Protein Kinase C and Regulatory Volume Decrease in Mudpuppy Red Blood Cells

This study examined whether protein kinase C (PKC) stimulates K⁺ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 μm), but not with the cyclic nucleotide-dependent kinase antagonists N-(2′-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 μm) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 μm). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 μm). The effect of these three antagonists and H-7 was reversed with gramicidin (5 μm in a choline Ringer), indicating PKC was linked to K⁺ efflux (gramicidin is a cationophore that was used to ensure a high K⁺ permeability). We also measured cell volume recovery from hypotonic shock (0.5× Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 μm), sphingosine (100 nm), chelerythrine (10 μm), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 μm) nor H-8 (5 μm). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 μm). Furthermore, using the patch clamp technique, we found H-7 (10 μm) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K⁺ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K⁺ loss during RVD is regulated, at least in part, by protein kinase C. Received: 20 January 1998/Revised: 2 September 1998     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.     

The Effect of an Extracellular Mucilage on the Response to Osmotic Shock in the Charophyte Alga Lamprothamnium papulosum

We have used current/voltage (I/V) analysis to investigate the role played by extracellular mucilage in the cellular response to osmotic shock in Lamprothamnium papulosum. Cells lacking extracellular mucilage originated in a brackish environment (1/3 seawater). These were compared, first with cells coated with thick (∼50 μm) extracellular mucilage, collected from a marine lake, and second, with equivalent mucilaginous marine cells, treated with heparinase enzyme to disrupt the mucilage layer. Histochemical stains Toluidine Blue and Alcian Blue at low pH identified the major component of the extracellular mucilage as sulfated polysaccharides. Treating mucilage with heparinase removed the capacity for staining with cationic dyes at low pH, although the mucilage was not removed, and remained as a substantial unstirred layer. Cells lacking mucilage responded to hypotonic shock with depolarization (by ∼95 mV), cessation of cyclosis, due to transient opening of Ca²⁺ channels, and opening of Ca²⁺-activated Cl⁻ channels and K⁺ channels. Cell conductance transiently increased tenfold, but after 60 min was restored to the conductance prior to hypotonic shock. Mucilaginous cells depolarized by a small amount (∼18 mV), but Ca²⁺ channels failed to open in large enough numbers for cyclosis to cease. Likewise most Ca²⁺-activated Cl⁻ channels failed to open and conductance increased only ∼1.2-fold above the prehypotonic level. After 60 min conductance was less than the conductance prior to hypotonic shock. Heparinased mucilaginous cells recovered several aspects of the hypotonic response in cells lacking mucilage. These cells depolarized (by ∼103 mV); cyclosis ceased, indicating that Ca²⁺ channels had opened, and conductance increased to ∼4 times the value prior to hypotonic shock, indicating that Ca²⁺-activated Cl⁻ channels opened. However, after 60 min, these cells had neither restored membrane potential (and remained at positive values), nor decreased their conductance. It was not possible to determine whether K⁺ channels had opened. The heparinased cells recovered the normal hypotonic response of mucilaginous cells when heparinase was washed out. Apical seawater cells, which lacked mucilage, were unaffected by heparinase treatment. The results demonstrate that the presence of extracellular sulfated polysaccharide mucilage impacts upon the electrophysiology of the response to osmotic shock in Lamprothamnium cells. The role of such sulfated mucilages in marine algae and animal cells is compared and discussed. Received: 24 March 1998/Revised: 28 April 1999     

Characterization of the Increase in [Ca2+] i During Hypotonic Shock and the Involvement of Ca2+-activated K+ Channels in the Regulatory Volume Decrease in Human Osteoblast-like Cells

The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, [Ca²⁺] _i , in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in [Ca²⁺] _i from ∼150 nm up to 1.3 μm. This increase in [Ca²⁺] _i was mainly due to an influx of extracellular Ca²⁺ since removing of extracellular Ca²⁺ reduced this increase to ∼250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca²⁺] _i on membrane currents in these cells. An increase in [Ca²⁺] _i revealed two types of Ca²⁺-activated K⁺ channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca²⁺] _i buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca²⁺] _i to 10 μm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from −160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than −40 mV from a holding potential of −80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in [Ca²⁺] _i elicited through hypotonic solutions. In combination with the volume-regulated Cl⁻ conductance in the same cells this K⁺ channel seems to play a vital role in volume regulation in human osteoblasts. Received: 8 February 2000/Revised: 13 July 2000     

Increases in Intracellular pH and Ca2+ are Essential for K+ Channel Activation After Modest `Physiological' Swelling in Villus Epithelial Cells

We studied the relationship between changes in intracellular pH (pH _i ), intracellular Ca²⁺([Ca²⁺] _i ) and charybdotoxin sensitive (CTX) maxi-K⁺ channels occurring after modest `physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was assessed by electronic cell sizing, and pH <sub>i</sub> and [Ca<sup>2+</sup>] <sub>i</sub> by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93 × isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase in a more hypotonic (0.80 × isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 × isotonic dilution, the pH <sub>i</sub> alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this ΔpH <sub>i</sub> and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH<sub>4</sub>Cl permitted complete RVD which could be inhibited by CTX. The rate of <sup>86</sup>Rb efflux which also increased after this 0.93 × isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na<sup>+</sup>-free medium. Modest swelling transiently increased [Ca<sup>2+</sup>] <sub>i</sub> and Ca<sup>2+</sup>-free medium or blocking alkalinization by MIA or Na<sup>+</sup>-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca<sup>2+</sup>-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca<sup>2+</sup>] <sub>i</sub> and volume changes became sensitive to CTX. We conclude that both alkalinization of pH <sub>i</sub> and increased [Ca<sup>2+</sup>] <sub>i</sub> observed with `physiological' volume increase are essential for the activation of CTX-sensitive maxi-K⁺ channels required for RVD. Received: 30 March 1999/Revised: 6 July 1999     

Mechanically Activated Currents in Chick Heart Cells

As predicted from stretch-induced changes of rate and rhythm in the heart, acutely isolated embryonic chick heart cells exhibit whole-cell mechanosensitive currents. These currents were evoked by pressing on cells with a fire polished micropipette and measured through a perforated patch using a second pipette. The currents were carried by Na⁺ and K⁺ but not Cl⁻, and were independent of external Ca²⁺. The currents had linear I/V curves reversing at −16 mV and were completely blocked by Gd³⁺≥ 30 μm and Grammostola spatulata venom at a dilution of 1:1000. Approximately 20% of cells showed time dependent inactivation. In contrast to direct mechanical stimulation, hypotonic volume stress produced an increase in conductance for anions rather than cations—the two stimuli are not equivalent. The cells had two types of stretch-activated ion channels (SACs): a 21 pS nonspecific cation-selective reversing at −2 mV and a 90 pS K⁺ selective reversing at −70 mV in normal saline. The activity of SACs was strongly correlated with the presence of whole-cell currents. Both the whole-cell currents and SACs were blocked by Gd³⁺ and by Grammostola spatulata spider venom. Mechanical stimulation of spontaneously active cells increased the beating rate and this effect was blocked by Gd³⁺. We conclude that physiologically active mechanosensitive currents arise from stretch activated ion channels. Received: 8 April 1996/Revised: 8 August 1996     

G Protein-mediated Activation of a Nonspecific Cation Current in Cultured Rat Retinal Pigment Epithelial Cells

We used whole-cell patch-clamp recording techniques to investigate G protein-activated currents in cultured rat retinal pigment epithelial (RPE) cells. Using 140 mm KCl intracellular and 130 mm NaCl extracellular solutions, rat RPE cells possessed both inward and outward K⁺ currents. Upon addition of the nonhydrolyzable guanine triphosphate analogue, guanosine-5′-O-(3-thiophosphate) (GTPγS, 0.1 mm), to the recording electrode, a nonspecific cation (NSC) current was elicited. The NSC current had a mean reversal potential of +5.7 mV in 130 mm extracellular NaCl with Cs⁺-aspartate in the pipette, and was not affected by alterations in the extracellular Ca²⁺ or Cl⁻ concentration. The GTPγS-activated current was found to be permeable to several monovalent cations (K⁺, Na⁺, choline, TRIS, and NMDG). Addition of fluoroaluminate, an activator of large molecular weight heterotrimeric GTP-binding proteins (G proteins), to the intracellular recording solution activated the NSC current. The G protein involved was pertussis toxin (PTX)-sensitive, since GTPγS failed to activate the NSC current in cells pretreated with PTX. Further investigation of second messenger molecules suggested that activation of the NSC current was not affected by alterations in intracellular Ca²⁺ or ATP. From these results, we conclude that a G protein-regulated NSC current is present in rat RPE cells. Activation of the NSC current may sufficiently depolarize RPE cells to activate outward K⁺ currents. This would provide a mechanism by which these cells could rid themselves of accumulated K⁺. Received: 25 January 1996/Revised: 24 April 1996     

Membrane Potassium Channels and Human Bladder Tumor Cells. I. Electrical Properties

These experiments were conducted to determine the membrane K⁺ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K⁺ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca²⁺-dependent outward K⁺ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca²⁺-activated, voltage-dependent K⁺ channel (K_Ca) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K_Ca activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K⁺ channel, 21 ± 1 pS at positive transmembrane potential (V _m ) and 145 ± 13 pS at negative V _m . Glibenclamide (50 μm), Ba²⁺ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K⁺ channels in inside/out patches in a dose-dependent manner, and the IC₅₀= 46 μm. The identity of this K⁺ channel with an ATP-sensitive K⁺ channel (K_ATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the K_Ca and a lower conductance K⁺ channel with properties consistent with a sulfonylurea receptor-linked K_ATP. Received: 12 June 1997/Revised: 21 October 1997     

Mechanism of Shrinkage Activation of Nonselective Cation Channels in M-1 Mouse Cortical Collecting Duct Cells

It has previously been shown that osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical collecting duct cells [54] and in a variety of other cell types [20]. In the present study we further characterized the shrinkage-activated NSC channel in M-1 cells and its mechanism of activation using whole-cell current recordings. Osmotic cell shrinkage induced by addition of 100 mm sucrose to the bath solution caused a 20-fold increase in whole-cell inward currents from −10.8 ± 1.5 pA to −211 ± 10.2 pA (n= 103). A similar response was observed when cell shrinkage was elicited using a hypo-osmotic pipette solution. This indicates that cell shrinkage and not extracellular osmolarity per se is the signal for current activation. Cation substitution experiments revealed that the activated channels discriminate poorly between monovalent cations with a selectivity sequence NH₄ (1.2) ≥ Na⁺ (1) ≈ K⁺ (0.9) ≈ Li⁺ (0.9). In contrast there was no measurable permeability for Ca²⁺ or Ba²⁺ and the cation-to-anion permeability ratio was about 14. The DPC-derivatives flufenamic acid, 4-methyl-DPC and DCDPC were the most effective blockers followed by LOE 908, while amiloride and bumetanide were ineffective. The putative channel activator maitotoxin had no effect. Current activation was dependent upon the presence of intracellular ATP and Mg²⁺ and was inhibited by staurosporine (1 μm) and calphostin C (1 μm). Moreover, cytochalasin D (10 μm) and taxol (2 μm) reduced the current response to cell shrinkage. These findings suggest that the activation mechanism of the shrinkage-activated NSC channel involves protein kinase mediated phosphorylation steps and cytoskeletal elements. Received: 3 May 2000/Revised: 6 July 2000     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998     

Kinetics of K-Cl Cotransport in Frog Erythrocyte Membrane: Effect of External Sodium

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO₃) has been shown to mediate a large fraction of the total K⁺ transport. In the present study, Cl⁻-dependent and Cl⁻-independent K⁺ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K⁺ ([K⁺] _e and [K⁺] _i ) concentration. The dependence of ouabain-resistant Cl⁻-dependent K⁺ (⁸⁶Rb) influx on [K⁺] _e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K _m ) of 8.2 ± 1.3 mm and maximal velocity (V _max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl⁻-dependent K⁺ influx increased both K _m (12.8 ± 1.7 mm, P < 0.05) and V _max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K⁺] _e above 20 mm in isotonic media significantly reduced the Cl⁻-dependent K⁺ influx due to a reciprocal decrease of the external Na⁺ ([Na⁺] _e ) concentration below 50 mm. Replacing [Na⁺] _e by NMDG⁺ markedly decreased V _max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K _m (15.7 ± 2.1 mm, P < 0.03) of Cl⁻-dependent K⁺ influx. Moreover, NMDG⁺ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb⁺ uptake and K⁺ loss from red cells. Cell swelling did not affect the Na⁺-dependent changes in Rb⁺ uptake and K⁺ loss. In a nominally K⁺(Rb⁺)-free medium, net K⁺ loss was reduced after lowering [Na⁺] _e below 50 mm. These results indicate that over 50 mm [Na⁺] _e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K⁺, Cl⁻-dependent K⁺ loss in K⁺-free media was a linear function of [K⁺] _i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr⁻¹ (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K⁺ ions. The residual, ouabain-resistant K⁺ fluxes in NO₃ were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K⁺ effluxes in isotonic (∼0.014 hr⁻¹) and hypotonic (∼0.022 hr⁻¹) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999     

Ca2+/Calmodulin Kinase II and Decreases in Intracellular pH are Required to Activate K+ Channels After Substantial Swelling in Villus Epithelial Cells

To assess the activation of the charybdotoxin-insensitive K⁺ channel responsible for Regulatory Volume Decrease (RVD) after substantial volume increases, we measured intracellular pH (pH _i ), intracellular calcium ([Ca²⁺] _i ) and inhibitors of kinases and phosphoprotein phosphatases in guinea pig jejunal villus enterocytes in response to volume changes. Fluorescence spectroscopy was used to measure pH _i and [Ca²⁺] _i of cells in suspension, loaded with 2,7,bis-carboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively, and cell volume was assessed using electronic cell sizing. A modest 7% volume increase or substantial 15 to 20% volume increase caused [Ca²⁺] _i to increase proportionately but the 7% increase caused alkalinization while the larger increases resulted in acidification of ≃0.14 pH units. Following a 15% volume increase, 1-N-0-bis (5-isoquinoline-sulfonyl)-N-methyl-l-4-phenyl-piperazine (KN-62, 50 μm), an inhibitor of Ca²⁺/calmodulin kinase II, blocked RVD. Gramicidin (0.5 μm) bypassed this inhibition suggesting that the K⁺ channel had been affected by the KN-62. RVD after a modest 7% volume increase was not influenced by KN-62 unless the cell was acidified. Okadaic acid, an inhibitor of phosphoprotein phosphatases 1 and 2A, accelerated RVD after a 20% volume increase; inhibition of RVD generated by increasing the K⁺ gradient was bypassed by okadaic acid. Tyrosine kinase inhibitor, genistein (100 μm) had no effect on RVD after 20% volume increases. We conclude that activation of charybdotoxin-insensitive K⁺ channels utilized for RVD after substantial (>7%) `nonphysiological' volume increases requires phosphorylation mediated by Ca²⁺/calmodulin kinase II and that increases in cytosolic acidification rather than larger increases in [Ca²⁺] _i are a critical determinant of this activation. Received: 30 March 1999/Revised: 6 July 1999     

Regulation of Cl− Secretion by Extracellular ATP in Cultured Mouse Endometrial Epithelium

The present study explored regulation of electrogenic ion transport across cultured mouse endometrial epithelium by extracellular ATP using the short-circuit current (I _SC ) and the patch-clamp techniques. The cultured endometrial monolayers responded to apical application of ATP with an increase in I _SC in a concentration-dependent manner (EC₅₀ at 3 μm). Replacement of Cl⁻ in the bathing solution or treatment of the cells with Cl⁻ channel blockers, DIDS and DPC, markedly reduced the I _SC , indicating that a substantial portion of the ATP-activated I _SC was Cl⁻-dependent. Amiloride at a concentration (10 μm) known to block Na⁺ channels was found to have no effect on the ATP-activated I _SC excluding the involvement of Na⁺ absorption. Adenosine was found to have little effect on the I _SC excluding the involvement of P₁ receptors. The effect of UTP, a potent P_2U receptor agonist on the I _SC was similar to that of ATP while potent P_2X agonist, α-β-Methylene adenosine 5′-triphosphate (α-β-M-ATP) and P_2Y agonist, 2-methylthio-adenosine triphosphate (2-M-ATP), were found to be ineffective. The effect of ATP on I _SC was mimicked by the Ca²⁺ ionophore, ionomycin, indicating a role of intracellular Ca²⁺ in mediating the ATP response. Confocal microscopic study also demonstrated a rise in intracellular Ca²⁺ upon stimulation by extracellular ATP. In voltage-clamped endometrial epithelial cells, ATP elicited a whole-cell Cl⁻ current which exhibited outward rectification and delayed activation and inactivation at depolarizing and hyperpolarizing voltages, respectively. The results of the present study demonstrate the presence of a regulatory mechanism involving extracellular ATP and P_2U purinoceptors for endometrial Cl⁻ secretion.     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

5-Lipoxygenase Metabolites of Arachidonic Acid Regulate Volume Decrease by Mudpuppy Red Blood Cells

We examined whether metabolites of arachidonic acid (AA) regulate K⁺ efflux during regulatory volume decrease (RVD) by mudpuppy red blood cells (RBCs). Volume regulation was inhibited by the phospholipase A₂ antagonists mepacrine (10 μm) and ONO-RS-082 (10 μm); the inhibitory effect of ONO-RS-082 was reversed by gramicidin (5 μm). Eicosatetraynoic acid (ETYA, 100 μm), a general antagonist of AA metabolism, also blocked RVD. In addition, volume regulation was inhibited by the lipoxygenase pathway antagonist nordihydroguaiaretic acid (NDGA, 10 μm), the 5 lipoxygenase antagonists AA-861 (5 μm) and curcumin (20 μm), and by the 5-lipoxygenase activating protein inhibitor L-655,298 (5 μm). Inhibition by all four of these agents was reversed with gramicidin. In contrast, the 12- and 15-lipoxygenase pathway inhibitor ethyl-3,4-dihydroxy-benzylidene-cyanoacetate (EDBCA, 1 μm) and the cytochrome P-450 monooxygenase pathway blocker ketoconazole (20 μm) had no effect. On the other hand, the cyclooxygenase pathway inhibitor aspirin (100 μm) slightly enhanced RVD. Consistent with these findings, a K⁺-selective whole cell conductance responsible for K⁺ efflux during cell swelling was inhibited by ONO-RS-082 (10 μm), NDGA (10 μm), AA-861 (5 μm), curcumin (20 μm), and l-655,298 (5 μm). In contrast, EDBCA (1 μm), ketoconazole (20 μm), and indomethacin (10 μm) did not block this whole cell conductance. These results indicate that a channel mediating K⁺ loss during RVD is regulated by a 5-lipoxygenase metabolite of arachidonic acid. Received: 12 December 1996/Revised: 28 February 1997     

On the Role of Calcium in the Regulatory Volume Decrease (RVD) Response in Ehrlich Mouse Ascites Tumor Cells

The putative role for Ca²⁺ entry and Ca²⁺ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P₃ content, as measured in extracts from [2-³H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca²⁺-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca²⁺-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca²⁺ (1% only at 0.1-mm Ca²⁺ and in Ca²⁺-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca²⁺ upon the volume-induced K⁺ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca²⁺ chelator BAPTA when the BAPTA-loading is performed in K⁺ equilibrium medium; (vi) an inhibition of the swelling-induced K⁺ loss (about 50%) at 1-mm Ba²⁺, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 μm), reported inhibitors of the K⁺ loss induced by Ca²⁺-mobilizing agonists. Thus, Ca²⁺signaling by Ca²⁺ release or Ca²⁺ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells. Received: 8 December 1996/Revised: 14 January 1997     

Selective Inhibition of Glucose-Stimulated β-Cell Activity by an Anion Channel Inhibitor

4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of the volume-sensitive anion channel, was used to investigate the role of this channel in the stimulation of rat pancreatic β-cells by glucose and by tolbutamide. Glucose-stimulated electrical activity in β-cells was markedly and reversibly inhibited by DIDS. The increase in cytosolic [Ca²⁺] and stimulated insulin release evoked by glucose were also inhibited by DIDS. In contrast to its inhibitory effect on glucose-induced β-cell activity, DIDS had no effect on electrical activity, the rise in [Ca²⁺] _i or insulin release induced by tolbutamide. DIDS failed to increase β-cell input conductance, an index of whole-cell K _ATP channel activity, or the rate of efflux of ⁸⁶Rb⁺ from perifused islets, a measure of net K⁺ permeability. Furthermore, DIDS had no effect on intracellular pH or on regulatory volume increase following exposure of cells to hypertonic solutions, indicating that the effects of DIDS were not the result of inhibition of Cl⁻ transport systems. It is suggested that the DIDS-induced repolarization is caused by inactivation of the volume-sensitive anion channel. The stimulation of β-cell electrical and secretory activity by glucose, but not tolbutamide, may therefore involve activation of the anion channel. Received: 30 November 1999/Revised: 23 June 2000     

Methylglyoxal Causes Swelling and Activation of a Volume-Sensitive Anion Conductance in Rat Pancreatic β-Cells

Membrane potential and whole-cell current were studied in rat pancreatic β-cells using the `perforated patch' technique and cell volume measured by a video-imaging method. Exposure of β-cells to the α-ketoaldehyde methylglyoxal (1 mm) resulted in depolarization and electrical activity. In cells voltage-clamped at −70 mV, this effect was accompanied by the development of inward current noise. In voltage-pulse experiments, methylglyoxal activated an outwardly rectifying conductance which was virtually identical to the volume-sensitive anion conductance previously described in these cells. Two inhibitors of this conductance, 4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), also inhibited the depolarization and inward current evoked by methylglyoxal. Methylglyoxal increased β-cell volume to a relative value of 1.33 after 10 min with a gradual return towards basal levels following withdrawal of the α-ketoaldehyde. None of the effects of methylglyoxal was observed in response to t-butylglyoxal which, unlike methylglyoxal, is a poor substrate for the glyoxalase pathway. Methylglyoxal had no apparent effect on β-cell K⁺ channel activity. It is suggested that the metabolism of methylglyoxal to d-lactate causes β-cell swelling and activation of the volume-sensitive anion channel, leading to depolarization. These findings could be relevant to the stimulatory action of d-glucose, the metabolism of which generates significant quantities of l-lactate. Received: 15 May 1998/Revised: 25 September 1998