator (CFTR). The channel-like nature of the cAMP-induced ATP-permeable pathway was also determined by assessing protein kinase A-activated single channel Cl(-) and ATP currents in excised inside-out patches of NRCM. Single channel currents were inhibited by DPC and the anti-CFTR R domain antibody. Thus the data in this report demonstrate the presence of a cAMP-inducible electrodiffusional ATP transport mechanism in NRCM. Based on the pharmacology, patch-clamping data, and luminometry studies, the data are most consistent with the role of a functional CFTR as the anion channel implicated in cAMP-activated ATP transport in NRCM.

Interaction Between Permeation and Gating in a Putative Pore Domain Mutant in the Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel with distinctive kinetics. At the whole-cell level, CFTR currents in response to voltage steps are time independent for wild type and for the many mutants reported so far. Single channels open for periods lasting up to tens of seconds; the openings are interrupted by brief closures at hyperpolarized, but not depolarized, potentials. Here we report a serine-to-phenylalanine mutation (S1118F) in the 11th transmembrane domain that confers voltage-dependent, single-exponential current relaxations and moderate inward rectification of the macroscopic currents upon expression in Xenopus oocytes. At steady state, the S1118F-CFTR single-channel conductance rectifies, corresponding to the whole-cell rectification. In addition, the open-channel burst duration is decreased 10-fold compared with wild-type channels. S1118F-CFTR currents are blocked in a voltage-dependent manner by diphenylamine-2-carboxylate (DPC); the affinity of S1118F-CFTR for DPC is similar to that of the wild-type channel, but blockade exhibits moderately reduced voltage dependence. Selectivity of the channel to a range of anions is also affected by this mutation. Furthermore, the permeation properties change during the relaxations, which suggests that there is an interaction between gating and permeation in this mutant. The existence of a mutation that confers voltage dependence upon CFTR currents and that changes kinetics and permeation properties of the channel suggests a functional role for the 11th transmembrane domain in the pore in the wild-type channel.

Bidirectional transepithelial water transport: chloride-dependent mechanisms.

We hypothesized that inhibition and activation of basolateral to luminal chloride transport mechanisms were associated with respective decreases and increases in basolateral to luminal water fluxes. The luminal to basolateral (J(W)(L-->B)) and basolateral to luminal (J(W)(B-->L)) water fluxes across ovine tracheal epithelia were measured simultaneously. The mean J(W)(L-->B) (6.5 microl/min/cm(2)) was larger than J(W)(B-->L) (6.1 microl/min/cm(2)). Furosemide reduced J(W)(B-->L) from 6.0 to 5.6 microl/min/cm(2). Diphenylamine-2-carboxylate (DPC) reduced J(W)(B-->L) from 7.9 to 7. 3 microl/min/cm(2) and reduced the membrane potential difference by 38%. Furosemide together with DPC decreased J(W)(L-->B) by 30% and J(W)(B-->L) by 15%. Norepinephrine increased J(W)(B-->L) from 4.9 to 6.0 microl/min/cm(2). Neuropeptide Y in the presence of norepinephrine decreased J(W)(L-->B) (6.4 to 5.2 microl/min/cm(2)) and returned J(W)(B-->L) to its baseline value. Vasopressin increased J(W)(B-->L) from 4.1 to 5.1 microl/min/cm(2). Endothelin-1 induced a simultaneous increase in J(W)(B-->L) (7.0 to 7.7 microl/min/cm(2)) and decrease in J(W)(L-->B) (7.4 to 6.4 microl/min/cm(2)); and decreased the membrane resistance. These data indicate that in tracheal epithelia under homeostatic conditions J(W)(B-->L) has a approximately 15% actively coupled component. Consistent with our hypothesis, inhibition and receptor-induced stimulation of chloride effluxes were associated with decreases and increases in J(W)(B-->L), respectively. However, as inhibition of transcellular chloride transport always decreased J(W)(L-->B) more than J(W)(B-->L), reducing transepithelial chloride transport did not result in less water being transported into the airway lumen.

Block of hippocampal CAN channels by flufenamate

Ca 2+-activated non-selective cation (CAN) channels are activated by cytoplasmic Ca 2+ and I CAN underlies many slow depolarizing processes in neurons including a putative role in excitotoxicity. CAN channels in many non-neuronal cells are blocked by non-steroidal antiinflammatory drugs that are derivatives of diphenylamine-2-carboxylate (DPC). The DPC derivative flufenamate (FFA) has a complex effect on certain neurons, whereby it blocks CAN channels and increases [Ca 2+] i. We report here that FFA, but not the parent compound, DPC, blocks CAN channels in hippocampal CA1 neurons. As was the case in other neurons, the effects of FFA are complex and include a maintained rise in [Ca 2+] i. Furthermore, the CAN channel blocking ability of FFA persists even when the channels have been potentiated by a Ca 2+-dependent process. The use of a CAN channel-blocking drug is important for delineating CAN channel-dependent processes and may provide a basis for therapy for CAN channel-dependent events in ischemia.

Activation of cystic fibrosis transmembrane conductance regulator in rat epididymal epithelium by genistein.

The effect of genistein on anion secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in cultured rat cauda epididymal epithelia was studied by short-circuit current (Isc) technique. Genistein added apically stimulated a concentration-dependent rise in Isc due to Cl(-) and HCO(3)(-) secretion. The genistein-induced Isc was observed in basolaterally permeabilized monolayers, suggesting that the Isc response was mediated by the apical anion channel. The response could be blocked by the nonspecific Cl(-) channel blocker, diphenylamine-2-carboxylate (DPC), but not by the Ca(2+)-activated Cl(-) channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Genistein did not increase intracellular cAMP, but H-89, a protein kinase A inhibitor, completely abolished the Isc response to genistein. Moreover, pretreatment of the tissues with MDL-12330A, an adenylate cyclase inhibitor, markedly attenuated the Isc response to genistein, but the response was restored upon the addition of exogenous cAMP. Ca(2+), protein kinase C, tyrosine kinase, and protein phosphatase signalling pathways were not involved in the action of genistein. It is speculated that genistein stimulates anion secretion by direct interaction with CFTR. This requires a low level of phosphorylation of CFTR by basal protein kinase A activity. It is suggested that genistein may provide therapeutic benefit to male infertility associated with cystic fibrosis.

Effects of chloride channel inhibitors on H(2)O(2)-induced renal epithelial cell injury.

Oxidative stress contributes to renal epithelial cell injury in certain settings. Chloride influx has also been proposed as an important component of acute renal epithelial cell injury. The present studies examined the role of Cl(-) in H(2)O(2)-induced injury to LLC-PK(1) renal epithelial cells. Exposure of LLC-PK(1) cells to 1 mM H(2)O(2) resulted in the following: depletion of intracellular ATP content; DNA damage; lipid peroxidation; and a loss of membrane integrity to both small molecules, e.g., trypan blue, and macromolecules, e.g., lactate dehydrogenase (LDH), and cell death. Substitution of Cl(-) by isethionate or the inclusion of certain Cl(-) channel blockers, e.g., diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino). benzoate (NPPB), and niflumic acid, prevented the H(2)O(2)-induced loss of membrane integrity to LDH. In addition, the H(2)O(2)-induced loss of membrane integrity was prevented by raising the osmolality of the extracellular solutions, by depletion of cell ATP, and by inhibitors of volume-sensitive Cl(-) channels. However, these maneuvers did not prevent the H(2)O(2)-induced permeability to small molecules or H(2)O(2)-induced ATP depletion, DNA damage, lipid peroxidation, or cell death. These results support the view that volume-sensitive Cl(-) channels play a role in the progressive loss of cell membrane integrity during injury.

Actin filament organization is required for proper cAMP-dependent activation of CFTR.

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl(-) channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl(-) channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

Chloride dependency of renal brush-border membrane phosphate transport.

In our present study, we examined the effect of Cl(-) on rabbit renal brush-border membrane (BBM) phosphate (P(i)) uptake. It was found that the Na(+)-dependent BBM (32)P uptake was significantly inhibited by Cl(-) replacement in the uptake solution with other anions, or by Cl(-) transport inhibitors, including DIDS, SITS, diphenylamine-2-carboxylate (DPC), niflumic acid (NF), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). Intravesicular formate or Cl(-) increased BBM (36)Cl(-) uptake but did not affect BBM (32)P uptake. BBM (22)Na(+) uptake was lowered by Cl(-) replacement in the uptake solution but not by Cl(-) transport inhibitors. Changes in transmembrane electrical potential altered BBM (36)Cl(-) and (32)P uptake in directions consistent with a net inward movement of negative and positive charges, respectively. However, the Cl(-)-dependent BBM P(i) uptake was not affected by changes in transmembrane electrical potential. Finally, a similar Cl(-) dependency of P(i) uptake was also found with BBM derived from rat and mouse kidneys. In summary, our study showed that a component of Na(+)-dependent P(i) uptake was also Cl(-) dependent in rabbit, rat, and mouse renal BBM. The mechanism underlying this Cl(-) dependency remains to be identified.

Role of chloride and inhibitory action of inorganic nitrate on gonadotropin-stimulated steroidogenesis in mouse Leydig tumor cells

The involvement of adenylate cyclase—cyclic adenosine monophosphate (AC-cAMP) in gonadotropin-stimulated testicular steroidogenesis is well known. Little is known about the role of guanylate cyclase—cyclic guanosine monophosphate (GC-cGMP) or early chloride conductance stimulated by gonadotropins in steroidogenesis. Human chorionic gonadotropin (hCG) 1 IU/L caused significant androgen secretion without a discernible effect on cAMP production. Despite negligible intracellular cAMP, the protein kinase A inhibitor H89 blocked basal and hCG-stimulated steroidogenesis. The GC inhibitors methylene blue (MB) and LY83583 decreased androgen secretion, but hCG did not stimulate cGMP production and there was not a steroidogenic response to exogenous cGMP. A chloride-channel inhibitor, diphenylamine-2-carboxylate (DPC), at concentrations up to 0.6 mmol/L stimulated basal steroid secretion and hCG 10 IU/L stimulated cAMP production, but higher concentrations had an inhibitory effect. Substitution of chloride by gluconate enhanced basal steroid secretion, but nitrate completely abolished the effect of 1 IU/L hCG on androgen secretion, which could be partially overcome by increasing the gonadotropin concentration. In conclusion, chloride, perhaps by activating AC-cAMP, mediates the steroidogenic action of gonadotropins in mouse Leydig tumor cells (MLTC-1). Inorganic nitrate probably inhibited steroidogenesis via conversion to nitric oxide (NO) without involving the GC-cGMP pathway. Nevertheless, the results obtained with GC inhibitors suggest a role for the GC-cGMP pathway in Leydig cell steroidogenesis.

Evidence for NO. redox form of nitric oxide as nitrergic inhibitory neurotransmitter in gut.

A nitric oxide (NO)-like product of the L-arginine NO synthase pathway has been shown to be a major inhibitory neurotransmitter that is involved in the slow component of the inhibitory junction potential (IJP) elicited by stimulation of nonadrenergic, noncholinergic nerves. However, the exact nature of the nitrergic transmitter, the role of cGMP, and the involvement of a potassium or a chloride conductance in the slow IJP remain unresolved. We examined the effects of soluble guanylate cyclase inhibitors LY-83583 and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), potassium-channel blockers and putative chloride-channel blockers diphenylamine-2-carboxylate (DPC) and niflumic acid (NFA) on the hyperpolarization elicited by an NO. donor, diethylenetriamine/NO adduct (DNO), NO in solution, and an NO+ donor, sodium nitroprusside (SNP), in the guinea pig ileal circular muscle. Effects of these blockers on purinergic (fast) and nitrergic (slow) IJP were also examined. DNO-induced hyperpolarization and nitrergic slow IJP were suppressed by LY-83583 or ODQ and DPC or NFA but not by the potassium-channel blocker apamin. In contrast, hyperpolarization caused by SNP or solubilized NO gas and purinergic fast IJP were antagonized by apamin but not by inhibitors of guanylate cyclase or chloride channels. These results demonstrate biological differences in the actions of different redox states of NO and suggest that NO. is the nitrergic inhibitory neurotransmitter.

Proton secretion in the male reproductive tract: involvement of Cl--independent HCO-3 transport.

The lumen of the epididymis is the site where spermatozoa undergo their final maturation and acquire the capacity to become motile. An acidic luminal fluid is required for the maintenance of sperm quiescence and for the prevention of premature activation of acrosomal enzymes during their storage in the cauda epididymis and vas deferens. We have previously demonstrated that a vacuolar H+-ATPase [proton pump (PP)] is present in the apical pole of apical and narrow cells in the caput epididymis and of clear cells in the corpus and cauda epididymis and that this PP is responsible for the majority of proton secretion in the proximal vas deferens. We now show that PP-rich cells in the vas deferens express a high level of carbonic anhydrase type II (CAII) and that acetazolamide markedly inhibits the rate of proton secretion by 46.2 +/- 6.1%. The rate of acidification was independent of Cl- and was strongly inhibited by SITS under both normal and Cl--free conditions (50.6 +/- 5.0 and 57. 5 +/- 6.0%, respectively). In the presence of Cl-, diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibited proton secretion by 63.7 +/- 11.3% when applied together with DPC. In Cl--free solution, DPC markedly inhibited proton efflux by 45.1 +/- 7.6%, SITS produced an additional inhibition of 18.2 +/- 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAII plays a major role in proton secretion by the proximal vas deferens. Acidification does not require the presence of Cl-, but DPC-sensitive Cl- channels might contribute to basolateral extrusion of HCO-3 under Cl--free conditions. The inhibition by SITS observed under both normal and Cl--free conditions indicates that a Cl-/HCO-3 exchanger is not involved and that an alternative HCO-3 transporter participates in proton secretion in the proximal vas deferens.

Regulated anion secretion in cultured epithelia from Sertoli cells of immature rats.

1. Cultured epithelia of Sertoli cells from prepubertal rats were grown on Matrigel-coated millipore filters for short-circuit current (Isc) measurements. Under basal conditions, these epithelia exhibited a 'zero' transepithelial potential difference, a 'zero' short-circuit current and a transepithelial resistance of 60 Omega cm2. 2. Forskolin (100 microM) and 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) (100 microM) added to the apical side stimulated the Isc (forskolin, peak DeltaIsc = 1.32 +/- 0.16 microA cm-1; cpt-cAMP, peak DeltaIsc = 0.88 +/- 0.16 microA cm-2). 3. ATP (100 microM) added apically elicited a Isc response (peak DeltaIsc = 6.45 +/- 0. 28 microA cm-2) which was similar in magnitude to that of 1 microM thapsigargin (peak DeltaIsc = 6.09 +/- 0.44 microA cm-2). The potency of the responses to other nucleotides: UTP >= ATP > ADP >> AMP = adenosine indicates the involvement of a mixture of P2Y receptors. 4. Removal of extracellular Cl- and HCO3- reduced the Isc response to ATP by 70 % and 40 %, respectively. Removal of K+ had no effect, whereas removal of Na+ attenuated the Isc response. 5. The response to ATP was insensitive to agents known to block anion secretion (except apical diphenylamine-2-carboxylate (DPC) and DIDS). The resistance to perturbation by pharmacological agents may be a unique property of the seminiferous epithelium. 6. Whole-cell current recordings in cultured rat Sertoli cells demonstrated a DIDS-sensitive outwardly rectifying Cl- conductance with activating and inactivating characteristics at depolarizing and hyperpolarizing voltages, respectively. 7. The stimulation of electrogenic ion transport by ATP may be part of a complex mechanism regulating fluid secretion by the testis. Cultured Sertoli cell epithelia are shown to provide a useful model to investigate transepithelial transport in the seminiferous epithelium.

Chloride transport across syncytiotrophoblast microvillous membrane of first trimester human placenta.

There are significant changes in the activity of some placental transporters between first trimester and term. However, chloride transport has previously been studied only in the term placenta. Therefore. in this study, we investigated chloride transport mechanisms in syncytiotrophoblast microvillous membrane (MVM) vesicles from first trimester human placentas and compared them with those in vesicles from term placentas. 36Cl- uptake into MVM vesicles was linear up to 45 s and had reached equilibrium by 1 h for both first trimester and term vesicles. In first trimester MVM at 0 mV, 0.1 mM diisothiocyano-2,2'-disulfonic stilbene (DIDS) blocked 25+/-3% (n=8) of 36Cl- uptake at 30 s (initial rate), which was similar to the 30+/-7% (n=6) inhibition by DIDS in term MVM. In the presence of a 25 mV inside-positive electrical potential difference, induced by imposition of a K+ gradient after preincubation with 200 microM valinomycin, 0.5 mM diphenylamine-2-carboxylate (DPC) significantly blocked 30+/-4% of 36Cl- uptake at 30 s by first trimester MVM (p < 0.01); 18+/-5% (n=8) of total uptake was inhibited by DPC but not by DIDS. There was a similar 15+/-3% (n=6) component of 36Cl- uptake by term MVM, which was inhibited by DPC but not by DIDS. Using Western blotting, it was shown that the anion exchanger-1 protein was expressed in first trimester MVM in quantitatively similar amounts to that in term MVM. This study suggests that there is both an anion exchanger and a DPC-sensitive conductance in MVM of first trimester placenta with activity similar to that of term human placenta.

Calcium-stimulated Cl- secretion in Calu-3 human airway cells requires CFTR.

Human airway serous cells secrete antibiotic-rich fluid, but, in cystic fibrosis (CF), Cl(-)-dependent fluid secretion is impaired by defects in CF transmembrane conductance regulator (CFTR) Cl- channels. Typically, CF disrupts adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion but spares Ca(2+)-mediated secretion. However, in CF airway glands, Ca(2+)-mediated secretion is also greatly reduced. To determine the basis of Ca(2+)-mediated Cl- secretion in serous cells, we used thapsigargin to elevate intracellular Ca2+ concentration ([Ca2+]i) in Calu-3 cells, an airway cell line bearing some similarities to serous cells. Cells were cultured using conventional and air interface methods. Short-circuit current (Isc) and transepithelial conductance (Gte) were measured in confluent cell layers. Thapsigargin stimulated large, sustained changes (delta) in Isc and Gte, whereas forskolin stimulated variable and smaller increases. delta Isc was decreased by basolateral bumetanide, quinidine, barium, or diphenylamine-2-carboxylate (DPAC) but was unaffected by high apical concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 4,4'-dinitrostilbene-2,2'-disulfonic acid, and calixarene. Isc was measured after permeabilizing the basolateral membrane and establishing transmembrane ion gradients. Unstimulated apical membranes displayed high Cl- conductance (GCl) that was decreased by DPAC but not by DIDS. Apical GCl could be increased by elevating intracellular cAMP concentration but not [Ca2+]i. We conclude that CFTR channels are the exclusive GCl pathway in the apical membrane and display approximately 60% of maximum conductance at rest. Thus elevated [Ca2+]i increases K+ conductance to force Cl- through open CFTR channels. We hypothesize that loss of CFTR channels causes diminution of cholinergically mediated gland secretions in CF.

KCl reabsorption by the lower malpighian tubule of rhodnius prolixus: inhibition by Cl − channel blockers and acetazolamide

Iono- and osmoregulation by the blood-feeding hemipteran Rhodnius prolixus involves co-ordinated actions of the upper and lower Malpighian tubules. The upper tubule secretes ions (Na +, K +, Cl −) and water, whereas the lower tubule reabsorbs K + and Cl − but not water. The extent of KCl reabsorption by the lower tubule in vitro was monitored by ion-selective microelectrode measurement of Cl − and/or K + concentration in droplets of fluid secreted by Malpighian tubules isolated under oil. An earlier study proposed that K + reabsorption involves an omeprazole-sensitive apical K +/H + ATPase and Ba 2+-sensitive basolateral K + channels. This paper examines the effects acetazolamide and of compounds that inhibit chloride channels, Cl −/HCO 3 − exchangers and Na +/K +/2Cl − or K +/Cl − co-transporters. The results suggest that Cl − reabsorption is inhibited by acetazolamide and by Cl − channel blockers, including diphenylamine-2-carboxylate(DPC) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by compounds that block Na +/K +/Cl − and K +/Cl − co-transporters. Measurements of transepithelial potential and basolateral membrane potential during changes in bathing saline chloride concentration indicate the presence of DPC- and NPPB-sensitive chloride channels in the basolateral membrane. A working hypothesis of ion movements during KCl reabsorption proposes that Cl − moves from lumen to cell through a stilbene-insensitive Cl −/HCO 3 − exchanger and then exits the cell through basolateral Cl − channels.

Effects of chloride channel blockers on hypoxic injury in rat proximal tubules

These studies examined the pathways and consequences of chloride uptake into proximal tubule cells during in vitro hypoxia. The chloride channel blocker diphenylamine-2-carboxylate (DPC) markedly reduced the degree of hypoxia-induced membrane damage as measured by the release of lactate dehydrogenase (LDH). DPC reduced the release of LDH from hypoxic tubules from 38 +/- 2.7% to 16 +/- 1.7% after 30 minutes of hypoxia (P < 0.001, N = 16) and also reduced 36Cl- uptake by hypoxic tubules. The reduction in LDH release was not associated with better preservation of cell ATP content or with protection against hypoxia-induced DNA damage. Other Cl- channel blockers, such as niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihyrdo-2-methyl-1-oxo-1H-in den-5-yl)oxy] acetic acid (IAA-94) provided even greater protection than DPC and were as effective as 2 mM glycine. The Cl- channel blockers appear to act late in the course of hypoxic injury since DNA damage, an early manifestation of injury, is not prevented by the blockers and since addition of the Cl- channel blocker after the hypoxic injury has begun reduces further membrane damage. These results support the conclusion that transport through Cl- channels contributes to hypoxic cell injury in proximal tubular cells.

A synthetic peptide derived from glycine-gated Cl- channel induces transepithelial Cl- and fluid secretion.

M2GlyR is a synthetic 23-amino acid peptide that mimics the second membrane-spanning region of the alpha-subunit of the postsynaptic glycine receptor. This peptide has been shown to form an anion-selective channel in phospholipid bilayers. We have investigated the possibility that the peptide may incorporate into the apical membrane of secretory epithelia and induce the secretion of Cl- and water. We improved the solubility of this peptide by adding four lysine residues to the carboxy terminus, C-K4-M2GlyR, and assayed its channel-forming activity using a subculture of Madin-Darby canine kidney (MDCK) cells. The addition of 100 microM C-K4-M2GlyR to the apical surface of MDCK monolayers significantly increased short-circuit current (Ise), hyperpolarized transepithelial potential difference, and induced fluid secretion. The increase in Ise was inhibited by 100 microM bumetanide and by Cl- channel inhibitors. The effectiveness of the channel blockers followed the sequence niflumic acid > or = 5-nitro-2-(3-phenylpropylamino)benzoate > diphenylamine-2-carboxylate (DPC) > glibenclamide. The effect of the peptide was not inhibited by 4.4'-diisothiocyanostilbene-2-2'-disulfonic acid. Removing Cl from the bathing solutions also inhibited the effect of the peptide. The Cl- efflux pathway induced by C-K4-M2GlyR differs from the native pathway activated by the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin. First, intracellular cAMP levels were unaffected. Second, the concentration of DPC required to inhibit the effect of the peptide was much lower than that needed to block the forskolin response (100 microM vs. 3 mM). These results support the hypothesis that the synthetic peptide C-K4-M2GlyR can from Cl -selective channels in the apical membrane of secretory epithelial cells and can induce sustained transepithelial secretion of Cl- and fluid.