Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore

The effects ofmaitotoxin (MTX) on plasmalemma permeability are similar to thosecaused by stimulation of P2Z/P2X₇ionotropic receptors, suggesting that1) MTX directly activatesP2Z/P2X₇ receptors or2) MTX andP2Z/P2X₇ receptor stimulationactivate a common cytolytic pore. To distinguish between these twopossibilities, the effect of MTX was examined in1) THP-1 monocytic cells before andafter treatment with lipopolysaccharide and interferon-, a maneuverknown to upregulate P2Z/P2X₇receptor, 2) wild-type HEK cells andHEK cells stably expressing theP2Z/P2X₇ receptor, and3) BW5147.3 lymphoma cells, a cellline that expresses functional P2Z/P2X₇ channels that are poorlylinked to pore formation. In control THP-1 monocytes, addition of MTXproduced a biphasic increase in the cytosolic freeCa²⁺ concentration([Ca²⁺]_i);the initial increase reflects MTX-inducedCa²⁺ influx, whereas the secondphase correlates in time with the appearance of large pores and theuptake of ethidium. MTX produced comparable increases in[Ca²⁺]_iand ethidium uptake in THP-1 monocytes overexpressing theP2Z/P2X₇ receptor. In bothwild-type HEK and HEK cells stably expressing theP2Z/P2X₇ receptor, MTX-inducedincreases in[Ca²⁺]_iand ethidium uptake were virtually identical. The response of BW5147.3cells to concentrations of MTX that produced large increases in[Ca²⁺]_ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- andBz-ATP-induced pores activate with similar kinetics and exhibit similarsize exclusion. Last, MTX-induced pore formation, but not channelactivation, is greatly attenuated by reducing the temperature to22°C, a characteristic shared by theP2Z/P2X₇-induced pore. Together,the results demonstrate that, although MTX activates channels that aredistinct from those activated byP2Z/P2X₇ receptor stimulation, thecytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

     

Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7 channel

Extracellular ATP is known to trigger apoptosis of thymocytesand lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel areCa²⁺ (andBa²⁺) selective but also passmolecules up to the size of ethidium cation (314 Da).RT-PCR showed identity between lymphocyte P2Z and thehP2X₇ gene recently cloned fromhuman monocytes. When human leukemic B lymphocytes were incubated withATP and¹³³Ba²⁺,an immediate influx of isotope occurred. It was augmented by 45% whenATP was added 10 min before isotope. Time-resolved flow cytometry wasused to examine kinetics of ethidium uptake in cells incubated withBzATP or the partial agonists ATP, 2-methylthioadenosine 5'-triphosphate, or adenosine5'-O-(3-thiotriphosphate).Maximally effective concentrations of BzATP (50 µM) induced immediateuptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition ofmaximally effective ATP concentrations (500 µM) at 37°C, and thedelay was longer at 24°C. ATP addition 2-10 min beforeethidium abolished the delay. The delay was longer with other partialagonists and inversely related to maximal flux produced by agonist. Adelay was also observed for submaximal BzATP concentrations (10-20µM). P2Z/P2X₇ inhibitors, KN-62and5-(N,N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened thedelay. The results support a model in which agonists forP2Z/P2X₇ receptor mediate animmediate channel opening allowing passage of small inorganic cations,followed by a slow further permeability increase allowing passage oflarger permeant cations like ethidium. The rate of the second stepdepends on time and temperature and the efficacy and concentration ofagonist and is slowed by antagonists, suggesting it depends on thefraction of P2Z/P2X₇ channels held in the initial openstate.

     

A P2X7 receptor stimulates plasma membrane trafficking in the FRTL rat thyrocyte cell line

Thyroid cells express a variety of P2Y and P2X purinergic receptor subtypes. G protein-coupled P2Y receptors influence a wide variety of thyrocyte-specific functions; however, functional P2X receptor-gated channels have not been observed. In this study, we used whole cell patch-clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line FRTL. We found a cation-selective current that was gated by ATP and 2',3'-O-(4-benzoylbenzoyl)-ATP but not by UTP. The ATP-evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, adenosine 5'-triphosphate-2',3'-dialdehyde, 100 µM Zn²⁺, and 50 µM Cu²⁺. Fluorescence imaging revealed pronounced, temperature-sensitive stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC₅₀ for ATP stimulation of internalization was 440 µM in saline containing 2 mM Ca²⁺ and 2 mM Mg²⁺, and 33 µM in low-Mg²⁺, nominally Ca²⁺-free saline. Overall, the results are most consistent with activation of a P2X₇ receptor by ATP^4–. However, low permeability to N-methyl-D-glucamine⁺ and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X₇ receptor activation. ATP stimulation of internalization occurs in Na⁺-free, Ca²⁺-free, or low-Mg²⁺ saline and therefore does not depend on cation influx through the ATP-gated channel. We conclude that ATP activation of a P2X₇ receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx. purinergic receptor; internalization; patch clamp     

N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X(7) receptors

Activation of membrane P2X₇ receptors by extracellular ATP [or its analog 2',3'-O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-D-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-D-glucamine and YO-PRO-1 enter through a common permeation pathway, the "dilating" P2X₇ receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2X₇ receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-D-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2X₇ receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-D-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-D-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway. ATP; cation channel; permeability; quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide     

Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors

Lymphocytes from normal subjects and patients with B-chroniclymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X₇ receptor (previously termedP2Z). These responses include opening of a cation-selectivechannel/pore that allows entry of the fluorescent dye ethidium andactivation of a membrane metalloprotease that sheds the adhesionmolecule L-selectin. The surface expression of P2X₇receptors was measured in normal leucocytes, platelets, and B-CLLlymphocytes and correlated with their functional responses. Monocytesshowed four- to fivefold greater expression of P2X₇ than B,T, and NK lymphocytes, whereas P2X₇ expression onneutrophils and platelets was weak. All cell types demonstratedabundant intracellular expression of this receptor. All 12 subjectswith B-CLL expressed lymphocyte P2X₇ at about the samelevel as B lymphocytes from normal subjects. P2X₇ function, measured by ATP-induced uptake of ethidium, correlated closely withsurface expression of this receptor in normal and B-CLL lymphocytes andmonocytes (n = 47, r = 0.70; P< 0.0001). However, in three patients the ATP-induced uptake ofethidium into the malignant B lymphocytes was low or absent. The lackof P2X₇ function in these B lymphocytes was confirmed bythe failure of ATP to induce Ba²⁺ uptake into theirlymphocytes. This lack of function of the P2X₇ receptorresulted in a failure of ATP-induced shedding of L-selectin, anadhesion molecule that directs the recirculation of lymphocytes fromblood into the lymph node.

     

Glu496Ala polymorphism of human P2X7 receptor does not affect its electrophysiological phenotype

A glutamateto alanine exchange at amino acid position 496 of the humanP2X₇ receptor was recently shown to be associated with aloss of function in human B lymphocytes in terms of ATP-induced ethidium⁺ uptake, Ba²⁺ influx, and induction ofapoptosis (Gu BJ, Zhang WY, Worthington RA, Sluyter R, Dao-UngP, Petrou S, Barden JA, and Wiley JS. J Biol Chem 276:11135-11142, 2001). Here we analyzed the effect of theGlu⁴⁹⁶ to Ala exchange on the channel properties of thehuman P2X₇ receptor expressed in Xenopus oocyteswith the two-microelectrode voltage-clamp technique. The amplitudes ofATP-induced whole cell currents characteristic of functionalexpression, kinetic properties including ATP concentration dependence,and permeation behavior were not altered by this amino acid exchange.Also in HEK293 cells, the Ala⁴⁹⁶ mutant mediated typicalP2X₇ receptor-dependent currents like the parentGlu⁴⁹⁶ hP2X₇ receptor. Because the function ofthe P2X₇ receptor as an ATP-gated channel for small cationsincluding Ba²⁺ remained unaffected by this mutation, weconclude that Glu⁴⁹⁶ plays a critical role in poreformation but does not determine the ion channel properties of thehuman P2X₇ receptor.

     

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

The plasmalemmal Na⁺-K⁺-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca²⁺. However, the ability of PTX to directly increase cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Na⁺ pump channels and to initiate Ca²⁺ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca²⁺]_i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca²⁺]_i that was dependent on extracellular Ca²⁺. The increase in [Ca²⁺]_i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC₅₀ < 1 µM. The elevation in [Ca²⁺]_i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca²⁺]_i occurs via the Na⁺ pump. Subsequent to the rise in [Ca²⁺]_i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca²⁺ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca²⁺ overload. necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2     

P2Z purinoceptor-associated pores induced by extracellular ATP in macrophages and J774 cells

Millimolarconcentrations of extracellular ATP(ATP_o) can induce thepermeabilization of plasma membranes of macrophages and other bonemarrow-derived cells to low-molecular-weight solutes, a phenomenon thatis the hallmark of P2Z purinoceptors. However, patch-clamp and wholecell electrophysiological experiments have so far failed to demonstratethe existence of any ATP_o-induced P2Z-associated pores underlying this permeabilization phenomenon. Here,we describe ATP_o-induced pores of409 ± 33 pS recorded using cell-attached patch-clamp experimentsperformed in macrophages and J774 cells. These pores are voltagedependent and display several properties of the P2Z-associatedpermeabilization phenomenon: they are permeable to both large cationsand anions, such as tris(hydroxymethyl)aminomethane, N-methyl-D-glucamine, andglutamate; their opening is favored at temperatures higher than30°C; they are blocked by oxidized ATP andMg²⁺; and they can be triggered by3'-O-(4-benzoylbenzoyl)-ATP but not by UTP or ADP. We conclude that the pores described in this reportare associated with the P2Z permeabilization phenomenon.

     

ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells

In human osteoblast-like MG-63cells, extracellular ATP increased [³H]thymidineincorporation and cell proliferation and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-induced[³H]thymidine incorporation. ATP-induced[³H]thymidine incorporation was mimicked by thenonhydrolyzable ATP analogs adenosine5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2purinoceptor antagonist suramin, suggesting involvement of P2purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptorantagonist reactive blue 2 did not affect [³H]thymidineincorporation, whereas the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced[³H]thymidine incorporation, suggesting that ATP-inducedDNA synthesis was mediated by P2X receptors. RT-PCR analysis revealedthat MG-63 cells expressed P2X₄, P2X₅,P2X₆, and P2X₇, but not P2X₁,P2X₂, and P2X₃, receptors. In fura 2-loadedcells, not only ATP, but also UTP, increased intracellularCa²⁺ concentration, and inhibitors for severalCa²⁺-activated protein kinases had no effect on ATP-inducedDNA synthesis, suggesting that an increase in intracellularCa²⁺ concentration is not indispensable for ATP-induced DNAsynthesis. ATP increased mitogen-activated protein kinase activity in aCa²⁺-independent manner and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-inducedkinase activity. Furthermore, the mitogen-activated protein kinasekinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors byactivating a mitogen-activated protein kinase pathway.

     

Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification

The mechanism involved inN-methyl-D-glucamine(NMDA)-induced Ca²⁺-dependentintracellular acidosis is not clear. In this study, we investigated indetail several possible mechanisms using cultured rat cerebellargranule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].When 100 µM NMDA or 40 mM KCl was added, a marked increase in theintracellular Ca²⁺ concentration([Ca²⁺]_i)and a decrease in the intracellular pH were seen. Acidosis wascompletely prevented by the use ofCa²⁺-free medium or1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca²⁺. The following fourmechanisms that could conceivably have been involved were excluded:1)Ca²⁺ displacement of intracellularH⁺ from common binding sites;2) activation of an acid loader or inhibition of acid extruders; 3)overproduction of CO₂ or lactate; and 4) collapse of the mitochondrialmembrane potential due to Ca²⁺uptake, resulting in inhibition of cytosolicH⁺ uptake. However,NMDA/KCl-induced acidosis was largely prevented by glycolyticinhibitors (iodoacetate or deoxyglucose in glucose-free medium) or byinhibitors of the Ca²⁺-ATPase(i.e.,Ca²⁺/H⁺exchanger), including La³⁺,orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca²⁺-ATPaseis involved in NMDA-induced intracellular acidosis in granule cells. Wealso provide new evidence that NMDA-evoked intracellular acidosisprobably serves as a negative feedback signal, probably with theacidification itself inhibiting the NMDA-induced[Ca²⁺]_i increase.

     

Mechanisms of P(i) regulation of the skeletal muscle SR Ca(2+) release channel

Inorganic phosphate(P_i) accumulates in the fibers of actively working musclewhere it acts at various sites to modulate contraction. To characterizethe role of P_i as a regulator of the sarcoplasmic reticulum(SR) calcium (Ca²⁺) release channel, we examined the actionof P_i on purified SR Ca²⁺ release channels,isolated SR vesicles, and skinned skeletal muscle fibers. In singlechannel studies, addition of P_i to the cis chamberincreased single channel open probability (P_o;0.079 ± 0.020 in 0 P_i, 0.157 ± 0.034 in 20 mMP_i) by decreasing mean channel closed time; mean channelopen times were unaffected. In contrast, the ATP analog,,-methyleneadenosine 5'-triphosphate (AMP-PCP), enhancedP_o by increasing single channel open time anddecreasing channel closed time. P_i stimulation of[³H]ryanodine binding by SR vesicles wassimilar at all concentrations of AMP-PCP, suggesting P_i andadenine nucleotides act via independent sites. In skinned musclefibers, 40 mM P_i enhanced Ca²⁺-inducedCa²⁺ release, suggesting an in situ stimulation ofthe release channel by high concentrations of P_i. Ourresults support the hypothesis that P_i may be an importantendogenous modulator of the skeletal muscle SR Ca²⁺ releasechannel under fatiguing conditions in vivo, acting via a mechanismdistinct from adenine nucleotides.

     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells

This study examines theCa²⁺ influx-dependent regulationof the Ca²⁺-activatedK⁺ channel(K_Ca) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theK_Ca current and cytosolic Ca²⁺ concentration([Ca²⁺]_i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa²⁺ and addition ofLa³⁺ orGd³⁺, but notZn²⁺, inhibited the increases inK_Ca current and[Ca²⁺]_i.Ca²⁺ influx during refill (i.e.,addition of Ca²⁺ to cells treatedwith CCh and then atropine inCa²⁺-free medium) failed to evokeincreases in the K_Ca current but achieved internal Ca²⁺ storerefill. When refill was prevented by thapsigargin,Ca²⁺ readdition induced rapidactivation of K_Ca. These dataprovide further evidence that intracellularCa²⁺ accumulation provides tightbuffering of[Ca²⁺]_iat the site of Ca²⁺ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca²⁺ influx-dependentregulation of the sustained K_Cacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa²⁺ into the internalCa²⁺ store and release via theinositol 1,4,5-trisphosphate-sensitive channel.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

Desensitization of P2Y2 receptor-activated transepithelial anion secretion

Desensitization ofP2Y₂ receptor-activated anionsecretion may limit the usefulness of extracellular nucleotides insecretagogue therapy of epithelial diseases, e.g., cystic fibrosis(CF). To investigate the desensitization process for endogenousP2Y₂ receptors, freshly excised orcultured murine gallbladder epithelia (MGEP) were mounted in Ussingchambers to measure short-circuit current (I_sc), an indexof electrogenic anion secretion. Luminal treatment with nucleotidereceptor agonists increased theI_sc with apotency profile of ATP = UTP > 2-methylthioATP >>,-methylene-ATP. RT-PCR revealed the expression ofP2Y₂ receptor mRNA in the MGEPcells. The desensitization of anion secretion required a 10-minpreincubation with the P2Y₂receptor agonist UTP and increased in aconcentration-dependent manner(IC₅₀  10⁶ M). Approximately 40%of the anion secretory response was unaffected by maximal desensitizingconcentrations of UTP. Recovery from UTP-induced desensitization wasrapid (<10 min) at preincubation concentrations less than theEC₅₀ (1.9 × 10⁶ M) but requiredprogressively longer time periods at greater concentrations.UTP-induced total inositol phosphate production and intracellularCa²⁺ mobilization desensitizedwith a concentration dependence similar to that of anion secretion. Incontrast, maximal anion secretion induced byCa²⁺ ionophore ionomycin wasunaffected by preincubation with a desensitizing concentration of UTP.It was concluded that 1)desensitization of transepithelial anion secretion stimulated by theP2Y₂ receptor agonist UTP is timeand concentration dependent; 2)recovery from desensitization is prolonged (>90 min) at UTPconcentrations >10⁵ M;and 3) UTP-induced desensitizationoccurs before the operation of the anion secretory mechanism.     

Mechanical signals and mechanosensitive modulation of intracellular [Ca(2+)] in smooth muscle

We testedthe hypothesis that strain is the primary mechanical signal in themechanosensitive modulation of intracellular Ca²⁺concentration ([Ca²⁺]_i) in airway smoothmuscle. We found that [Ca²⁺]_i wassignificantly correlated with muscle length during isotonic shorteningagainst 20% isometric force (F_iso). When the isotonic loadwas changed to 50% F_iso, data points from the 20 and 50% F_iso experiments overlapped in thelength-[Ca²⁺]_i relationship. Similarly, datapoints from the 80% F_iso experiments clustered near thosefrom the 50% F_iso experiments. Therefore, despite 2.5- and4-fold differences in external load, [Ca²⁺]_idid not deviate much from the length-[Ca²⁺]_irelation that fitted the 20% F_iso data. Maximal inhibition of sarcoplasmic reticular (SR) Ca²⁺ uptake by 10 µMcyclopiazonic acid (CPA) did not significantly change[Ca²⁺]_i in carbachol-induced isometriccontractions and isotonic shortening. CPA also did not significantlychange myosin light-chain phosphorylation or force redevelopment whencarbachol-activated muscle strips were quickly released from optimallength (L_o) to 0.5 L_o. These results are consistent with thehypothesis and suggest that SR Ca²⁺ uptake is not theunderlying mechanism.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

E-C coupling failure in mouse EDL muscle after in vivo eccentric contractions

The objectives of this research were to determine thecontribution of excitation-contraction (E-C) coupling failure to the decrement in maximal isometric tetanic force(P_o) in mouse extensor digitorumlongus (EDL) muscles after eccentric contractions and to elucidatepossible mechanisms. The left anterior crural muscles of femaleICR mice (n = 164) wereinjured in vivo with 150 eccentric contractions.P_o, caffeine-,4-chloro-m-cresol-, andK⁺-induced contracture forces,sarcoplasmic reticulum (SR) Ca²⁺release and uptake rates, and intracellularCa²⁺ concentration([Ca²⁺]_i)were then measured in vitro in injured and contralateral control EDLmuscles at various times after injury up to 14 days. On the basis ofthe disproportional reduction inP_o (~51%) compared with caffeine-induced force (~11-21%), we estimate that E-C coupling failure can explain 57-75% of theP_o decrement from 0 to 5 days postinjury. Comparable reductions inP_o andK⁺-induced force (51%), and minorreductions (0-6%) in the maximal SRCa²⁺ release rate, suggest thatthe E-C coupling defect site is located at the t tubule-SR interfaceimmediately after injury. Confocal laser scanning microscopy indicatedthat resting[Ca²⁺]_iwas elevated and peak tetanic[Ca²⁺]_iwas reduced, whereas peak4-chloro-m-cresol-induced[Ca²⁺]_iwas unchanged immediately after injury. By 3 days postinjury, 4-chloro-m-cresol-induced[Ca²⁺]_ibecame depressed, probably because of decreased SRCa²⁺ release and uptake rates(17-31%). These data indicate that the decrease inP_o during the first several daysafter injury primarily stems from a failure in the E-C couplingprocess.

     

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification

In this study, we test the hypothesisthat in newborn hearts (as in adults) hypoxia and acidificationstimulate increased Na⁺ uptake, in part via pH-regulatoryNa⁺/H⁺ exchange. Resulting increases inintracellular Na⁺ (Na_i) alter the force drivingthe Na⁺/Ca²⁺ exchanger and lead to increasedintracellular Ca²⁺. NMR spectroscopy measuredNa_i and cytosolic Ca²⁺ concentration([Ca²⁺]_i) and pH (pH_i) inisolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. AfterNa⁺/K⁺ ATPase inhibition, hypoxic hearts gainedNa⁺, whereas normoxic controls did not [19 ± 3.4 to139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg drywt, respectively]. In normoxic hearts acidified using theNH₄Cl prepulse, pH_i fell rapidly and recovered,whereas Na_i rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]_i;however, [Ca]_i increased less in newborn hearts than inadults (P < 0.05). Increases in Na_i and[Ca]_i were inhibited by theNa⁺/H⁺ exchange inhibitormethylisobutylamiloride (MIA, 40 µM; P < 0.05), aswell as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data supportthe hypothesis that in newborn hearts, like adults, increases inNa_i and [Ca]_i during hypoxia and afternormoxic acidification are in large part the result of increased uptakevia Na⁺/H⁺ and Na⁺/Ca²⁺exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]_i is less in newborn thanadult hearts.