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J W Putney

Publications and source records attributed to J W Putney.

At least 37 records · Page 2Linked to original sources
PubMed1999

Capacitative calcium entry channels.

In the phospholipase C signaling system, Ca(2+) is mobilized from intracellular stores by an action of inositol 1,4,5-trisphosphate. The depletion of intracellular calcium stores activates a calcium entry mechanism at the plasma membrane called capacitative calcium entry. The signal for activating the entry is unknown but likely involves either the generation or release, or both, from the endoplasmic reticulum of some diffusible signal. Recent research has focused on mammalian homologues of the Drosophila TRP protein as potential candidates for capacitative calcium entry channels. This review summarizes current knowledge about the nature of capacitative calcium entry signals, as well as the potential role of mammalian TRP proteins as capacitative calcium entry channel molecules.

Animals↗
PubMed1998

Effect of adenophostin A on Ca2+ entry and calcium release-activated calcium current (Icrac) in rat basophilic leukemia cells.

In most non-excitable cells, calcium influx is signaled by depletion of intracellular calcium stores, a process known as capacitative calcium entry. Adenophostin A, a potent activator of the inositol 1, 4,5-trisphosphate receptor, has been reported to activate Ca2+ entry in Xenopus oocytes to a greater extent than expected on the basis of its ability to release calcium stores. In this study, we compared the abilities of adenophostin A and inositol 2,4,5-trisphosphate ((2, 4,5)IP3) to release Ca2+ from intracellular stores, to activate Ca2+ entry, and to activate calcium release-activated calcium current (Icrac) in rat basophilic leukemia cells. Under conditions of low intracellular Ca2+ buffering (0.1 mM BAPTA), adenophostin A-induced Ca2+ release and activation of Icrac could be monitored simultaneously. However, other reagents that would be expected to deplete Ca2+ stores ((2,4,5)IP3, 3-fluoro-inositol 1,4, 5-trisphosphate, thapsigargin, and ionomycin) were unable to activate Icrac under this low Ca2+ buffering condition. Adenophostin A activated Icrac after a significant delay, longer than the delay for Ca2+ release. Thus, adenophostin A activates Icrac as a consequence of release of intracellular Ca2+, rather than directly acting on store-operated channels. The unique ability of adenophostin A to activate Icrac under conditions of low intracellular Ca2+ buffering suggests an additional site of action, perhaps in preventing or reducing rapid Ca2+-dependent inactivation of store-operated Ca2+ channels.

Adenosine↗
PubMed1998

Relationship between intracellular calcium store depletion and calcium release-activated calcium current in a mast cell line (RBL-1).

The kinetic relationship between depletion of endoplasmic reticulum calcium stores and the activation of a calcium release-activated calcium current (Icrac) was investigated in the RBL-1 mast cell line. The inositol trisphosphate receptor activator, inositol 2,4, 5-trisphosphate ((2,4,5)IP3), the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, and the calcium ionophore, ionomycin, were used to deplete stored calcium. For (2,4,5)IP3 and thapsigargin, a significant delay was observed between the initiation of calcium store depletion and the activation of Icrac. However, for ionomycin, little or no delay was observed. This may indicate that a specialized subcompartment of the endoplasmic reticulum functions as a regulator of calcium entry and that this compartment is relatively resistant to depletion by (2,4,5)IP3 and thapsigargin but not to depletion by ionomycin. For all three calcium-depleting agents, the rate of development of Icrac, once initiated, was relatively constant, suggesting an all-or-none mechanism. However, there were also clear experimental situations in which submaximal, graded depletion of stored calcium resulted in submaximal activation of Icrac. This complex behavior could also result from the existence of a specific subcompartment of endoplasmic reticulum regulating Icrac. The kinetic behavior of this compartment may not be accurately reflected by the kinetics of calcium changes in the bulk of endoplasmic reticulum. These findings add to the growing body of evidence suggesting specialization of the endoplasmic reticulum calcium stores with regard to the control of capacitative calcium entry.

Animals↗
PubMed1998

Calcium signalling in exocrine glands.

In exocrine gland cells, stimulation of a variety of surface receptors initiates a Ca2+ signalling system through activation of a polyphosphoinositide-specific phospholipase C. One product of phospholipase C activity, inositol 1,4,5-trisphosphate ((1,4,5)IP3), signals the release of intracellular Ca2+. Release of intracellular Ca2+ is followed by entry of Ca2+ into the cell across the plasma membrane. The mechanism by which Ca2+ entry is regulated is not well understood, although it is clear that (1,4,5)IP3 plays an important role. One hypothesis suggests that Ca2+ entry is triggered by the depletion of intracellular Ca2+ stores by (1,4,5)IP3, a process termed 'capacitative calcium entry'. The purpose of these studies is to gain understanding into the processes controlling capacitative calcium entry in exocrine gland cells.

Animals↗
PubMed1997

Role of the cytoskeleton in calcium signaling in NIH 3T3 cells. An intact cytoskeleton is required for agonist-induced [Ca2+]i signaling, but not for capacitative calcium entry.

Treatment of NIH 3T3 cells with cytochalasin D (10 microM, 1 h at 37 degrees C) disrupted the actin cytoskeleton and changed the cells from a planar, extended morphology, to a rounded shape. Calcium mobilization by ATP or by platelet-derived growth factor was abolished, while the ability of thapsigargin (2 microM) to empty calcium stores and activate calcium influx was unaffected. Similar experiments with nocodazole to depolymerize the tubulin network yielded identical results. Platelet-derived growth factor induced an increase in inositol phosphates, and this increase was undiminished in the presence of cytochalasin D. Therefore, the blockade of agonist responses by this drug does not result from decreased phospholipase C. Injection of inositol 1,4,5-trisphosphate (IP3) released calcium to the same extent in control and cytochalasin D-treated cells. Confocal microscopic studies revealed a significant rearrangement of the endoplasmic reticulum after cytochalasin D treatment. Thus, disruption of the cytoskeleton blocks agonist-elicited [Ca2+]i mobilization, but this effect does not result from a lower calcium storage capacity, impaired function of the IP3 receptor, or diminished phospholipase C activity. We suggest that cytoskeletal disruption alters the spatial relationship between phospholipase C and IP3 receptors, impairing phospholipase C-dependent calcium signaling. Capacitative calcium entry was not altered under these conditions, indicating that the coupling between depletion of intracellular calcium stores and calcium entry does not depend on a precise structural relationship between intracellular stores and plasma membrane calcium channels.

3T3 Cells↗
PubMed1997

Effect of cytoplasmic Ca2+ on (1,4,5)IP3 formation in vasopressin-activated hepatocytes.

The role of cytoplasmic calcium as a regulator of phospholipase C in vasopressin-activated hepatocytes was examined. According to models in which calcium spiking arises because of a positive feedback by calcium on phospholipase C, Ca2+ is seen as a positive modulator of phospholipase C under conditions of submaximal receptor activation. However, in hepatocytes whose precursor lipids had been labeled by incubation in [3H]-inositol, no increase in [3H]-(1,4,5)IP3 was detected in response to thapsigargin, in either unstimulated cells, or in cells stimulated with 1 nM vasopressin. Addition of a maximal concentration of vasopressin (1 microM) caused a rapid and substantial increase in [3H]-(1,4,5)IP3. These results indicate that changes in cytoplasmic calcium do not influence phospholipase C activity in hepatocytes, even under conditions of submaximal agonist activation. These findings also support models that provide for calcium spiking at constant levels of (1,4,5)IP3 at least in the case of the rat hepatocyte.

Animals↗
PubMed1997

Type 3 inositol 1,4,5-trisphosphate receptor and capacitative calcium entry.

It is well established that depletion of intracellular stores of calcium can activate calcium influx across the plasma membrane, a process known as "capacitative calcium entry'. Recent research and reviews have focussed on homologs of the Drosophila mutant, trp, as candidates for the channels underlying this phenomenon. However, there is recent evidence that the type 3 inositol 1,4,5-trisphosphate {(1,4,5)IP3} receptor can also function as a capacitative calcium entry channel, specifically: (i) in some cell types, the properties of Ca2+ store depletion-activated currents and channels resemble those of an (1,4,5)IP3 receptor;(ii) in these same cell types, (1,4,5)IP3 directly activates channels in the plasma membrane (including in one study, the same channels activated by depletion of Ca2+ stores); and (iii) expression of the type 3 receptor in Xenopus oocytes resulted in the association of this receptor with the plasma membrane, and facilitation of Ca2+ entry across the plasma membrane. The type 3 receptor may represent one type of capacitative calcium entry channel expressed in some cell types and, in other cell types, channels with markedly different properties (i.e. trp) may carry out this function. Furthermore, as there is little, if any, homology between the IP3 and trp protein families, this may represent an example of convergent evolution of function.

Animals↗
PubMed1997

Roles of cytoplasmic Ca2+ and intracellular Ca2+ stores in induction and suppression of apoptosis in S49 cells.

The Ca2+-ATPase inhibitors, thapsigargin and cyclopiazonic acid, depleted intracellular Ca2+ stores, induced large increases in intracellular Ca2+ concentration, and caused apoptosis in S49 cells. Removal of extracellular Ca2+ augmented apoptosis due to thapsigargin, indicating that depletion of Ca2+ from intracellular stores is responsible for apoptosis with this agent. Overexpression of the apoptosis suppressor, Bcl-2, inhibited apoptosis due to thapsigargin but did not affect thapsigargin-induced Ca2+ signaling. Dexamethasone induced apoptosis, diminished the size of the endoplasmic reticulum Ca2+ pool, and caused a small elevation of intracellular Ca2+. However, this elevation was not due to Ca2+ influx because the increase was similar in the presence or absence of Ca2+ in the medium. Furthermore, in contrast to the results with thapsigargin, apoptosis due to dexamethasone was unchanged in a Ca2+-free medium. These results indicate that depletion of Ca2+ stores initiates a pathway leading to apoptosis. Elevations in cytoplasmic Ca2+ appears to play a lesser role than previously thought in the actions of Bcl-2 and glucocorticoids.

Apoptosis↗
PubMed1996

Differential effects of protein kinase C activation on calcium storage and capacitative calcium entry in NIH 3T3 cells.

In NIH 3T3 cells, treatment with phorbol 12-myristate 13-acetate (PMA) reduced the release of Ca2+ by thapsigargin, but did not activate Ca2+ entry; Ca2+ influx was triggered after the residual pool was emptied by thapsigargin, and this Ca2+ influx was similar to that induced by thapsigargin in control cells. The effect of PMA was due to decreased Ca2+ storage because 1) Ca2+ release by ionomycin was similarly affected by PMA, and in both control and PMA-treated cells, ionomycin did not release Ca2+ following thapsigargin treatment; 2) PMA reduced 45Ca2+ accumulation; and 3) studies with Ca2+ indicator compartmentalized into the endoplasmic reticulum indicated that stored Ca2+ was reduced by PMA. Although PMA did not itself activate Ca2+ entry, PMA potentiated Ca2+ entry with low concentrations of cyclopiazonic acid. With a somewhat higher concentration of cyclopiazonic acid, PMA had no effect on calcium entry. Thus, protein kinase C has two apparent actions on calcium signaling in NIH 3T3 cells: 1) reduced intracellular Ca2+ storage capacity and 2) augmented calcium entry with submaximal intracellular Ca2+ pool depletion. These actions indicate a complex and potentially important role for the protein kinase C system in calcium homeostasis in this cell type.

3T3 Cells↗
PubMed1996

Cell type-specific modes of feedback regulation of capacitative calcium entry.

The Ca2+-ATPase inhibitor, thapsigargin, activated Ca2+ entry into pancreatic acinar cells, a process known as capacitative calcium entry. In cells loaded with the calcium chelator BAPTA, the transient Ca2+ release was blunted and the rise of [Ca2+]i on readdition of Ca2+ was slowed. However, the steady-state [Ca2+]i due to Ca2+ entry was substantially augmented compared with control cells. This indicates that [Ca2+]i exerts a negative feedback on Ca2+ entry from a compartment buffered by BAPTA and separated from the bulk of cytoplasmic Ca2+. This interaction probably occurs close to the calcium channel where [Ca2+] is higher than in the bulk of the cytoplasm. In support of this interpretation, the slower Ca2+ chelator, EGTA, also blunted the release of Ca2+ and slowed the rise of the sustained [Ca2+]i phase but failed to augment steady-state [Ca2+]i. In contrast, Ca2+ entry in NIH 3T3 cells was characterized by a transient rise of [Ca2+]i that decays to near prestimulus levels. This decay in Ca2+ entry also results from negative feedback by Ca2+ because the decrease in Ca2+ entry was reversed by incubation in a Ca2+-deficient medium. However, unlike its effects in acinar cells, BAPTA neither augmented steady-state [Ca2+]i nor prevented the inactivation of entry. Rather, in BAPTA-loaded cells, [Ca2+]i failed to increase substantially suggesting that negative regulation by Ca2+ may occur at a site distinct from the cytoplasmic compartment and inaccessible to cytoplasmic BAPTA. These two distinct types of feedback behavior may indicate subtypes of store-operated calcium channels expressed in different cells or a single type of channel which is differentially regulated in a cell type-specific manner.

3T3 Cells↗
PubMed1996

Effect of inositol 1,3,4,5-tetrakisphosphate on inositol trisphosphate-activated Ca2+ signaling in mouse lacrimal acinar cells.

In mouse lacrimal acinar cells, microinjection of the metabolically stable analog of inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate ((2,4,5)IP3), stimulated both intracellular Ca2+ mobilization and Ca2+ entry. Microinjection of inositol 1,3,4,5-tetrakisphosphate ((1,3,4,5)IP4), the inositol 1,4,5-trisphosphate-3-kinase product, was ineffective at mobilizing intracellular Ca2+ or activating Ca2+ entry. In lacrimal cells previously microinjected with submaximal levels of (2,4,5)IP3, the subsequent microinjection of low to moderate concentrations of (1,3,4,5)IP4 did not result in additional release of intracellular Ca2+, nor did it potentiate the Ca2+ entry phase attributable to (2,4,5)IP3. However, as previously demonstrated (Bird, G. S. J., Rossier, M. F., Hughes, A. R., Shears, S. B., Armstrong, D. L., and Putney, J. W., Jr. (1991) Nature 352, 162-165), additional injections of (2,4,5)IP3 induced further mobilization of intracellular Ca2+ and increased the elevated and sustained Ca2+ entry phase. Introduction of high concentrations of (1,3,4,5)IP4 appeared to inhibit or block the (2,4,5)IP3-induced Ca2+ entry phase. These results were consistent with the observed effect of (1,3,4,5)IP4 in permeabilized lacrimal cells, where (1,3,4,5)IP4 did not release cellular 45Ca2+ but at high concentrations inhibited the ability of submaximal concentrations of (2,4,5)IP3 to release 45Ca2+. Likewise, injection of a high concentration of (1,3,4,5)IP4 prior to injection of (2,4,5)IP3 blocked both release and influx of Ca2+. The inhibitory action of (1,3,4,5)IP4 on Ca2+ signaling observed in intact cells occurred at concentrations that might be obtained in agonist-stimulated cells. However, in permeabilized cells, (1,3,4,5)IP4 inhibited Ca2+ mobilization at concentrations exceeding those likely to occur in agonist-stimulated cells. These results suggest that physiologically relevant levels of (1,3,4,5)IP4 in the cell cytoplasm do not release Ca2+, nor do they potentiate inositol trisphosphate-induced Ca2+ entry across the plasma membrane. Rather, the possibility is raised that (1,3,4,5)IP4 or one of its metabolites could function as a negative feedback on Ca2+ mobilization by inhibiting inositol 1,4,5-trisphosphate-induced Ca2+ release.

Animals↗
PubMed1996

cGMP is not required for capacitative Ca2+ entry in Jurkat T-lymphocytes.

In this study, we have addressed the potential role of cGMP in regulating calcium entry in Jurkat T-lymphocytes. These cells display capacitative Ca(2+)-entry in response to the intracellular Ca(2+)-ATPase inhibitor, thapsigargin (TG). In the presence of extracellular Ca2+, TG stimulates a sustained elevation of intracellular cGMP levels. In the absence of extracellular Ca2+, TG induces no apparent increase in the levels of cGMP. However, experiments using Mn2+, as a surrogate for Ca2+, demonstrated that TG increased the rate of divalent cation entry in the absence of extracellular Ca2+. Treatment of Jurkat cells with the guanylyl cyclase inhibitor, LY83583 (20 microM), completely blocked cGMP formation in response to TG. However, LY83583 treated cells still exhibited a sustained, albeit partially reduced, Ca2+ response induced by TG. These data demonstrate that, in Jurkat cells, the sustained formation of cGMP is dependent on elevated intracellular Ca2+, and that elevated levels of cGMP are not necessary for the activation of capacitative Ca2+ entry.

Aminoquinolines↗
PubMed1996

Constitutive activation of a phosphoinositidase C-linked G protein in murine fibroblasts decreases agonist-stimulated Ca2+ mobilization.

We compared Ca2+ signaling and inositol polyphosphate metabolism in NIH-3T3 cells stably transfected with cDNA encoding either the wild-type G protein G16 alpha subunit or a GTPase-deficient alpha 16 subunit (Q212L-alpha 16). Constitutive activation of phosphoinositidase C (PIC) in cells expressing Q212L-alpha 16 was demonstrated by 1) an increased basal level of [3H]inositol polyphosphates, 2) an enhanced rate of [3H]inositol polyphosphate accumulation in cells treated with 10 mM LiCl, and 3) an increased rate of incorporation of [3H]inositol into cell lipids. Q212L-alpha 16 cells had a diminished cell growth rate. Basal intracellular Ca2+ concentration was equivalent in Fura-2 acetoxymethyl ester-loaded Q212L-alpha 16 cells compared with controls; however, calcium release in Q212L-alpha 16 cells exposed to ionomycin, ATP (a G protein-linked agonist), or platelet-derived growth factor (a tyrosine kinase-linked agonist) was decreased. Permeabilized, 45Ca-loaded Q212L-alpha 16 cells released less 45Ca at each concentration of inositol-1,4,5-trisphosphate than did control cells. Accordingly, the total amount of inositol trisphosphate (IP3) receptor protein was decreased in Q212L-alpha 16 cells relative to controls. These data demonstrate that Q212L-alpha 16 cells maintain physiological levels of cytoplasmic calcium and partially loaded Ca2+ stores in the face of constitutively active PIC. This is accomplished, at least in part, by down-regulation of IP3 receptor number. Thus, diminution in cell growth rate in Q212L-alpha 16 cells seems to be attributable to a combination of at least two effects: a direct effect of PIC activation leading to partial depletion of Ca2+ stores and an indirect, adaptive response resulting in a decreased IP3 receptor number.

3T3 Cells↗
PubMed1996

Spatial and temporal aspects of cellular calcium signaling.

Cytosolic Ca2+ signals are often organized in complex temporal and spatial patterns, even under conditions of sustained stimulation. In this review we discuss the mechanisms and physiological significance of this behavior in nonexcitable cells, in which the primary mechanism of Ca2+ mobilization is through (1,4,5)IP3-dependent Ca2+ release from intracellular stores. Oscillations of cytosolic free Ca2+ ([Ca2+]i) are a common form of temporal organization; in the spatial domain, these [Ca2+]i oscillations may take the form of [Ca2+]i waves that propagate throughout the cell or they may be restricted to specific subcellular regions. These patterns of Ca2+ signaling result from the limited range of cytoplasmic Ca2+ diffusion and the feedback regulation of the pathways responsible for Ca2+ mobilization. In addition, the spatial organization of [Ca2+]i changes appears to depend on the strategic distribution of Ca2+ stores within the cell. One type of [Ca2+]i oscillation is baseline spiking, in which discrete [Ca2+]i spikes occur with a frequency, but not amplitude, that is determined by agonist dose. Most current evidence favors a model in which baseline [Ca2+]i spiking results from the complex interplay between [Ca2+]i and (1,4,5)IP3 in regulating the gating of (1,4,5)IP3-sensitive intracellular Ca2+ channels. Sinusoidal [Ca2+]i oscillations represent a mechanistically distinct type of temporal organization, in which agonist dose regulates the amplitude but has no effect on oscillation frequency. Sinusoidal [Ca2+]i oscillations can be explained by a negative feedback effect of protein kinase C on the generation of (1,4,5)IP3 at the level of phospholipase C or its activating G-protein. The physiological significance of [Ca2+]i oscillations and waves is becoming more established with the observation of this behavior in intact tissues and by the recognition of Ca2+-dependent processes that are adapted to respond to frequency-modulated oscillatory [Ca2+]i signals. In some cells, these [Ca2+]i signals are targeted to control processes in limited cytoplasmic domains, and in other systems [Ca2+]i waves can be propagated through gap junctions to coordinate the function of multicellular systems.

Animals↗
PubMed1995

Role of cyclic GMP in the control of capacitative Ca2+ entry in rat pancreatic acinar cells.

We have investigated the possible roles of cyclic GMP (cGMP) in initiating or regulating capacitiative Ca2+ entry in rat pancreatic acinar cells. In medium containing 1.8 mM external Ca2+, thapsigargin activated Ca2+ entry and slightly but significantly increased intracellular cGMP concentration. This rise in cGMP levels was prevented by pretreating the cells with the guanylate cyclase inhibitor, LY-83583, or by omitting Ca2+ during stimulation by thapsigargin or methacholine. LY-83583 and NG-nitro-L-arginine (L-NA, an inhibitor of NO synthase) both had a small inhibitory effect on Ca2+ entry when they were added after thapsigargin in Ca2(+)-containing medium, and they reduced by 32 and 48% respectively the thapsigargin-induced capacitative Ca2+ entry when added to the cells during a 20 min preincubation period. However, neither dibutyryl cGMP (Bt2cGMP) nor sodium nitroprusside, an NO mimic, affected either basal intracellular Ca2+ concentration [Ca2+]i or thapsigargin-induced capacitative Ca2+ entry. Further, the inhibitory effects observed after preincubation with LY-83583 or L-NA could not be prevented by preincubation with Bt2cGMP, nor could they be reversed by adding Bt2cGMP, 8-bromo-cGMP or sodium nitroprusside acutely after activation of capacitative Ca2+ entry by thapsigargin. Finally, pretreatment of cells with LY-83583 or L-NA did not affect Ca2+ signalling in response to 1 microM methacholine, including the pattern of [Ca2+]i oscillations. In conclusion, in pancreatic acinar cells, the rise in cellular cGMP levels appears to depend on, rather than cause, the increase in [Ca2+]i with agonist stimulation.

Aminoquinolines↗
PubMed1995

The Ca(2+)-mobilizing actions of a Jurkat cell extract on mammalian cells and Xenopus laevis oocytes.

Randriamampita and Tsien (Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814) suggested that an acid-extracted fraction from a Jurkat cell line contains a messenger responsible for the coupling of calcium entry to the depletion of intracellular stores, i.e. capacitative calcium entry. We found that the extract, prepared as described by Randriamampita and Tsien, caused Ca2+ entry in 1321N1 astrocytoma cells which was not blocked by the D-myo-1,4,5-trisphosphate-receptor antagonist, heparin. In contrast to astrocytoma cells, when applied to mouse lacrimal acinar cells and rat hepatocytes the Jurkat extract always caused the release of intracellular Ca2+, followed by Ca2+ entry across the plasma membrane. This activity of the extract on lacrimal cells was blocked by either intracellular injection of heparin or extracellular atropine. Similarly prepared lacrimal cell extracts gave Ca2+ responses when applied to astrocytoma cells or lacrimal cells which were similar to those for Jurkat-derived extract. However, extracts from hepatocytes had no effect. In most Xenopus oocytes, the Jurkat extract had no effect, while in a few oocytes, the extract gave a [Ca2+]i response similar to that seen in lacrimal cells, that is, release of Ca2+ followed by Ca2+ entry. We conclude that the actions of the Jurkat cell extract are not consistent with its containing the long sought messenger for capacitative calcium entry. It is likely that this fraction contains a number of factors that mediate Ca2+ response in different cell types, possibly through receptor-mediated mechanisms.

Animals↗