Ca-independent effects of BAPTA and EGTA on single-channel Cl currents in brown adipocytes

The Cl⁻ channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl⁻ channels (ORCC). To study tentative Ca²⁺ regulation of these channels, we attempted to control Ca²⁺ levels at the cytoplasmic side of the inside-out membrane patches with Ca²⁺-chelating agents. However, we found that the commonly used Ca²⁺-chelators EGTA and BAPTA by themselves influenced the Cl⁻ channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca²⁺-chelators (acting from the cytoplasmic side) on the single Cl⁻ channel currents in patch-clamp experiments. Using fixed (1-2 mM) concentrations of chelators, two types of Cl⁻ channels were identified, as discriminated by their reaction to the Ca²⁺-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca²⁺. The anionic classical Cl⁻ channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca²⁺ level control in themselves could fundamentally affect the behaviour of Cl⁻ channels.     

Ca(2+)-dependent and Ca(2+)-independent mechanisms modulate whole-cell cationic currents in human neutrophils.

We used whole-cell, voltage-clamp methodology to study the activation and inhibition of cationic currents in neutrophil. Cationic channels involved were impermeable to N-methyl-D-glucamine and to choline, but permeable to Na+, K+, Cs+, tris(hydroxymethyl)amino-ethane, and tetraethylammonium. N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the Ca(2+)-ionophore A23187, and phorbol myristate acetate activated the cationic current. Activated currents showed voltage dependence and outward rectification. The Ca(2+)-chelator 1,2 bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate markedly inhibited A23187-induced currents, but only partially decreased phorbol ester- or chemoattractant-induced currents. Dibutyryl cAMP diminished only the chemoattractant-induced currents. The adenosine analogs 5'N-ethylcarboxamidoadenosine and N6-cyclohexyladenosine blocked the currents induced by all agents. Thus, we conclude that activation and inhibition of cationic channels in human neutrophils involve both Ca(2+)-dependent and Ca(2+)-independent mechanisms.     

Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage

Ca²⁺-activatedCl currents (I_Cl,Ca) wereexamined using fluorescence confocal microscopy to monitorintracellular Ca²⁺ liberation evoked by flash photolysis ofcaged inositol 1,4,5-trisphosphate (InsP₃) involtage-clamped Xenopus oocytes. Currents at +40 mV exhibited asteep dependence on InsP₃ concentration([InsP₃]), whereas currents at140 mV exhibited a higher threshold and more graded relationshipwith [InsP₃]. Ca²⁺ levelsrequired to half-maximally activate I_Cl,Ca wereabout 50% larger at 140 mV than at +40 mV, and currents evokedby small Ca²⁺ elevations were reduced >25-fold. Thehalf-decay time of Ca²⁺ signals shortened at increasinglypositive potentials, whereas the decay of I_Cl,Calengthened. The steady-state current-voltage (I-V) relationshipfor I_Cl,Ca exhibited outward rectification withweak photolysis flashes but became more linear with stronger stimuli.Instantaneous I-V relationships were linear with both strongand weak stimuli. Current relaxations following voltage steps duringactivation of I_Cl,Ca decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at 160 mV. We conclude that InsP₃-mediated Ca²⁺liberation activates a single population of Clchannels, which exhibit voltage-dependent Ca²⁺ activationand voltage-independent instantaneous conductance.

     

Two bestrophins cloned from Xenopus laevis oocytes express Ca(2+)-activated Cl(-) currents

Ca2+-activated Cl- channels play important diverse roles from fast block to polyspermy to olfactory transduction, but their molecular identity has not been firmly established. By searching sequence databases with the M2 pore domain of ligand-gated anion channels, we identified potential Ca2+-activated Cl- channels, which included members of the bestrophin family. We cloned two bestrophins from Xenopus oocytes, which express high levels of Ca2+-activated Cl- channels. The Xenopus bestrophins were expressed in a variety of tissues. We predict that bestrophin has six transmembrane domains with the conserved RFP domain playing an integral part in ionic selectivity. When Xenopus bestrophins were heterologously expressed in human embryonic kidney-293 cells, large Ca2+-activated Cl- currents were observed. The currents are voltage- and time-independent, do not rectify, have a Kd for Ca2+ of approximately 210 nm, and exhibit a permeability ratio of I- > Br- > Cl- > aspartate. The W93C and G299E mutations produce non-functional channels that exert a dominant negative effect on wild type channels. We conclude that bestrophins are the first molecularly identified Cl- channels that are dependent on intracellular Ca2+ in a physiological range.     

Anion permeation in Ca(2+)-activated Cl(-) channels

Ca(2+)-activated Cl channels (Cl(Ca)Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca(2+)]. Here, we examine the mechanisms of anion permeation through Cl(Ca)Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl(Ca)Cs exhibited moderate selectivity for Cl over Na: P(Na)/P(Cl) = 0.1. The apparent affinity of Cl(Ca)Cs for Cl was low: K(d) = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E(rev) were as follows: C(CN)(3) > SCN > N(CN)(2) > ClO(4) > I > N(3) > Br > Cl > formate > HCO(3) > acetate = F > gluconate. The conductance sequence was as follows: N(3) > Br > Cl > N(CN)(2) > I > SCN > COOH > ClO(4) > acetate > HCO(3) = C(CN)(3) > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)(3) > SCN = ClO(4) > N(CN)(2) > I > N(3) > Br > HCO(3) > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl(Ca)Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca(2+) depended on the permeant anion at low [Ca(2+)] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca(2+). The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = approximately 9.2. The channel may be blocked by OH(-) ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl(Ca)Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl(Ca)C pore.     

Differences in Ca(2+) signaling underlie age-specific effects of secretagogues on colonic Cl(-) transport

Taurodeoxycholic acid (TDC) stimulates Cl transport inadult (AD), but not weanling (WN) and newborn (NB), rabbit colonic epithelial cells (colonocytes). The present study demonstrates thatstimuli like neurotensin (NT) are also age specific and identifies theage-dependent signaling step. Bile acid actions are segment and bileacid specific. Thus although TDC and taurochenodeoxycholate stimulateCl transport in AD distal but not proximal colon,taurocholate has no effect in either segment. TDC increasesintracellular Ca²⁺ concentration([Ca²⁺]_i) in AD, but not in WN and NB,colonocytes. In AD cells, TDC (5 min) action on Cltransport needs intra- but not extracellular Ca²⁺. NT,histamine, and bethanechol increase Cl transport and[Ca²⁺]_i in AD, but not WN, distalcolonocytes. However, A-23187 increased [Ca²⁺]_i and Cl transport in allage groups, suggesting that Ca²⁺-sensitive Cltransport is present from birth. Study of the proximal steps inCa²⁺ signaling revealed that NT, but not TDC, activates aGTP-binding protein, G_q, in AD and WN cells. Inaddition, although WN and AD colonocytes had similar levels ofphosphatidylinositol 4,5-bisphosphate, NT and TDC increased1,4,5-inositol trisphosphate content only in AD cells.Nonresponsiveness of WN cells to Ca²⁺-dependent stimuli,therefore, is due to the absence of measurable phospholipase Cactivity. Thus delays in Ca²⁺ signaling afford a crucialprotective mechanism to meet the changing demands of the developing colon.

     

Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals

Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.     

Ca(2+)-activated Cl(-) current in sheep lymphatic smooth muscle

Freshly dispersed sheep mesenteric lymphaticsmooth muscle cells were studied at 37°C using the perforatedpatch-clamp technique with Cs⁺- and K⁺-filledpipettes. Depolarizing steps evoked currents that consisted ofL-type Ca²⁺ [I_Ca(L)]current and a slowly developing current. The slow current reversed at1 ± 1.5 mV with symmetrical Cl concentrationscompared with 23.2 ± 1.2 mV (n = 5) and34.3 ± 3.5 mV (n = 4) when externalCl was substituted with either glutamate (86 mM) orI (125 mM). Nifedipine (1 µM) blocked and BAY K 8644 enhanced I_Ca(L), the slow-developing sustainedcurrent, and the tail current. The Cl channel blockeranthracene-9-carboxylic acid (9-AC) reduced only the slowly developinginward and tail currents. Application of caffeine (10 mM) tovoltage-clamped cells evoked currents that reversed close to theCl equilibrium potential and were sensitive to 9-AC.Small spontaneous transient depolarizations and larger actionpotentials were observed in current clamp, and these were blocked by9-AC. Evoked action potentials were triphasic and had a prominentplateau phase that was selectively blocked by 9-AC. Similarly, fluidoutput was reduced by 9-AC in doubly cannulated segments ofspontaneously pumping sheep lymphatics, suggesting that theCa²⁺-activated Cl current plays an importantrole in the electrical activity underlying spontaneous activity in this tissue.

     

Ca(2+)- and phospholipase D-dependent and -independent pathways activate mTOR signaling

The mammalian target of rapamycin (mTOR) promotes increased protein synthesis required for cell growth. It has been suggested that phosphatidic acid, produced upon activation of phospholipase D (PLD), is a common mediator of growth factor activation of mTOR signaling. We used Rat-1 fibroblasts expressing the alpha(1A) adrenergic receptor to study if this G(q)-coupled receptor uses PLD to regulate mTOR signaling. Phenylephrine (PE) stimulation of the alpha(1A) adrenergic receptor induced mTOR autophosphorylation at Ser2481 and phosphorylation of two mTOR effectors, 4E-BP1 and p70 S6 kinase. These PE-induced phosphorylations were greatly reduced in cells depleted of intracellular Ca(2+). PE activation of PLD was also inhibited in Ca(2+)-depleted cells. Incubation of cells with 1-butanol to inhibit PLD signaling attenuated PE-induced phosphorylation of mTOR, 4E-BP1 and p70 S6 kinase. By contrast, platelet-derived growth factor (PDGF)-induced phosphorylation of these proteins was not blocked by Ca(2+) depletion or 1-butanol treatment. These results suggest that the alpha(1A) adrenergic receptor promotes mTOR signaling via a pathway that requires an increase in intracellular Ca(2+) and activation of PLD. The PDGF receptor, by contrast, appears to activate mTOR by a distinct pathway that does not require Ca(2+) or PLD.     

Pyrimidinoceptors-mediated activation of Ca(2+)-dependent Cl(-) conductance in mouse endometrial epithelial cells

Previous studies have demonstrated the activation of endometrial Cl(-) secretion through P(2Y2) (P(2U)) purinoceptors by extracellular ATP. The present study further explored the presence of pyrimidine-sensitive receptors in the primary cultured mouse endometrial epithelial cells using the short-circuit current (I(SC)) and whole-cell patch-clamp techniques. UDP induced a transient increase in I(SC) in a concentration-dependent manner (EC(50) approximately 8.84 microM). The UDP-induced I(SC) was abolished after pretreating the epithelia with a calcium chelator, 1, 2-bis-(2-aminophenoxy)-ethane-N,N,N'N'tetraacetic acid-acetomethyl ester (BAPTA-AM), suggesting the dependence of the I(SC) on cytosolic free Ca(2+). The type of receptor involved was studied by cross-desensitization between ATP and UDP. ATP or UDP desensitized its subsequent I(SC) response. However, when ATP was added after UDP, or vice versa, a second I(SC) response was observed, indicating the activation of distinct receptors, possibly pyrimidine-sensitive receptors in addition to P(2Y2) (P(2U)) receptors. Similar results were observed in the patch-clamp experiments where UDP and ATP were shown to sequentially activate whole-cell current in the same cell. The UDP-activated whole-cell current exhibited outward rectification with delay activation and inactivation at depolarizing and hyperpolarizing voltages, respectively. In addition, the UDP-evoked whole-cell current reversed near the equilibrium potential of Cl(-) in the presence of a Cl(-) gradient across the membrane, and was sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), indicating the activation of Ca(2+)-activated Cl(-) conductance. These characteristics were very similar to that of the ATP-activated whole-cell current. Taken together, our findings indicate the presence of distinct receptors, pyrimidinoceptors and P(2Y2) (P(2U)) receptors in mouse endometrial epithelial cells. These distinct receptors appear to converge on the same Ca(2+)-dependent Cl(-) channels.     

Expression of Ca(2+)-independent and Ca(2+)-dependent phospholipases A(2) and cyclooxygenases in human melanocytes and malignant melanoma cell lines

We provide novel evidence that human melanoma cell lines (M10, M14, SK-MEL28, SK-MEL93, 243MEL, 1074MEL, OCM-1, and COLO38) expressed, at mRNA and protein levels, either Ca(2+)-independent phospholipase A(2) (iPLA(2)) or cytosolic phospholipase A(2) (cPLA(2)) and its phosphorylated form. Normal human melanocytes contained the lowest levels of both PLA(2)s. Cyclooxygenase-1 and -2 (COX-1 and COX-2) were also expressed in cultured tumor cells as measured by Western blots. The most pronounced overexpression of iPLA(2) and COX-1 was found in two melanoma-derived cells, M14 and COLO38. Normal human melanocytes and the M10 melanoma cell line displayed no COX-2 expression. Using subcellular fractionation, Western blot and confocal microcopy analyses, in paradigmatic SK-MEL28 and SK-MEL93 cells we showed that iPLA(2), COX-1 and even cPLA(2) were equally located in the cytosol, membrane structures and perinuclear region while COX-2 was preferentially associated with the cytosol. Specific inhibitors of these three enzymes significantly reduced the basal proliferation rate either in melanocytes or in melanoma cell lines. These results, coupled with the inhibition of the cell proliferation by electroporation of melanoma cells with cPLA(2) or COX-2 antibodies, demonstrate that a possible correlation between PLA(2)-COX expression and tumor cell proliferation in the melanocytic system does exist. In addition, the high expression level of both PLA(2)s and COXs suggests that eicosanoids modulate cell proliferation and tumor invasiveness.     

Bestrophin 1 and 2 are components of the Ca(2+) activated Cl(-) conductance in mouse airways

Ca(2+) activated Cl(-) transport is found in airways and other organs and is abnormal in cystic fibrosis, polycystic kidney disease and infectious diarrhea. The molecular identity of Ca(2+) activated Cl(-) channels (CaCC) in the airways is still obscure. Bestrophin proteins were described to form CaCC and to regulate voltage gated Ca(2+) channels. The present Ussing chamber recordings on tracheas of bestrophin 1 knockout (vmd2(-/-)) mice indicate a reduced Cl(-) secretion when activated by the purinergic agonist ATP (0.1-1 muM). As two paralogs, best1 and best2, are present in mouse tracheal epithelium, we examined the contribution of each paralog to Ca(2+) activated Cl(-) secretion. In whole cell patch-clamp measurements on primary airway epithelial cells from vmd2(-/-) tracheas, ATP activated Cl(-) currents were reduced by 50%. Additional knockdown of mbest2 in vmd2(-/-) cells by short interfering RNA further suppressed ATP-induced Cl(-) currents down to 20% of that observed in cells from vmd2(+/+) animals. Moreover, RNAi-suppression of both mbest1 and mbest2 reduced CaCC in vmd2(+/+) cells. Direct activation of CaCC by increase of intracellular Ca(2+) was also reduced in whole cell recordings of vmd2(-/-) cells. These results clearly suggest a role of bestrophin 1 and 2 for Ca(2+) dependent Cl(-) secretion in mouse airways.     

Selective plasmalogen substrate utilization by thrombin-stimulated Ca(2+)-independent PLA(2) in cardiomyocytes

Thrombin stimulation of rabbit ventricular myocytes activates a membrane-associated, Ca(2+)-independent phospholipase A(2) (PLA(2)) capable of hydrolyzing plasmenylcholine (choline plasmalogen), plasmanylcholine (alkylacyl choline phospholipid), and phosphatidylcholine substrates. To identify the endogenous phospholipid substrates, we quantified the effects of thrombin stimulation on diradyl phospholipid mass and arachidonic acid and lysophospholipid production. Thrombin stimulation resulted in a selective decrease in arachidonylated plasmenylcholine, with no change in arachidonylated phosphatidylcholine. The decrease in arachidonylated plasmenylcholine was accompanied by an increase in plasmenylcholine species containing linoleic and linolenic acids at the sn-2 position. A decrease in arachidonylated plasmenylethanolamine was also observed after thrombin stimulation, with no concomitant change in arachidonylated phosphatidylethanolamine. Thrombin stimulation resulted in the selective production of lysoplasmenylcholine, with no increase in lysophosphatidylcholine content. There was no evidence for significant acetylation of lysophospholipids to form platelet-activating factor. Arachidonic acid released after thrombin stimulation was rapidly oxidized to prostacyclin. Thus thrombin-stimulated Ca(2+)-independent PLA(2) selectively hydrolyzes arachidonylated plasmalogen substrates, resulting in production of lysoplasmalogens and prostacyclin as the principal bioactive products.     

Low extracellular Ca(2+) activates a transient Cl(-) current in chicken ovarian granulosa cells

The effects of low Ca²⁺ on ion currents in hen ovariangranulosa cells were examined. A fast activating and inactivatingtransient outward current (TOC) and a slowly activating outward current (SOC) could be observed. In the presence of normal Ca²⁺concentration (2.5 mM) and with a holding potential of 80 mV, SOC wasactivated in all cells with command pulses more positive than 20 mV.In 2.5 mM Ca²⁺, TOC appeared in 10% of cells at thecommand pulse of +80 mV and in 60-85% of cells at +100 to +120mV. In low-Ca²⁺ solution and command potential of +80 mV(holding potential of 80 mV), the amplitude of TOC was enhanced incells that expressed it in normal Ca²⁺, and TOC appeared in43% of the cells that did not express it initially in normalCa²⁺. At both normal and low Ca²⁺ levels, TOCdecreased as the holding potential became more positive. TOC wasreduced in Cl-deficient solution and in the presence of5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl channelblocker. These findings suggest that chicken granulosa cells express aCa²⁺-inactivated TOC carried by Cl. Thiscurrent may serve as a signal for some of the reduced metabolic functions of granulosa cells associated with Ca²⁺ deficiency.

     

Ca(2+)- and volume-sensitive chloride currents are differentially regulated by agonists and store-operated Ca2+ entry

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca(2+)-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca(2+)-activated (I(Cl(Ca))) and volume-regulated (I(Cl, swell)) chloride currents. NPPB and DIDS more efficiently inhibited I(Cl(Ca)) and I(Cl, swell), respectively. Cell swelling caused by hypotonic solution invariably activated I(Cl, swell) while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling-induced I(Cl, swell), while its inactive analogue U-73343 had no effect. I(Cl(Ca)) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, I(Cl, swell) could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced I(Cl, swell) in a nonadditive manner, suggesting their convergence on a common pathway. I(Cl, swell) and I(Cl(Ca)) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated I(Cl(Ca)), but abolished I(Cl, swell), thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that I(Cl, swell) can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.     

Hydrogen peroxide activation of Ca(2+)-independent phospholipase A(2) in uterine stromal cells

In rat uterine stromal cells (U(III) cells), an oxidative stress induced by H(2)O(2) caused a dose-dependent release of arachidonic acid (AA) that was independent of intracellular Ca(2+) concentration and was not inhibited by Ca(2+)-dependent phospholipase A(2) (cPLA(2)) inhibitors, nor by protein kinase C (PKC) inhibitors or by PKC down-regulation. H(2)O(2) treatment did not impair AA esterification but significantly increased Ca(2+)-independent PLA(2) (iPLA(2)) activity. Since iPLA(2) specific inhibitor bromoenollactone almost completely suppressed the release of AA induced by H(2)O(2), we conclude that iPLA(2) activity represents the major mechanism by which H(2)O(2) increases the availability of non-esterified AA in U(III) cells. Moreover, PKC inhibitors sphingosine and calphostin C markedly potentiated the release of AA trigger by H(2)O(2), suggesting a regulatory mechanism of iPLA(2) by PKC that remains to be clarified.     

pH and external Ca(2+) regulation of a small conductance Cl(-) channel in kidney distal tubule

A single channel characterization of the Cl(-) channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl(-) channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca(2+) regulation of a small conductance Cl(-) channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability P(O). Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel P(O) with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca(2+) concentration, with a dose-dependent increase in channel activity as a function of the cis Ca(2+) concentration. It is concluded on the basis of these results that the small conductance Cl(-) channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca(2+).     

Alpha 1- and beta-adrenergic regulation of intracellular Ca2+ levels in brown adipocytes

In order to monitor changes in cytosolic Ca2+ levels, brown-fat cells were incubated with the fluorescent Ca2+-indicator fura-2 and the fluorescence intensity ratio followed. The addition of norepinephrine led to a rapid and persistent increase in the cytosolic Ca2+ level, which was dose-dependent with a maximal effect at about 1 microM. The response was diminished in the absence of extracellular Ca2+ and was inhibited more efficiently by phentolamine and prazosin than by propranolol or yohimbine, indicating alpha 1-adrenergic mediation. Accordingly, selective alpha 1-adrenergic stimulation also increased the cytosolic Ca2+ level. However, selective beta-adrenergic stimulation, as well as the adenylate cyclase activator forskolin, were also able to increase the cytosolic Ca2+ level in these cells to a certain extent. It was concluded that the major part of the increase in cytosolic Ca2+ was mediated, as in other cell types, via alpha 1-adrenergic receptors, but that Ca2+ levels were also positively modulated by a cAMP-mediated process. These observations are discussed in relation to known alpha 1/beta synergisms in brown adipose tissue.     

Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism

Coexpression of the beta(1) subunit with the alpha subunit (mSlo) of BK channels increases the apparent Ca(2+) sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca(2+) sensitivity requires Ca(2+), by comparing the gating in 0 Ca(2+)(i) of BK channels composed of alpha subunits to those composed of alpha+beta(1) subunits. The beta(1) subunit increased burst duration approximately 20-fold and the duration of gaps between bursts approximately 3-fold, giving an approximately 10-fold increase in open probability (P(o)) in 0 Ca(2+)(i). The effect of the beta(1) subunit on increasing burst duration was little changed over a wide range of P(o) achieved by varying either Ca(2+)(i) or depolarization. The effect of the beta(1) subunit on increasing the durations of the gaps between bursts in 0 Ca(2+)(i) was preserved over a range of voltage, but was switched off as Ca(2+)(i) was increased into the activation range. The Ca(2+)-independent, beta(1) subunit-induced increase in burst duration accounted for 80% of the leftward shift in the P(o) vs. Ca(2+)(i) curve that reflects the increased Ca(2+) sensitivity induced by the beta(1) subunit. The Ca(2+)-dependent effect of the beta(1) subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the beta(1) subunit are independent of Ca(2+)(i) suggests that the beta(1) subunit mainly alters the energy barriers of Ca(2+)-independent transitions. The changes in gating induced by the beta(1) subunit differ from those induced by depolarization, as increasing P(o) by depolarization or by the beta(1) subunit gave different gating kinetics. The complex gating kinetics for both alpha and alpha+beta(1) channels in 0 Ca(2+)(i) arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca(2+)(i).