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Anant B Parekh

Publications and source records attributed to Anant B Parekh.

17 recordsLinked to original sources
PubMed2006

Ca2+ influx through CRAC channels activates cytosolic phospholipase A2, leukotriene C4 secretion, and expression of c-fos through ERK-dependent and -independent pathways in mast cells.

Cytosolic phospholipase A2 (cPLA2) is a Ca2+-dependent enzyme that mediates agonist-dependent arachidonic acid release in most cell types. Arachidonic acid can then be metabolized by the 5-lipoxygenase enzyme to generate the proinflammatory signal leukotriene C4 (LTC4). Here we report that Ca2+ entry through store-operated CRAC (Ca2+ release-activated Ca2+) channels activates the extracellular signal-regulated kinases (ERKs), members of the mitogen-activated protein kinase family, within minutes and this is necessary for stimulation of cPLA2. Ca2+ entry activates ERK indirectly, via recruitment of Ca2+-dependent protein kinase C alpha and betaI. Ca2+ influx also promotes translocation of cytosolic 5-lipoxygenase to the nuclear membrane, a key step in the activation of this enzyme. Translocation is dependent on ERK activation. A role for gene activation is shown by the finding that CRAC channel opening results in increased transcription and translation of c-fos. Inhibition of ERK activation failed to prevent c-fos expression. Our results show that CRAC channel activation elicits short-term effects through the co-coordinated regulation of two metabolic pathways (cPLA2 and 5-lipoxygenase), which results in the generation of both intra- and intercellular messengers within minutes, as well as longer term changes involving gene activation. These short-term effects are mediated via ERK, whereas, paradoxically, c-fos expression is not. Ca2+ influx through CRAC channels can therefore activate different signaling pathways at the same time, culminating in a range of temporally diverse responses.

Animals↗
PubMed2006

Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration.

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses including neurotransmitter release, muscle contraction, and cell growth and proliferation [1]. During intracellular Ca(2+) signaling, mitochondria rapidly take up significant amounts of Ca(2+) from the cytosol, and this stimulates energy production, alters the spatial and temporal profile of the intracellular Ca(2+) signal, and triggers cell death [2-10]. Mitochondrial Ca(2+) uptake occurs via a ruthenium-red-sensitive uniporter channel found in the inner membrane [11]. In spite of its critical importance, little is known about how the uniporter is regulated. Here, we report that the mitochondrial Ca(2+) uniporter is gated by cytosolic Ca(2+). Ca(2+) uptake into mitochondria is a Ca(2+)-activated process with a requirement for functional calmodulin. However, cytosolic Ca(2+) subsequently inactivates the uniporter, preventing further Ca(2+) uptake. The uptake pathway and the inactivation process have relatively low Ca(2+) affinities of approximately 10-20 microM. However, numerous mitochondria are within 20-100 nm of the endoplasmic reticulum, thereby enabling rapid and efficient transmission of Ca(2+) release into adjacent mitochondria by InsP(3) receptors on the endoplasmic reticulum. Hence, biphasic control of mitochondrial Ca(2+) uptake by Ca(2+) provides a novel basis for complex physiological patterns of intracellular Ca(2+) signaling.

Animals↗
PubMed2006

On the activation mechanism of store-operated calcium channels.

The development of the patch clamp technique has revolutionised our understanding of the life sciences. One area in which it has made an enormous contribution is cellular signalling. In many cell types, calcium influx across the plasma membrane is essential for the regulation of a wide range of critical physiological responses including secretion, gene transcription and cell growth. For many years the calcium influx pathways in non-excitable cells remained unknown, despite their importance in physiological and pathophysiological states. Very careful and insightful work by James Putney led to the formulation of the capacitative calcium entry (store-operated calcium influx) model, in which the process of emptying intracellular calcium stores resulted in the activation of calcium entry channels. Unequivocal evidence for this revolutionary model was provided by patch clamp studies carried out by Markus Hoth and Reinhold Penner, who demonstrated that store depletion activated a novel class of calcium channel called the CRAC channel. This review provides a historical perspective on the development of store-operated calcium influx and how patch clamping resolved a long-standing controversy in cell physiology. The review also discusses current ideas relating to how store emptying opens channels in the plasma membrane.

Animals↗
PubMed2005

Store-operated calcium channels.

In electrically nonexcitable cells, Ca(2+) influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and proliferation, and apoptosis. The major Ca(2+) entry pathway in these cells is the store-operated one, in which the emptying of intracellular Ca(2+) stores activates Ca(2+) influx (store-operated Ca(2+) entry, or capacitative Ca(2+) entry). Several biophysically distinct store-operated currents have been reported, but the best characterized is the Ca(2+) release-activated Ca(2+) current, I(CRAC). Although it was initially considered to function only in nonexcitable cells, growing evidence now points towards a central role for I(CRAC)-like currents in excitable cells too. In spite of intense research, the signal that relays the store Ca(2+) content to CRAC channels in the plasma membrane, as well as the molecular identity of the Ca(2+) sensor within the stores, remains elusive. Resolution of these issues would be greatly helped by the identification of the CRAC channel gene. In some systems, evidence suggests that store-operated channels might be related to TRP homologs, although no consensus has yet been reached. Better understood are mechanisms that inactivate store-operated entry and hence control the overall duration of Ca(2+) entry. Recent work has revealed a central role for mitochondria in the regulation of I(CRAC), and this is particularly prominent under physiological conditions. I(CRAC) therefore represents a dynamic interplay between endoplasmic reticulum, mitochondria, and plasma membrane. In this review, we describe the key electrophysiological features of I(CRAC) and other store-operated Ca(2+) currents and how they are regulated, and we consider recent advances that have shed insight into the molecular mechanisms involved in this ubiquitous and vital Ca(2+) entry pathway.

Animals↗
PubMed2004

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels.

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.

Animals↗
PubMed2004

Close functional coupling between Ca2+ release-activated Ca2+ channels, arachidonic acid release, and leukotriene C4 secretion.

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types, particularly of hemopoietic origin, store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. However, little is known about the downstream consequences of CRAC channel activation. Here, we report that Ca2+ entry through CRAC channels stimulates arachidonic acid production, whereas Ca2+ release from the stores is ineffective even though the latter evokes a robust intracellular Ca2+ signal. We find that arachidonic acid released by Ca2+ entering through CRAC channels is used to synthesize the potent paracrine proinflammatory signal leukotriene C4 (LTC4). Both pharmacological inhibitors of CRAC channels and mitochondrial depolarization, which impairs CRAC channel activity, suppress arachidonic acid release and LTC4 secretion. Thus, arachidonic acid release is preferentially stimulated by elevated subplasmalemmal Ca2+ levels due to open CRAC channels, suggesting that the enzyme is located close to the CRAC channels. Our results also identify a novel role for CRAC channels, namely the activation of a downstream signal transduction pathway resulting in the secretion of LTC4. Finally, mitochondria are key determinants of the generation of both intracellular (arachidonic acid) and paracrine (LTC4) signals through their effects on CRAC channel activity.

Animals↗
PubMed2004

Arf-1 (ADP-ribosylation factor-1) is involved in the activation of a mammalian Na+-selective current.

Stimulation of mammalian cells often results in an increase in the intracellular Na(+) concentration, brought about by Na(+) influx into the cell via Na(+)-permeable ion channels. In some cell types, particularly renal epithelia and mast cells, non-hydrolysable analogues of GTP, such as GTP[S] (guanosine 5'-[gamma-thio]triphosphate), activate a non-voltage-activated Na(+)-selective current. We have carried out whole-cell patch-clamp experiments to examine how GTP[S] activates the Na(+) current in a rat mast cell line. The ability of GTP[S] to activate Na(+) influx was prevented by including GTP in the pipette solution, indicating the involvement of small G-proteins. Brefeldin A and Arf-1-(2-17), inhibitors of Arf-1 (ADP-ribosylation factor-1) proteins, suppressed the activation of Na(+) entry by GTP[S]. However, non-active succinylated Arf-1-(2-17) or an N-terminal myristoylated peptide directed towards Arf-5 were ineffective. Arf proteins modulate the cytoskeleton, and disruption of the cytoskeleton with cytochalasin D or its stabilization with phalloidin impaired the development of the Na(+) current. Disaggregation of microtubules was without effect. Dialysis with cAMP or inhibition of cAMP phosphodiesterase with caffeine both decreased the extent of Na(+) entry, and this was not prevented by pre-treatment with broad-spectrum protein kinase inhibitors. Collectively, our results suggest that the mechanism of activation of a mammalian non-voltage-activated Na(+)-selective current requires an Arf small G-protein, most probably Arf-1.

ADP-Ribosylation Factor 1↗
PubMed2003

Activation of the store-operated calcium current ICRAC can be dissociated from regulated exocytosis in rat basophilic leukaemia (RBL-1) cells.

In many cell types, the emptying of intracellular Ca2+ stores results in the opening of store-operated Ca2+ channels in the plasma membrane. However, the nature of the signal that couples store content to the opening of these Ca2+ channels is unclear. One model proposes that the Ca2+ channels are initially stored in cytoplasmic vesicles but inserted into the plasma membrane upon store depletion via a regulated exocytoytic mechanism (vesicular fusion model). Using the whole-cell patch-clamp technique to measure the store-operated Ca2+ current ICRAC and the capacitance method to monitor vesicular fusion, an indicator of exocytosis, we have investigated the effects of interfering with regulated exocytosis on the ability of ICRAC to activate. We find that the recombinant protein alpha-SNAP1-285, an inhibitor of exocytosis in many systems, suppresses such fusion but has no impact on the activation of ICRAC. A variety of other manoeuvres that interfere with vesicle trafficking and exocytosis were also without effect on ICRAC. Impairing constitutive exocytosis with brefeldin A reduced the extent of ICRAC, but this effect was less pronounced when current density was considered instead. Activation of ICRAC can therefore be clearly dissociated from an exocytotic mechanism, a finding that is not easily reconcilable with the vesicular fusion model.

Animals↗
PubMed2003

Store-operated Ca2+ entry: dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.

In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a biphasic increase in intracellular free Ca2+ concentration: an initial transient release of Ca2+ from intracellular stores is followed by a sustained phase of Ca2+ influx. This influx is generally store-dependent and is required for controlling a host of Ca2+-dependent processes ranging from exocytosis to cell growth and proliferation. In many cell types, store-operated Ca2+ entry is manifest as a non-voltage-gated Ca2+ current called ICRAC (Ca2+ release-activated Ca2+ current). Just how store emptying activates CRAC channels remains unclear, and some of our recent experiments that address this issue will be described. No less important from a physiological perspective is the weak Ca2+ buffer paradox: whereas macroscopic (whole cell) ICRAC can be measured routinely in the presence of strong intracellular Ca2+ buffer, the current is generally not detectable under physiological conditions of weak buffering following store emptying with the second messenger InsP3. In this review, I describe some of our experiments aimed at understanding just why InsP3 is ineffective under these conditions and which lead us to conclude that respiring mitochondria are essential for the activation of ICRAC in weak intracellular Ca2+ buffer. Mitochondrial Ca2+ uptake also increases the dynamic range over which InsP3 functions as the second messenger that controls Ca2+ influx. Finally, we find that Ca2+-dependent slow inactivation of Ca2+ influx, a widespread but poorly understood phenomenon that helps shape the profile of an intracellular Ca2+ signal, is regulated by mitochondrial Ca2+ buffering. Thus, by enabling macroscopic store-operated Ca2+ current to activate and then by controlling its extent and duration, mitochondria play a crucial role in all stages of store-operated Ca2+ influx. Store-operated Ca2+ entry reflects therefore a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.

Animals↗
PubMed2003

Mitochondrial regulation of intracellular Ca2+ signaling: more than just simple Ca2+ buffers.

Mitochondrial Ca(2+) uptake shapes the profile of intracellular Ca(2+) signals, both spatially and temporally. In addition, such uptake controls the gating of Ca(2+) release and store-operated Ca(2+) entry channels, partitions cells into subcellular Ca(2+) hotspots, and can result in the release of diffusible signals into the cytosol that subsequently regulate protein function.

Animals↗
PubMed2002

Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake.

Store-operated Ca(2+) channels, which are activated by the emptying of intracellular Ca(2+) stores, provide one major route for Ca(2+) influx. Under physiological conditions of weak intracellular Ca(2+) buffering, the ubiquitous Ca(2+) releasing messenger InsP(3) usually fails to activate any store-operated Ca(2+) entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca(2+) that has been released by InsP(3), enabling stores to empty sufficiently for store-operated channels to activate. Here, we report a novel role for mitochondria in regulating store-operated channels under physiological conditions. Mitochondrial depolarization suppresses store-operated Ca(2+) influx independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP(3)-sensitive store depletion, can be distinguished from Ca(2+)-dependent inactivation of the store-operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidification, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca(2+) uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store-operated influx and raise the possibility of bidirectional Ca(2+)-dependent crosstalk between mitochondria and store-operated Ca(2+) channels.

Adenosine↗
PubMed2002

Inwardly rectifying potassium currents in rat basophilic leukaemia (RBL-1) cells: regulation by spermine and implications for store-operated calcium influx.

In non-excitable cells, the major Ca(2+) influx pathway is the store-operated one. Store-operated Ca(2+) entry is intimately related to the prevalent membrane potential, in that hyperpolarisation enhances Ca(2+) influx and depolarisation reduces it. Inwardly rectifying potassium channels are important determinants of the membrane potential and hence will regulate, indirectly, the rate and extent of Ca(2+) entry through store-operated channels. Here we investigated inwardly rectifying potassium currents ( I(RK)) in rat basophilic leukaemia (RBL-1) cells, a model system for studying store-operated Ca(2+) influx. I(RK) was voltage dependent in that the current decays during strong hyperpolarisations. Recovery from this decay was both time and voltage dependent. Close to the resting potential of RBL-1 cells, however, I(RK) was stable. Neither store depletion per se nor the subsequent rise in intracellular [Ca(2+)] appeared to alter I(RK) activity. Receptor stimulation reduced the current only weakly. Unexpectedly, intracellular spermine inhibited I(RK) quite strongly and via a mechanism that seemed distinct from that responsible for current rectification. The relevance of these findings to store-operated Ca(2+) influx is discussed.

Animals↗
PubMed2002

Effects of inhibitors of the lipo-oxygenase family of enzymes on the store-operated calcium current I(CRAC) in rat basophilic leukaemia cells.

In non-excitable cells, the major Ca2+ entry pathway is the store-operated pathway in which emptying of intracellular Ca2+ stores activates Ca2+ channels in the plasma membrane. In many cell types, store-operated influx gives rise to a Ca2+-selective current called I(CRAC) (Ca2+ release-activated Ca2+ current). Using both the whole-cell patch clamp technique to measure I(CRAC) directly and fluorescent Ca2+ imaging, we have examined the role of the lipo-oxygenase pathway in the activation of store-operated Ca2+ entry in the RBL-1 rat basophilic leukaemia cell-line. Pretreatment with a variety of structurally distinct lipo-oxygenase inhibitors all reduced the extent of I(CRAC), whereas inhibition of the cyclo-oxygenase enzymes was without effect. The inhibition was still seen in the presence of the broad protein kinase blocker staurosporine, or when Na+ was used as the charge carrier through CRAC channels. The lipo-oxygenase blockers released Ca2+ from intracellular stores but this was not associated with subsequent Ca2+ entry. Lipo-oxygenase blockers also reduced both the amount of Ca2+ that could subsequently be released by the combination of thapsigargin and ionomycin in Ca2+-free solution and the Ca2+ influx component that occurred when external Ca2+ was re-admitted. The inhibitors were much less effective if applied after I(CRAC) had been activated. This inhibition of I(CRAC) could not be rescued by dialysis with 5(S)-hydroxyperoxyeicosa-6E,8Z,11Z,14Z,tetraenoic acid (5-HPETE), the first product of the 5-lipo-oxygenase pathway. Our findings indicate that exposure to pharmacological tools that inhibit the lipo-oxygenase enzymes all decrease the extent of activation of the current. Our results raise the possibility that a lipo-oxygenase might be involved in the activation of I(CRAC). Alternative explanations are also discussed.

Adenosine Triphosphate↗
PubMed2002

Monovalent cation permeability and Ca(2+) block of the store-operated Ca(2+) current I(CRAC )in rat basophilic leukemia cells.

Like voltage-operated Ca(2+) channels, store-operated CRAC channels become permeable to monovalent cations in the absence of external divalent cations. Using the whole-cell patch-clamp technique, we have characterized the permeation and selectivity properties of store-operated channels in the rat basophilic leukemia (RBL-1) cell line. Store depletion by dialysis with InsP(3) and 10 mM EGTA resulted in the rapid development of large inward currents in Na(+)- and Li(+)-based divalent-free solutions. Cs(+) permeated the channels poorly (P(Cs)/ P(Na)=0.01). Trimethylamine (TMA(+)), tetramethylammonium (TeMA(+)), tetraethylammonium (TEA(+)), N-methyl- D-glucamine (NMDG(+)) and TRIS(+) were not measurably permeant. NH(4)(+) was conducted well. We estimated the minimum pore diameter under divalent-free conditions to be between 0.32 nm and 0.55 nm. When cells were dialysed with buffered Ca(2+) solution and I(CRAC) activated by application of thapsigargin, P(Cs)/ P(Na) was still low (0.08). Outward currents through CRAC channels were carried by intracellular Na(+), K(+) and, to a much lesser extent, by Cs(+). Currents were unaffected by dialysis with Mg(2+)-free solution. The Na(+) current was inhibited by external Ca(2+) (half-maximal blocking concentration of 10 microM). This Ca(2+)-dependent block could be alleviated by hyperpolarization. The monovalent Na(+) current was voltage dependent, increasing as the holding potential depolarized above 0 mV. Our results suggest that CRAC channels in RBL-1 cells have a smaller pore diameter than voltage-operated Ca(2+) channels, discriminate between Group I cations, and differ markedly in their selectivity from CRAC channels reported in lymphocytes.

Animals↗
PubMed2002

Adenophostin A and ribophostin, but not inositol 1,4,5-trisphosphate or manno-adenophostin, activate the Ca2+ release-activated Ca2+ current, I(CRAC), in weak intracellular Ca2+ buffer.

Under physiological conditions of weak intracellular Ca(2+) buffering (0.1 mM EGTA), the second messenger Ins(1,4,5)P(3) often fails to activate any detectable store-operated Ca(2+) current. However, it has been reported that the fungal metabolite adenophostin A [which has a severalfold higher affinity than Ins(1,4,5)P(3) for Ins(1,4,5)P(3) receptors] consistently activates the current under similar conditions. Here, whole-cell patch clamp experiments have been performed to examine how adenophostin A can activate the store-operated Ca(2+) current (I(CRAC)) in RBL-1 (rat basophilic leukaemia) cells. In a strong intracellular Ca(2+) buffer, saturating concentrations of adenophostin A activated I(CRAC) maximally and the current amplitude and kinetics were indistinguishable from those obtained with high concentrations of Ins(1,4,5)P(3). In a weak Ca(2+) buffer, adenophostin A consistently activated I(CRAC), but the current was submaximal. High concentrations of Ins(1,4,5)P(3) or the non-metabolizable analogue Ins(2,4,5)P(3) were largely ineffective under these conditions. The size of I(CRAC) to adenophostin A in weak Ca(2+) buffer could be significantly increased by either inhibiting sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase ('SERCA') pumps with thapsi-gargin or enhancing mitochondrial Ca(2+) uptake, although blocking the mitochondrial Ca(2+) uniporter with Ruthenium Red did not suppress the activation of the current. Changing the levels of free ATP in the recording pipette did not enhance the size of I(CRAC) evoked by adenophostin A. We also examined two structurally distinct analogues of adenophostin A (manno-adenophostin and ribophostin), for which the affinities for the Ins(1,4,5)P(3) receptor are similar to that of Ins(1,4,5)P(3) in equilibrium binding experiments. Although these analogues were able to activate I(CRAC) to its maximal extent in strong buffer, ribophostin, but not manno-adenophostin, consistently activated the current in weak buffer. We conclude that adenophostin A and ribophostin are able to activate I(CRAC) in weak buffer through a mechanism that is quite distinct from that employed by Ins(1,4,5)P(3) and manno-adenophostin and is not related to equilibrium affinities.

Adenosine↗