Removal Of ATP Research Articles

Lysophosphatidic Acid-induced Ca2+ Mobilization Requires Intracellular Sphingosine 1-Phosphate Production: POTENTIAL INVOLVEMENT OF ENDOGENOUS EDG-4 RECEPTORS

Lysophosphatidic acid (LPA)-mediated Ca(2+) mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4, 5-trisphosphate (Ins(1,4,5)P(3))- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca(2+) signaling cascade based upon the lipid second messenger molecule, sphingosine 1-phosphate. Both LPA and direct G-protein activation increase [(3)H]sphingosine 1-phosphate levels in SH-SY5Y cells. Measurements of (45)Ca(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine 1-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca(2+). Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca(2+) "puffs," which resulted from an interaction between the sphingolipid Ca(2+) release pathway and Ins(1,4,5)P(3) receptors. Down-regulation of Ins(1,4,5)P(3) receptors uncovered a Ca(2+) response to LPA, which was manifest as a progressive increase in global cellular Ca(2+) with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine 1-phosphate to stimulate Ca(2+) mobilization in SH-SY5Y cells. Unlike traditional Ca(2+) release processes, this novel pathway does not require the progressive recruitment of elementary Ca(2+) events.

Stabilization of calcium uptake in rat rod outer segments by taurine and ATP.

Calcium ion (Ca2+) uptake was measured in rod outer segments (ROS) isolated from rat retina in the presence of varying concentrations of CaCl2 in the incubation buffer (1.0-2.5 mM). It is known that taurine increases Ca2+ uptake in rat ROS in the presence of ATP and at low concentrations of CaCl2 (Lombardini, 1985a); taurine produces no significant effects when CaCl2 concentrations are increased to 1.0 and 2.5mM. With the removal of both taurine and ATP, Ca2+ uptake in rat ROS increased significantly in the presence of 2.5mM CaCl2. Taurine treatment in the absence of ATP was effective in decreasing Ca2+ uptake at the higher levels of CaCl2 (2.0 and 2.5 mM). Similar effects were observed with ATP treatment. The data suggest that taurine and ATP, alone or in combination, limit the capacity of the rat ROS to take up Ca2+ to the extent that a stable uptake level is achieved under conditions of increasing extracellular Ca2+, indicating a protective role for both agents against calcium toxicity.

Dual role of ATP in supporting volume-regulated chloride channels in mouse fibroblasts

The effects of inhibitors of protein tyrosine kinases (PTKs) on the Cl − current ( I Cl(vol)) through volume-regulated anion/chloride (VRAC) channels whilst manipulating cellular ATP have been studied in mouse fibroblasts using the whole-cell patch clamp technique. Removal of ATP from the pipette-filling solution prevented activation of the current during osmotic cell swelling and when the volume of patched cells was increased by the application of positive pressure through the patch pipette to achieve rates exceeding 100%/min. Equimolar substitution of ATP in the pipette solution with its non-hydrolyzable analogs, adenosine 5′- O-(3-thiotriphosphate) (ATPγS) or adenylyl-(β,γ-methylene)-diphosphonate (AMP-PCP), not only supported activation of the current but also maintained its amplitude. The PTK inhibitors, tyrphostins A25, B46, 3-amino-2,4-dicyano-5-(4-hydroxyphenyl)penta-2,4-dienonitrile and genistein (all at 100 μM), inhibited I Cl(vol) in a time-dependent manner. Tyrphostin A1, which does not inhibit PTK activity, did not affect the current amplitude. The PTK inhibitors also inhibited I Cl(vol) under conditions where ATP in the pipette was substituted with ATPγS or AMP-PCP. We conclude that in mouse fibroblasts ATP has a dual role in the regulation of the current: it is required for protein phosphorylation to keep VRAC channels operational and, through non-hydrolytic binding, determines the magnitude of I Cl(vol). We also suggest that tyrosine-specific protein kinases and phosphatases exhibit an interdependent involvement in the regulation of VRAC channels.

Hyperpolarization-activated, mixed-cation current (I(h)) in octopus cells of the mammalian cochlear nucleus.

Octopus cells in the posteroventral cochlear nucleus of mammals detect the coincidence of synchronous firing in populations of auditory nerve fibers and convey the timing of that coincidence with great temporal precision. Earlier recordings in current clamp have shown that two conductances contribute to the low input resistance and therefore to the ability of octopus cells to encode timing precisely, a low-threshold K(+) conductance and a hyperpolarization-activated mixed-cation conductance, g(h). The present experiments describe the properties of g(h) in octopus cells as they are revealed under voltage clamp with whole-cell, patch recordings. The hyperpolarization-activated current, I(h), was blocked by extracellular Cs(+) (5 mM) and 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (50-100 nM) but not by extracellular Ba(2+) (2 mM). The reversal potential for I(h) in octopus cells under normal physiological conditions was -38 mV. Increasing the extracellular potassium concentration from 3 to 12 mM shifted the reversal potential to -26 mV; lowering extracellular sodium concentration from 138 to 10 mM shifted the reversal potential to -77 mV. These pharmacological and ion substitution experiments show that I(h) in octopus cells is a mixed-cation current that resembles I(h) in other neurons and in heart muscle cells. Under control conditions when cells were perfused intracellularly with ATP and GTP, I(h) had an activation threshold between about -35 to -40 mV and became fully activated at -110 mV. The maximum conductance associated with hyperpolarizing voltage steps to -112 mV ranged from 87 to 212 nS [150 +/- 30 (SD) nS, n = 36]. The voltage dependence of g(h) obtained from peak tail currents is fit by a Boltzmann function with a half-activation potential of -65 +/- 3 mV and a slope factor of 7. 7 +/- 0.7. This relationship reveals that g(h) was activated 41% at the mean resting potential of octopus cells, -62 mV, and that at rest I(h) contributes a steady inward current of between 0.9 and 2.1 nA. The voltage dependence of g(h) was unaffected by the extracellular application of dibutyryl cAMP but was shifted in hyperpolarizing direction, independent of the presence or absence of dibutyryl cAMP, by the removal of intracellular ATP and GTP.

SWI-SNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state.

SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling-restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.

Direct actions of nitric oxide on rat neurohypophysial K+ channels.

1. Nitric oxide (NO) has been shown to modulate neuropeptide secretion from the posterior pituitary. Here we show that NO activates large-conductance Ca2+-activated K+ (BK) channels in posterior pituitary nerve terminals. 2. NO, generated either by the photolysis of caged-NO or with chemical donors, irreversibly enhanced the component of whole-terminal K+ current due to BK channels and increased the activity of BK channels in excised patches. NO also inhibited the transient A-current. The time courses of these effects on K+ current were very different; activation of BK channels developed slowly over several minutes whereas inhibition of A-current immediately followed NO uncaging. 3. Activation of BK channels by NO occurred in the presence of guanylyl cyclase inhibitors and after removal of ATP or GTP from the pipette solution, suggesting a cGMP-independent signalling pathway. 4. The sulfhydryl alkylating agent N-ethyl maleimide (NEM) increased BK channel activity. Pretreatment with NEM occluded NO activation. 5. NO activation of BK channels occurred independently of voltage and cytoplasmic Ca2+ concentration. In addition, NO removed the strict Ca2+ requirement for channel activation, rendering channels highly active even at nanomolar Ca2+ levels. 6. These results suggest that NO, or a reactive nitrogen byproduct, chemically modifies nerve terminal BK channels or a closely associated protein and thereby produces an increase in channel activity. Such activation is likely to inhibit impulse activity in posterior pituitary nerve terminals and this may explain the inhibitory action of NO on secretion.

The activation of an apical Cl- conductance by extracellular ATP is potentiated by genistein in Necturus gallbladder epithelium.

Necturus gallbladder epithelium (NGE) expresses a CFTR-homologous apical Cl- conductance (Ga,Cl) which can be activated either by elevation of intracellular cAMP or by extracellular ATP. Here we show by microelectrode experiments and impedance analysis that genistein (50 microM), which is known to potentiate the stimulation of Ga,Cl in several cell culture models, also potentiates the stimulation of Ga,Cl by low doses of forskolin in NGE. Moreover, we show that genistein also potentiates the stimulation of Ga,Cl by ATP. In addition genistein renders gallbladders that initially do not respond to ATP sensitive to this stimulant, and it delays the conductance inactivation after ATP removal. Under control conditions Ga,Cl inactivates within < 5 min, but in the presence of genistein a significant Ga,Cl persists even after 60 min. These effects of genistein are not related to inhibition of protein tyrosine kinases, since structurally different inhibitors of the tyrphostin family do not mimic the genistein effects. The data support our conclusion that stimulation of Ga,Cl by ATP is mediated by activation of the cAMP pathway and involves a CFTR-homologous protein. They also favour the view that genistein acts via inhibition of protein phosphatases which dephosphorylate CFTR, but cannot exclude the possibility of a direct interaction with CFTR.

Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed in Xenopus laevis oocytes.

The rat kidney Na+-HCO3- cotransporter (rkNBC) was expressed in Xenopus laevis oocytes and transport via rkNBC was studied with the patch-clamp technique in giant inside/out (i/o) or outside/out (o/o) membrane patches. The current/voltage (I/V) relation(s) of individual patches was(were) determined in solutions containing only Na+ and HCO3- as permeable ions. The current carried by rkNBC (INBC) was identified by its response to changing bath Na+ concentration(s) and quantified as the current blocked by 4, 4'-diisothiocyanatostilbene disulfonate (DIDS). The stoichiometric ratio (q) of HCO3- to Na+ transport was determined from zero-current (reversal) potentials. The results and conclusions are as follows. First, DIDS (250 micromol/l) blocks INBC irreversibly from both the extracellular and the intracellular surface. Second, in the presence of Na+ and HCO3- concentration gradients similar to those which rkNBC usually encounters in tubular cells, q was close to 2. The same value was also observed when the HCO3- concentration was 25 mmol/l throughout, but the Na+ concentration was either high (100 mmol/l) or low (10 mmol/l) on the extracellular or intracellular surface of the patch. These data demonstrate that in the oocyte cell membrane rkNBC works with q=2 as previously observed in a study of isolated microperfused tubules (Seki et al., Pflügers Arch 425:409, 1993), however, they do not exclude the possibility that in a different membrane and cytoplasmic environment rkNBC may operate with a different stoichiometry. Third, in most experiments bath application of up to 2 mmol/l ATP increased the DIDS-inhibitable conductance of i/o patches by up to twofold with a half saturation constant near 0.5 mmol/l. This increase was not associated with a change in q, nor with a shift in the I/V relationship which would suggest induction of active transport (pump current). Since the effect persisted after ATP removal and was not observed with the non-hydrolysable ATP analogue AMP-PNP, it is possible that rkNBC is activated by phosphorylation via protein kinases that might adhere to the cytoplasmic surface of the membrane patch.

Disassembly of the Cytosolic Chaperonin in Mammalian Cell Extracts at Intracellular Levels of K+ and ATP

The eukaryotic, cytoplasmic chaperonin, CCT, is essential for the biogenesis of actin- and tubulin-based cytoskeletal structures. CCT purifies as a doubly toroidal particle containing two eight-membered rings of approximately 60-kDa ATPase subunits, each encoded by an essential and highly conserved gene. However, immunofluorescence detection with subunit-specific antibodies has indicated that in cells CCT subunits do not always co-localize. We report here that CCT ATPase activity is highly dependent on K+ ion concentration and that in cell extracts, at physiological levels of K+ and ATP, there is considerable dissociation of CCT to a smaller oligomeric structure and free subunits. This dissociation is consequent to ATP hydrolysis and is readily reversed on removal of ATP. The ranking order for ease with which subunits can exit the chaperonin particle correlates well with the length of a loop structure, identified by homology modeling, in the intermediate domain of CCT subunits. K+-ATP-induced disassembly is not an intrinsic property of purified CCT over a 40-fold concentration range and requires the presence of additional factor(s) present in cell extracts.

Endogenous ATP release regulates Cl- secretion in cultured human and rat biliary epithelial cells.

P2Y receptor stimulation increases membrane Cl- permeability in biliary epithelial cells, but the source of extracellular nucleotides and physiological relevance of purinergic signaling to biliary secretion are unknown. Our objectives were to determine whether biliary cells release ATP under physiological conditions and whether extracellular ATP contributes to cell volume regulation and transepithelial secretion. With the use of a sensitive bioluminescence assay, constitutive ATP release was detected from human Mz-ChA-1 cholangiocarcinoma cells and polarized normal rat cholangiocyte monolayers. ATP release increased rapidly during cell swelling induced by hypotonic exposure. In Mz-ChA-1 cells, removal of extracellular ATP (apyrase) and P2 receptor blockade (suramin) reversibly inhibited whole cell Cl- current activation and prevented cell volume recovery during hypotonic stress. Moreover, exposure to apyrase induced cell swelling under isotonic conditions. In intact normal rat cholangiocyte monolayers, hypotonic perfusion activated apical Cl- currents, which were inhibited by addition of apyrase and suramin to bathing media. These findings indicate that modulation of ATP release by the cellular hydration state represents a potential signal coordinating cell volume with membrane Cl- permeability and transepithelial Cl- secretion.

The structure and dynamics of partially folded actin.

Steady-state and time-resolved intrinsic fluorescence, fluorescence quenching by acrylamide, and surface testing by hydrophobic label ANS were used to study the structure of inactivated alpha-actin. The results are discussed together with that of earlier experiments on sedimentation, anisotropy of fluorescence, and CD spectrum in the near- and far-UV regions. A dramatic increase in ANS binding to inactivated actin in comparison with native and unfolded protein indicates that the inactivated actin has solvent-exposed hydrophobic clusters on the surface. It results in specific association of actin macromolecules (sedimentation constants for native and inactivated actin are 3 and 20 S, respectively) and, consequently, in irreversibility of native-inactivated actin transition. It was found that, though the fluorescence spectrum of inactivated actin is red-shifted, the efficiency of the acrylamide collision quenching is even lower than that of the intact protein. It suggests that tryptophan residues of inactivated actin are located in the inner region of protein formed by polar groups, which are highly packed. It correlates with the pronounced near-UV CD spectrum of inactivated actin. The experimentally found tryptophan fluorescence lifetimes allowed evaluation rotational correlation times on the basis of Perrin plots. It is found that oscillations of tryptophan residues in inactivated actin are restricted in comparison with native one. The inactivated actin properties were invariant with experimental conditions (ionic strength, the presence of reducing agents), the way of inactivation (Ca2+ and/or ATP removal, heating, 3-5 M urea or 1.5 M GdmCl treatment), and protein concentration (within the limits 0.005-1.0 mg/mL). The same state of actin appears on the refolding from the completely unfolded state. Thermodynamic stability, pronounced secondary structure, and the existing hydrophobic clusters, tested by ANS fluorescence and reversibility of transition inactivated-unfolded forms, allowed us to suggest that inactivated actin can be intermediate in the folding-unfolding pathway.

Stable remodeling of tailless nucleosomes by the human SWI-SNF complex.

The histone N-terminal tails have been shown previously to be important for chromatin assembly, remodeling, and stability. We have tested the ability of human SWI-SNF (hSWI-SNF) to remodel nucleosomes whose tails have been cleaved through a limited trypsin digestion. We show that hSWI-SNF is able to remodel tailless mononucleosomes and nucleosomal arrays, although hSWI-SNF remodeling of tailless nucleosomes is less effective than remodeling of nucleosomes with tails. Analogous to previous observations with tailed nucleosomal templates, we show both (i) that hSWI-SNF-remodeled trypsinized mononucleosomes and arrays are stable for 30 min in the remodeled conformation after removal of ATP and (ii) that the remodeled tailless mononucleosome can be isolated on a nondenaturing acrylamide gel as a novel species. Thus, nucleosome remodeling by hSWI-SNF can occur via interactions with a tailless nucleosome core.

Activation of the P2Z/P2X7Receptor in Human Lymphocytes Produces a Delayed Permeability Lesion: Involvement of Phospholipase D

Leukemic lymphocytes possess a cytolytic P2Z/P2X7receptor which, when activated by extracellular ATP, opens a Ca2+- and Ba2+-permeable ion channel. This ATP-stimulated influx of divalent cations has been shown to activate an intracellular phospholipase D (PLD) which hydrolyzes membrane phosphatidylcholine. Lymphocytes that were exposed to ATP for 20 min at 37°C, washed, and then incubated without ATP for 2 h showed an increased uptake of propidium2+, a dye widely used to measure cytotoxicity. The potent P2Z/P2X7receptor inhibitor, KN-62, which is known to prevent the channel opening when added with ATP, did not block development of the permeability lesion when added 15 min before dye addition. The activity of lymphocyte PLD was stimulated fourfold by ATP and a proportion of this increased activity persisted for several hours after removal of ATP. Loading lymphocytes with intracellular choline+by prior incubation of cells with ATP in isotonic choline chloride abolished both ATP-stimulated PLD activity and the ATP-induced permeability lesion. Addition of PLD but not phospholipase C to the extracellular medium increased lymphocyte permeability to propidium2+and this effect was not observed in a choline medium. The cytolytic effect of exogenous PLD together with the inhibitory effect of choline, a product of the PLD reaction, suggests that sustained activation of intracellular PLD may be involved in the ATP-initiated cytolytic pathway.

ATP hydrolysis-dependent formation of a dynamic ternary nucleoprotein complex with MutS and MutL.

Functional interactions of Escherichia coli MutS and MutL in mismatch repair are dependent on ATP. In this study, we show that MutS and MutL associate with immobilised DNA in a manner dependent on ATP hydrolysis and with an ATP concentration near the solution K m of the ATPase of MutS. After removal of MutS, MutL and ATP, much of the protein in this ternary complex is not stably associated, with MutL leaving the complex more rapidly than MutS. The rapid dissociation reveals a dynamic interaction with concurrent rapid association and dissociation of proteins from the DNA. Analysis by surface plasmon resonance showed that the DNA interacting with dynamically bound protein was more resistant to nuclease digestion than the DNA in MutS-DNA complexes. Non-hydrolysable analogs of ATP inhibit the formation of this dynamic complex, but permit formation of a second type of ternary complex with MutS and MutL stably bound to the immobilised DNA.

Measurement of SR Ca2+ content in the presence of caffeine in permeabilised rat cardiac trabeculae.

This study was designed to measure the Ca2+ content of rat cardiac sarcoplasmic reticulum (SR) after equilibration with normal diastolic levels of Ca2+ (100 nM), in the absence and presence of caffeine. Measurements of [Ca2+] based on Fura-2 fluorescence were made from a limited bath volume (230 nl) containing individual saponin-permeabilised rat cardiac trabeculae. Injection of caffeine (5-40 mM) into this volume caused an initial release of Ca2+ from the SR, but within 30 s the SR was able to re-accumulate a significant proportion of the Ca2+. Ca2+ re-accumulation into the SR could be prevented by removal of ATP to inhibit the SR Ca2+ pump. Incubation of the preparation in an ATP-containing solution containing caffeine (5-40 mM) and 100 nM Ca2+ indicated that the SR's ability to retain Ca2+ depends inversely on the dose of caffeine. The relative Ca2+ content of the SR after preincubation with caffeine was 86.7+/-3.5% at a caffeine concentration of 5 mM, 62.5+/-5.1% at 10 mM caffeine, 37.8+/-8.1% at 20 mM caffeine and 7. 1+/-1.9% at 40 mM caffeine. Measurement of the SR Ca2+ release in the presence of different BAPTA concentrations was used to calculate (1) the Ca2+-binding capacity of the preparation (equivalent to 245+/-10 microM BAPTA) and (2) the Ca2+ content of the SR accessed by caffeine after equilibration with 100 nM Ca2+ (186+/-11 micromol/l cell volume or 5.6 mmol/l SR volume).

Activation of cAMP-dependent C1- currents in guinea-pig paneth cells without relevant evidence for CFTR expression.

1. To determine whether Paneth cells exhibit functional expression of cAMP-activated Cl- currents and molecular expression of the cystic fibrosis transmembrane conductance regulator (CFTR), we applied whole-cell patch clamp and single-cell mRNA analysis by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification to single Paneth cells in crypts isolated from the guinea-pig small intestine. 2. Prominent activation of Cl- currents was consistently observed after stimulation with dibutyryl cAMP and forskolin or with vasoactive intestinal polypeptide (VIP). The cAMP-activated Cl- current was inhibited by removal of intracellular ATP or administration of an inhibitor of protein kinase A. 3. Many of the biophysical and pharmacological properties of the currents were phenotypically similar to those of the CFTR Cl- channel, such as the ohmic current-voltage relationship, the anion selectivity with a Type III sequence (Br- > Cl- > I- >> F- >= gluconate-), I--induced blockage, insensitivity to a stilbene-derivative Cl- channel blocker, and sensitivity to a carboxylate analogue Cl- channel blocker. The sensitivity of the current to glibenclamide was, however, much weaker than that reported for the CFTR Cl- channel current. In contrast to the time independence of CFTR currents, the inward component of the Paneth cell Cl- currents exhibited inactivation kinetics. 4. Expression of CFTR mRNA could not be detected by RT-PCR analysis in almost all single Paneth cells, although its expression was consistently detected at the whole-crypt level. The presence of a small number of CFTR-expressing epithelial cells, which were scattered both in villi and crypts but not at the crypt base where Paneth cells were located, was demonstrated by immunocytochemistry. 5. Taken together, it appears that guinea-pig Paneth cells functionally express cAMP-activated Cl- conductance without relevant evidence for molecular expression of CFTR. Functional expression of VIP receptors in the Paneth cells was also demonstrated.

Mechanisms underlying phosphate-induced failure of Ca2+ release in single skinned skeletal muscle fibres of the rat.

1. Single mechanically skinned fibres from rat extensor digitorum longus (EDL) muscles were used to investigate the mechanisms underlying inorganic phosphate (Pi) movements between the myoplasm and the sarcoplasmic reticulum (SR). Force transients elicited by caffeine/low Mg2+ application were used to assess the rate of Pi-induced inhibition of SR Ca2+ release and the subsequent recovery of Ca2+ release following removal of myoplasmic Pi. 2. Myoplasmic Pi reduced SR Ca2+ release in a concentration- and time-dependent manner. A 10 s exposure to 10, 20 and 50 mM myoplasmic Pi reduced SR Ca2+ release by 12 +/- 9, 29 +/- 5 and 82 +/- 5 %, respectively. 3. Removal of myoplasmic ATP at the time of Pi exposure significantly increased the rate and extent of SR Ca2+ release inhibition. For example, Ca2+ release was reduced by 86 +/- 6 % (n = 6) after 20 s exposure to 20 mM Pi in the absence of ATP compared with only 47 +/- 5 % (n = 5) in the presence of ATP. 4. The half and full recovery times for SR Ca2+ release following washout of myoplasmic Pi were 35 s and approximately 7 min, respectively. Recovery of Ca2+ release was unaffected by the absence of ATP during washout of Pi but was prevented when fibres were washed in the presence of high myoplasmic Pi (30 mM). Neither the Pi transporter blocker phenylphosphonic acid (PHPA) nor the anion channel blockers anthracene-9-carboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) affected the rate of recovery of SR Ca2+ release. 5. These results show that Pi entry and exit from the SR occur primarily through a passive pathway that is insensitive to well-known anion channel blockers. Pi inhibition of SR Ca2+ release appears to be a complicated phenomenon influenced by the rate of Pi movement across the SR as well as by the rate, extent and species of Ca2+-Pi precipitate formation in the SR lumen. The more rapid inhibitory effect of Pi in the absence of myoplasmic ATP suggests that Pi may inhibit SR Ca2+ release more efficiently during the later stages of fatigue.

Luminal Ca2+ regulates passive Ca2+ efflux from the intracellular stores of hepatocytes.

Ca2+ uptake into the intracellular stores of permeabilized hepatocytes was entirely dependent on ATP and substantially inhibited by either ionomycin or thapsigargin, although both were required for complete inhibition. Unidirectional efflux of 45Ca2+ after removal of ATP from cells loaded to steady state (1.60+/-0.12 nmol/10(6) cells) was monoexponential and occurred with a half-time of 103+/-10 s. However, the 45Ca2+ content of the stores did not return to their pre-ATP level, but reached a plateau at 0.12+/-0.04 nmol/10(6) cells. A similar amount of Ca2+ was trapped within the stores when Ca2+ uptake was prevented by thapsigargin and chelation of Ca2+; at all temperatures between 2 degreesC and 37 degreesC; and after stores had first been loaded with unlabelled Ca2+. Simultaneous addition of inositol 1,4,5-trisphosphate (InsP3) and inhibition of Ca2+ uptake reduced the amount of trapped Ca2+ to a level consistent with InsP3 rapidly and more completely emptying a fraction of the stores that could be only partially emptied by the passive leak. After dilution of the specific activity of the 45Ca2+ under conditions that maintained the steady-state activities of the pumps and leaks, the stores rapidly lost their entire 45Ca2+ content. We conclude that the channel responsible for mediating the leak of Ca2+ abruptly closes when the luminal [Ca2+] of the intracellular stores falls below a critical threshold corresponding to about 7% of their steady-state loading. Whereas InsP3 is capable of completely emptying a fraction of the stores, regulation of the passive leak by luminal [Ca2+] is likely to prevent it from completely emptying them; such regulation may ensure that the many other functions of Ca2+ within the endoplasmic reticulum are not compromised.

GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor.

In a cell-free system from neutrophil cytosol GTP(&ggr ;)S can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTP(&ggr ;)S stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPgammaS.Gialpha2 or Gbeta1gamma2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPgammaS. When recombinant, constitutively activated and GTPgammaS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPgammaS. The action of GTPgammaS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPgammaS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPgammaS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPgammaS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.

Abscisic acid maintains S-type anion channel activity in ATP-depleted Vicia faba guard cells

The plant hormone abscisic acid (ABA) regulates important developmental and stress responses. Recent data show that ABA activates phosphorylation events, but whether dephosphorylation events are post-translationally regulated by ABA or whether these are constitutive remains unknown. Slow anion channels in the plasma membrane of guard cells have been proposed to play an important role during ABA-induced stomatal closing. Anion channels are deactivated by removal of cytosolic ATP. However, when guard cells were treated with ABA and depleted of ATP, anion currents remained active. Subsequent removal of extracellular ABA caused deactivation of currents. Deactivation of currents was reversed by reintroduction of cytosolic MgATP. These data show that anion channels are regulated by ABA even in the absence of cytosolic ATP required for kinase-induced phosphorylation events and that anion channel activity is maintained by ABA under conditions that favor dephosphorylation-induced deactivation. Furthermore, channel activation proceeded at high ATP concentrations with nanomolar cytosolic Ca 2+ showing a Ca 2+-independent final step in anion channel activation.