Calcium mobilization evoked by hepatocellular swelling is linked to activation of phospholipase Cgamma

Recovery from swelling of hepatocytes and selected other epithelia is triggered by intracellular Ca(2+) release from the endoplasmic reticulum, which leads to fluid and electrolyte efflux through volume-sensitive K(+) and Cl(-) channels. The aim of this study was to determine the mechanisms responsible for swelling-mediated hepatocellular Ca(2+) mobilization. Swelling of HTC rat hepatoma cells, evoked by exposure to hypotonic medium, elicited transient increases in intracellular levels of inositol 1,4,5-trisphosphate (IP(3)) and cytosolic [Ca(2+)]. The latter was attenuated by inhibition of phospholipase C (PLC) with and by IP(3) receptor blockade with 2-aminoethoxydiphenyl borate, but it was unaffected by ryanodine, an inhibitor of intracellular Ca(2+)-induced Ca(2+) release channels. Hypotonic swelling was associated with a transient increase in tyrosine phosphorylation of PLCgamma, with kinetics that paralleled the increases in intracellular IP(3) levels and cytosolic [Ca(2+)]. Confocal imaging of HTC cells exposed to hypotonic medium revealed a swelling-induced association of tyrosine-phosphorylated PLCgamma with the plasma membrane. These findings suggest that activation of PLCgamma by hepatocellular swelling leads to the generation of IP(3) and stimulates discharge of Ca(2+) from the endoplasmic reticulum via activation of IP(3) receptors. By extension, these data support the concept that tyrosine phosphorylation of PLCgamma represents a critical step in adaptive responses to hepatocellular swelling.     

Swelling-induced, CFTR-independent ATP release from a human epithelial cell line: lack of correlation with volume-sensitive cl(-) channels.

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(-) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(-) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56-80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 microM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 microM. Glibenclamide (500 microM) and arachidonic acid (100 microM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(-) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(-) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach approximately 13 microM by a biosensor technique using P2X(2) receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl(-) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 microM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(-) channel; and third, the ATP release is not a prerequisite to activation of the Cl(-) channel.     

The role of P2Y1 purinergic receptors and cytosolic Ca2+ in hypotonically activated osmolyte efflux from a rat hepatoma cell line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca(2+) concentration ([Ca(2+)](i)) to increase transiently by approximately 90 nm. This rise in [Ca(2+)](i) was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca(2+)](i) increase. The hypotonically induced rise in [Ca(2+)](i) was prevented by the prior discharge of the intracellular Ca(2+) store of the cells by thapsigargin. Removal of extracellular Ca(2+) or inhibition of Ca(2+) influx by 1-10 microm Gd(3+) depleted the thapsigargin-sensitive Ca(2+) stores and thereby diminished the rise in [Ca(2+)](i). The hypotonically induced rise in [Ca(2+)](i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca(2+)](i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca(2+)](i) elicited by the addition of the P2Y(2) and P2Y(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y(1), P2Y(2), and P2Y(6) receptors. Inhibition of the hypotonically induced rise in [Ca(2+)](i) blocked hypotonically induced K(+) ((86)Rb(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl(-) ((125)I-) efflux. The interaction of extracellular ATP and/or ADP with P2Y(1) purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.     

Ca2+ nanodomain-mediated component of swelling-induced volume-sensitive outwardly rectifying anion current triggered by autocrine action of ATP in mouse astrocytes

The volume-sensitive outwardly rectifying (VSOR) anion channel provides a major pathway for anion transport during cell volume regulation. It is typically activated in response to cell swelling, but how the channel senses the swelling remains unclear. Meanwhile, we recently found that in mouse astrocytes the channel is activated by an inflammatory chemical mediator, bradykinin, without cell swelling and that the activation is regulated via high concentration regions of intracellular Ca(2+) ([Ca(2+)](i)) in the immediate vicinity of open Ca(2+)-permeable channels, so-called Ca(2+) nanodomains. Here we investigated whether a similar mechanism is involved in the swelling-induced VSOR channel activation in the astrocytes. A hypotonic stimulus (25% reduction in osmolality) caused the [Ca(2+)](i) rises in the astrocytes, and the rises were abolished in the presence of an ATP-degrading enzyme, apyrase (10 U/ml). Application of ATP (100 μM) under isotonic conditions generated the current through VSOR channels via Ca(2+) nanodomains, as bradykinin does. The current induced by the hypotonic stimulus was suppressed by ~40% in the Ca(2+)-depleted condition where the ATP-induced VSOR current was totally prevented. Thus the swelling-induced VSOR channel activation in mouse astrocytes is partly regulated via Ca(2+) nanodomains, whose generation is triggered by an autocrine action of ATP.     

ClC-2 chloride channels contribute to HTC cell volume homeostasis

Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.     

Ca2+-activated IK1 channels associate with lipid rafts upon cell swelling and mediate volume recovery

Restoration of cell volume in the continued presence of osmotic stimuli is essential, particularly in hepatocytes, which swell upon nutrient uptake. Responses to swelling involve the Ca2+-dependent activation of K+ channels, which promote fluid efflux to drive volume recovery; however, the channels involved in hepatocellular volume regulation have not been identified. We found that hypotonic exposure of HTC hepatoma cells evoked the opening of 50 pS K+-permeable channels, consistent with intermediate conductance (IK) channels. We isolated from rat liver and HTC cells a cDNA with sequence identity to the coding region of IK1. Swelling-activated currents were inhibited by transfection with a dominant interfering IK1 mutant. The IK channel blockers clotrimazole and TRAM-34 inhibited whole cell swelling-activated K+ currents and volume recovery. To determine whether IK1 underwent volume-sensitive localization, we expressed a green fluorescent protein fusion of IK1 in HTC cells. The localization of IK1 was suggestive of distribution in lipid rafts. Consistent with this, there was a time-dependent increase in colocalization between IK1 and the lipid raft ganglioside GM1 on the plasma membrane, which subsequently decreased with volume recovery. Pharmacological disruption of lipid rafts altered the plasma membrane distribution of IK1 and inhibited volume recovery after hypotonic exposure. Collectively, these findings support the hypothesis that IK1 regulates compensatory responses to hepatocellular swelling and suggest that regulation of cell volume involves coordination of signaling from lipid rafts with IK1 function.     

Activation of P2Y receptors causes strong and persistent shrinkage of C11-MDCK renal epithelial cells

Purinergic receptors activate diverse signaling cascades and regulate the activity of cell volume-sensitive ion transporters. However, the effects of ATP and other agonists of P2 receptors on cell volume dynamics are only scarcely studied. In the present work, we used the recently developed dual-image surface reconstruction technique to explore the influence of purinergic agonists on cell volume in the C11-Madin-Darby canine kidney cell line resembling intercalated cells from kidney collecting ducts. Unexpectedly, we found that ATP and UTP triggered very robust (55-60%) cell shrinkage that lasted up to 2 h after agonist washout. Purinergic regulation of cell volume required increases in intracellular Ca(2+) and could be partially mimicked by the Ca(2+)-ionophore ionomycin or activation of protein kinase C by 4β-phorbol 12-myristate 13-acetate. Cell shrinkage was accompanied by strong reductions in intracellular K(+) and Cl(-) content measured using steady-state (86)Rb(+) and (36)Cl(-) distribution. Both shrinkage and ion efflux in ATP-treated cells were prevented by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and by the BK(Ca) channel inhibitors charybdotoxin, iberiotoxin, and paxilline. To evaluate the significance of cell-volume changes in purinergic signaling, we measured the impact of ATP on the expression of the immediate-early gene c-Fos. Thirty-minute treatment with ATP increased c-Fos immunoreactivity by approximately fivefold, an effect that was strongly inhibited by charybdotoxin and completely prevented by NPPB. Overall, our findings suggest that ATP-induced cell-volume changes are partially responsible for the physiological actions of purinergic agonists.     

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPβS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αβMeATP, and βγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.     

Purinergic activation of Ca2+-permeable TRPV4 channels is essential for mechano-sensitivity in the aldosterone-sensitive distal nephron

Mechanical forces are known to induce increases of [Ca(2+)](i) in the aldosterone-sensitive distal nephron (ASDN) cells to regulate epithelial transport. At the same time, mechanical stress stimulates ATP release from ASDN cells. In this study, we combined ratiometric Fura-2 based monitoring of [Ca(2+)](i) in freshly isolated split-opened ASDN with targeted deletion of P2Y2 and TRPV4 in mice to probe a role for purinergic signaling in mediating mechano-sensitive responses in ASDN cells. ATP application causes a reproducible transient Ca(2+) peak followed by a sustained plateau. Individual cells of the cortical collecting duct (CCD) and the connecting tubule (CNT) respond to purinergic stimulation with comparative elevations of [Ca(2+)](i). Furthermore, ATP-induced Ca(2+)-responses are nearly identical in both principal (AQP2-positive) and intercalated (AQP2-negative) cells as was confirmed using immunohistochemistry in split-opened ASDN. UTP application produces elevations of [Ca(2+)](i) similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca(2+)](i) response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca(2+) responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca(2+) appeared to contribute to the generation of ATP-induced Ca(2+) response in ASDN cells. Importantly, flow- and hypotonic-induced Ca(2+) elevations are markedly blunted in P2Y2 -/- mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca(2+)](i) elevation during purinergic stimulation. Consistent with this, ATP-induced Ca(2+) plateau are dramatically attenuated in TRV4 -/- mice. Inhibition of TRPC channels with 10 μM BTP2 also decreased ATP-induced Ca(2+) plateau whilst to a lower degree than that observed with TRPV4 inhibition/genetic deletion. We conclude that stimulation of purinergic signaling by mechanical stimuli leads to activation of TRPV4 and, to a lesser extent, TRPCs channels, and this is an important component of mechano-sensitive response of the ASDN.     

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.     

Ca2+ signaling in mouse mesenteric small arteries: myogenic tone and adrenergic vasoconstriction

Arteries that have developed myogenic tone (MT) are in a markedly different physiological state compared with those that have not, with higher cytosolic [Ca(2+)] and altered activity of several signal transduction pathways. In this study, we sought to determine whether alpha(1)-adrenoceptor-induced Ca(2+) signaling is different in pressurized arteries that have spontaneously developed MT (the presumptive physiological state) compared with those that have not (a common experimental state). At 32 degrees C and intraluminal pressure of 70 mmHg, cytoplasmic [Ca(2+)] was steady in most smooth muscle cells (SMCs). In a minority of cells (34%), however, at least one propagating Ca(2+) wave occurred. alpha(1)-Adrenoceptor activation (phenylephrine, PE; 0.1-10.0 microM) caused strong vasoconstriction and markedly increased the frequency of Ca(2+) waves (in virtually all cells). However, when cytosolic [Ca(2+)] was elevated experimentally in these arteries ([K(+)] 20 mM), PE failed to elicit Ca(2+) waves, although it did elevate [Ca(2+)] (F/F(0)) further and caused further vasoconstriction. During development of MT, the cytosolic [Ca(2+)] (F/F(0)) in individual SMCs increased, Ca(2+) waves disappeared (from SMCs that had them), and small Ca(2+) ripples (frequency approximately 0.05 Hz) appeared in approximately 13% of cells. PE elicited only spatially uniform increases in [Ca(2+)] and a smaller change in diameter (than in the absence of MT). Nevertheless, when cytosolic [Ca(2+)] and MT were decreased by nifedipine (1 microM), PE did elicit Ca(2+) waves. Thus alpha(1)-adrenoceptor-mediated Ca(2+) signaling is markedly different in arteries with and without MT, perhaps due to the elevated [Ca(2+)], and may have a different molecular basis. alpha(1)-Adrenoceptor-induced vasoconstriction may be supported either by Ca(2+) waves or by steady elevation of cytoplasmic [Ca(2+)], depending on the amount of MT.     

Parathyroid hormone-activated volume-sensitive calcium influx pathways in mechanically loaded osteocytes

This paper documents for the first time a volume-sensitive Ca(2+) influx pathway in osteocytes, which transmits loading-induced signals into bone formation. Stretch loading by swelling rat and chicken osteocytes in hypo-osmotic solution induced a rapid and progressive increase of cytosolic calcium concentration, [Ca(2+)](i). The influx of extracellular Ca(2+) explains the increased [Ca(2+)](i) that paralleled the increase in the mean cell volume. Gadolinium chloride (Gd(3+)), an inhibitor of stretch- activated cation channels, blocked the [Ca(2+)](i) increase caused by hypotonic solutions. Also, the expression of alpha1C subunit of voltage-operated L-type Ca(2+) channels (alpha1C) is required for the hypotonicity-induced [Ca(2+)](i) increase judging from the effect of alpha1C antisense oligodeoxynucleotides. Parathyroid hormone (PTH) specifically potentiated the hypotonicity-induced [Ca(2+)](i) increase in a dose-dependent manner through the activation of adenyl cyclase. The increases induced by both PTH and hypotonicity were observed primarily in the processes of the osteocytes. In cyclically stretched osteocytes on flexible-bottomed plates, PTH also synergistically elevated the insulin-like growth factor-1 mRNA level. Furthermore, Gd(3+) and alpha1C antisense significantly inhibited the stretch-induced insulin-like growth factor-1 mRNA elevation. The volume-sensitive calcium influx pathways of osteocytes represent a mechanism by which PTH potentiates mechanical responsiveness, an important aspect of bone formation.     

Interaction of luminal calcium and cytosolic ATP in the control of type 1 inositol (1,4,5)-trisphosphate receptor channels

Ca(2+) within intracellular stores (luminal Ca(2+)) is believed to play a role in regulating Ca(2+) release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P(3))-gated Ca(2+) channel (or Ins(1,4,5)P(3) receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P(3) receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca(2+)] using K(+) as the current carrier. At a high luminal [Ca(2+)] and in the presence of optimal [Ins(1,4,5)P(3)] and cytosolic [Ca(2+)], a short burst of Ins(1,4,5)P(3) receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca(2+)] caused the channel to reactivate indefinitely. At luminal [Ca(2+)], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca(2+)] caused reactivation. We provide evidence that luminal Ca(2+) is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca(2+) overload is prevented when the energy state of the cell is compromised.     

Streptomycin and its analogues are potent inhibitors of the hypotonicity-induced Ca2+ entry and Cl- channel activity

Streptomycin is a common antibiotic used in culture media. It is also a known blocker of stretch-activated and mechanosensitive ion channels in neurons and cardiac myocytes. But very little information is available on its effect in the regulation of epithelial ion channels. Osmotic swelling is a kind of mechanical stretch. The opening of stretch-activated Ca(2+) channels contributes to hypotonicity-induced Ca(2+) influx which is necessary for the activation of volume-regulated Cl(-) channels in human cervical cancer cells. This study aimed to investigate the role of streptomycin in cell volume regulation. Treatment of cervical cancer SiHa cells with streptomycin and its analogues (gentamicin and netilmicin) did not affect the basal cytosolic Ca(2+) ([Ca(2+)](i)) level. But it attenuated the hypotonicity-stimulated increase of [Ca(2+)](i) in a dose-dependent manner with half-maximal inhibitory concentrations (IC(50)) of 25, 90 and 200 microM for streptomycin, gentamicin and netilmicin, respectively, when measured at room temperature. In contrast, under free extracellular Ca(2+) condition, hypotonic stress only induced a small, progressive increase of [Ca(2+)](i), while 500 microM streptomycin did not affect this Ca(2+) signaling. Streptomycin and its analogues (gentamicin and netilmicin) also inhibited the activation of volume-regulated Cl(-) channels in a dose-dependent manner with IC(50) of 30, 95 and 250 microM at room temperature, respectively. Chronic culture with 50 microM streptomycin downregulates the activity of volume-regulated Cl(-) channels and retards the process of regulatory volume decrease in SiHa cells and MDCK cells. We suggest that using cells chronically cultured with streptomycin to study epithelial ion channels risks studying cellular and molecular pathology rather than physiology.     

Intracellular signalling involved in activation of the volume-sensitive K+ current in Ehrlich ascites tumour cells

The cell swelling-activated K+ channel in Ehrlich ascites tumour cells has a conductance of 5 pS estimated from noise analysis of the volume-sensitive whole-cell K+ current (I(K,vol)). I(K,vol) exhibits Goldman-Hodgkin-Katz type behaviour and is insensitive to clotrimazole, apamin and charybdotoxin (ChTX), but inhibited by clofilium. Its small conductance, lack of intrinsic voltage-dependence and peculiar pharmacological profile are similar to properties described for the two-pore domain background K+ TASK channels. Neither Ca2+ nor ATP work as initiators in the activation of I(K,vol). In contrast, several investigations in Ehrlich cells suggest an important role for leukotriene D4 (LTD4) in the activation of I(K,vol). Under isotonic conditions, LTD4 activates Ca2+-dependent, ChTX-sensitive K+ channels as well as Ca2+-independent. ChTX-insensitive K+ channels. The LTD4-activated, ChTX-insensitive K+ current exhibits a current-voltage relation, pharmacological profile and single channel conductance similar to that of I(K,vol), indicating that LTD4 is the signalling molecule responsible for activation of the volume-sensitive K+ channels in Ehrlich cells. Hypotonic swelling of Ehrlich cells results in translocation of the 85-kDa cytosolic (c) PLA2alpha to the nucleus where it is activated. This activation leads to an increase in arachidonic acid release followed by an increased release of leukotrienes, and is essential in cell swelling-induced activation of I(K,vol) and of the organic osmolyte channels.     

A new mode of Ca2+ signaling by G protein-coupled receptors: gating of IP3 receptor Ca2+ release channels by Gbetagamma

The most common form of Ca(2+) signaling by Gq-coupled receptors entails activation of PLCbeta2 by Galphaq to generate IP(3) and evoke Ca(2+) release from the ER. Another form of Ca(2+) signaling by G protein-coupled receptors involves activation of Gi to release Gbetagamma, which activates PLCbeta1. Whether Gbetagamma has additional roles in Ca(2+) signaling is unknown. Introduction of Gbetagamma into cells activated Ca(2+) release from the IP(3) Ca(2+) pool and Ca(2) oscillations. This can be due to activation of PLCbeta1 or direct activation of the IP(3)R by Gbetagamma. We report here that Gbetagamma potently activates the IP(3) receptor. Thus, Gbetagamma-triggered [Ca(2+)](i) oscillations are not affected by inhibition of PLCbeta. Coimmunoprecipitation and competition experiments with Gbetagamma scavengers suggest binding of Gbetagamma to IP(3) receptors. Furthermore, Gbetagamma inhibited IP(3) binding to IP(3) receptors. Notably, Gbetagamma activated single IP(3)R channels in native ER as effectively as IP(3). The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca(2+) signals evoked by Gi- and Gq-coupled receptors to Gbetagamma scavenging and PLCbeta inhibition. We propose that gating of IP(3)R by Gbetagamma is a new mode of Ca(2+) signaling with particular significance for Gi-coupled receptors.     

ATP induces intracellular calcium increases and actin cytoskeleton disaggregation via P2x receptors

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.     

Disturbances in purinergic [Ca2+]i signaling pathways in a transformed rat thyroid cell line

It was previously shown that in rat thyroid PC-Cl3 cell line, a purinergic P2Y receptor increases the concentration of free cytosolic Ca(2+) ([Ca(2+)](i)) via phospholipase C activation. We here studied whether in a transformed cell line (PC-E1Araf) derived from parental PC-Cl3 cells, ATP is still able to transduce the [Ca(2+)](i)-based intracellular signal.We demonstrate the expression of mRNA for P2Y2 in both PC-Cl3 and PC-E1Araf cells; mRNAs for P2Y1, P2Y4, P2Y6 and P2Y11 were absent. In both cell lines activation of P2Y2 receptor provokes a transient increase in [Ca(2+)](i) followed by a lower sustained phase persisting for over 5min in PC-Cl3 and only 1.5 min in PC-E1Araf cells. In both cell lines the [Ca(2+)](i) reached a plateau level significantly higher than the basal [Ca(2+)](i) level persisting for over 10 min. Removal of extracellular Ca(2+) reduced the initial transient response to ATP in PC-Cl3, but not in PC-E1Araf cells, and completely abolished the plateau phase in both cell lines.In the presence of extracellular Ca(2+) thapsigargin (TG) caused a rise in [Ca(2+)](i) significantly higher in PC-Cl3 than transformed PC-E1Araf cells, while in Ca(2+)-free medium the effect of TG was similar in both cell lines. The capacitative Ca(2+)-entry in PC-Cl3 resulted significantly higher than in PC-E1Araf cells.Further studies were performed in order to investigate whether the different effects of ATP on [Ca(2+)](i) was due to variation in divalent cation plasma membrane permeability. PC-E1Araf cells showed a much lower permeability to Ca(2+), Ba(2+), Sr(2+), Mn(2+), and Co(2+) that may be responsible for the differences in purinergic Ca(2+) signaling pathway with respect to parental PC-Cl3 cells.     

Autocrine regulation of volume-sensitive anion channels in airway epithelial cells by adenosine

The activity of volume-sensitive Cl- channels was studied in human tracheal epithelial cells (9HTEo-) by taurine efflux experiments. The efflux elicited by a hypotonic shock was partially inhibited by adenosine receptor antagonists, by alpha,beta-methyleneadenosine 5'-diphosphate (alphabetaMeADP), an inhibitor of the 5'-ectonucleotidase, and by adenosine deaminase. On the other hand, dipyridamole, a nucleoside transporter inhibitor, increased the swelling-induced taurine efflux. Extracellular ATP and adenosine increased taurine efflux by potentiating the effect of hypotonic shock. alphabetaMeADP strongly inhibited the effect of extracellular ATP but not that of adenosine. These results suggest that anion channel activation involves the release of intracellular ATP, which is then degraded to adenosine by specific ectoenzymes. Adenosine then binds to purinergic receptors, causing the activation of the channels. To directly demonstrate ATP efflux, cells were loaded with [3H]AMP, and the release of radiolabeled molecules was analyzed by high performance liquid chromatography. During hypotonic shock, cell supernatants showed the presence of ATP, ADP, and adenosine. alphabetaMeADP inhibited adenosine formation and caused the appearance of AMP. Under hypotonic conditions, elevation of intracellular Ca2+ by ionomycin caused an increase of ATP and adenosine in the extracellular solution. Our results demonstrate that volume-sensitive anion channels are regulated with an autocrine mechanism involving swelling-induced ATP release and then hydrolysis to adenosine.