Biomedical subjects
J W Putney
Publications and source records attributed to J W Putney.
Delayed "all-or-none" activation of inositol 1,4,5-trisphosphate-dependent calcium signaling in single rat hepatocytes.
When single rat hepatocytes were stimulated with the phospholipase C-activating hormone, vasopressin (from 300 pM to 1 microM), the [Ca2+]i signals were always "all-or-none" responses. At low concentrations of vasopressin, Ca2+ release was maximal because liberation of additional inositol 1,4,5-trisphosphate (IP3) by photolysis of its caged precursor at the top of the [Ca2+]i spike failed to increase [Ca2+]i further. However, if IP3 was generated by photolysis of caged IP3 in previously unstimulated cells, [Ca2+]i increased immediately, and the magnitude of the response was a graded function of the quantity of IP3 released. We also analyzed the kinetics of activation of intracellular IP3 receptor/Ca2+ channels by monitoring the quench of sequestered dye by the entry of cytoplasmic Mn2+ into fura-2-loaded intracellular IP3-sensitive organelles. This Mn(2+)-induced quench was precipitous and always preceded by a delay inversely related to the vasopressin concentration. In hepatocytes stimulated with 10 nM vasopressin, IP3 increased slowly, and the half-time of the IP3 rise was comparable with the latency for the release of intracellular calcium. The slow rise in IP3 would be predicted to produce accelerating Ca2+ release. This is consistent with the results of the Mn2+ quench experiments, which revealed accelerating activation of intracellular IP3-regulated calcium channels. We conclude that this accelerating release of Ca2+, which does not occur with instantaneous increases in IP3 due to flash photolysis, is likely to be important for generating the all-or-none Ca2+ mobilization that initiates the processes of intracellular [Ca2+]i oscillations.
The inositol phosphate-calcium signalling system in lacrimal gland cells.
From the above discussion, it is clear that the regulation of Ca2+ signalling in exocrine cells is a complex process involving activation of both intracellular Ca2+ release as well as the entry of Ca2+ across the plasma membrane. A poorly understood mechanism links these two phases of Ca2+ signalling thereby providing both rapid as well as sustained signals for the initiation and maintenance of appropriate exocrine responses. Further work is needed to better understand the mechanisms controlling this important and ubiquitous signalling system.
Excitement about calcium signaling in inexcitable cells.
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The signal for capacitative calcium entry.
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Inhibition of thapsigargin-induced calcium entry by microinjected guanine nucleotide analogues. Evidence for the involvement of a small G-protein in capacitative calcium entry.
Injection of mouse lacrimal acinar cells with the non-hydrolyzable analogue of GTP, GTP gamma S (guanosine 5'-3-O-(thio)triphosphate), caused a rapid release of intracellular calcium but failed to activate calcium entry. Injection of GTP gamma S together with the inositol 1,4,5-trisphosphate receptor inhibitor, heparin, did not induce calcium release but blocked the activation of capacitative calcium entry by thapsigargin. Injection of GDP beta S (guanosine 5'-O-(thio)diphosphate) produced similar effects. The inhibitor effects of GDP beta S were prevented if an equal concentration of GTP was included in the injection pipette. These findings suggest that one of the steps linking the depletion of intracellular calcium pools to calcium entry across the plasma membrane requires the hydrolysis of GTP and may involve a small G-protein.
Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes.
Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.
Sinusoidal oscillations in intracellular calcium requiring negative feedback by protein kinase C.
Stimulation of mouse lacrimal acinar cells with submaximal concentrations of the muscarinic agonist, methacholine, resulted in an increase in intracellular calcium ([Ca2+]i), which took the form of sinusoidal oscillations. These oscillations were relatively constant (approximately 4-5/min) regardless of the methacholine concentration, suggesting that the oscillations arise from an oscillating negative feedback in the signal transduction pathway. This negative feedback appears to involve oscillations in protein kinase C activity because the oscillations were prevented by activation, inhibition, or down-regulation of protein C. Activation of protein kinase C with phorbol esters inhibited the methacholine-induced [Ca2+]i signal and formation of the Ca2+ mobilizing messenger, inositol 1,4,5-trisphosphate. [Ca2+]i signals elicited by intracellular introduction of inositol phosphates did not oscillate and were not affected by activators or inhibitors of protein kinase C. Thus, the constant frequency [Ca2+]i oscillations appear to result from a negative feedback loop involving inhibition of inositol trisphosphate production by protein kinase C.
Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells.
There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.
Inositol phosphates and cell signaling: new views of InsP5 and InsP6.
Ins(1,3,4,5,6)P5 and InsP6 comprise the bulk of the inositol phosphate content of mammalian cells, but their intracellular functions are unknown. Until recently it seemed that these compounds were metabolically lethargic; this has diverted attention away from their possible role in short-term regulation of physiological processes. Interest in the idea that these polyphosphates play more dynamic roles is now increasing, following recent demonstrations that they are precursors of several inositol phosphates that turnover rapidly.
The inositol phosphate-calcium signaling system in nonexcitable cells.
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Inhibition of agonist-induced activation of phospholipase C following poxvirus infection.
Recent studies indicate that viruses may influence polyphosphoinositide levels. This study has examined the effects of vaccinia virus infection on phospholipase C activity. Infection of BS-C-1 cells, an African Green Monkey kidney cell line, or A431 cells, a human carcinoma cell line, with vaccinia virus inhibits receptor-mediated phospholipase C activation. As a consequence, agonist-mediated Ca2+ mobilization in BS-C-1 cells also was inhibited by vaccinia virus infection. Alleviation of the inhibition of phospholipase C activation was observed in vaccinia virus-infected cells treated with cycloheximide without influencing uninfected cells. Treatment of infected cells with alpha-amanitin, an inhibitor of host mRNA synthesis but not virus mRNA synthesis, failed to alleviate the inhibition of phospholipase C activation. Together these results suggest that a virus-encoded gene product mediates the inhibition of phospholipase C activation without the need of a virus-induced host factor. Analysis of the processes involved in the formation of inositol (1,4,5)-trisphosphate and mobilization of intracellular Ca2+ indicate that the vaccinia virus gene product exerts its inhibitory effects at the level of phospholipase C activity. This may occur by either directly reducing the amount of phospholipase C, reducing the specific activity of phospholipase C, or by inhibiting the association of phospholipase C with its substrate, phosphatidylinositol 4,5-bisphosphate.
In situ imaging of agonist-sensitive calcium pools in AR4-2J pancreatoma cells. Evidence for an agonist- and inositol 1,4,5-trisphosphate-sensitive calcium pool in or closely associated with the nuclear envelope.
The activation of phospholipase C by hormones and neurotransmitters activates a complex combination of Ca2+ release and accumulation by intracellular organelles. Previously, we demonstrated that, in some cell types, the fluorescent Ca2+ indicator, fura-2, can be loaded into intracellular, agonist-sensitive Ca2+ pools (Glennon, M. C., Bird, G. St. J., Kwan, C.-Y., and Putney, J. W., Jr. (1992) J. Biol. Chem. 267, 8230-8233). In the current study, we have attempted to exploit this phenomenon by employing digital fluorescence imaging of compartmentalized fura-2 to investigate the localization and function of the major intracellular sites of Ca2+ regulation in AR4-2J pancreatoma cells. By judicious use of a surface receptor agonist together with the Ca(2+)-ATPase inhibitor, thapsigargin, cellular regions were identified whose behavior indicates that they contain the sites of agonist- and inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ release. These regions were located throughout the cell and may include the nuclear envelope. They were distinct in locus and behavior from two other regions, which counterstained with fluorescent markers for nuclei and mitochondria. Fura-2 in mitochondrial regions reported low resting levels of [Ca2+], and revealed that organelles in these regions accumulate and retain Ca2+ after agonist activation. These findings demonstrate that fluorescent Ca2+ indicators can be employed to directly monitor changes in [Ca2+] in the major Ca(2+)-regulating organelles, and provide the first in situ visualization and localization of the major sites of Ca2+ regulation in cells.
The interconversion of inositol 1,3,4,5,6-pentakisphosphate and inositol tetrakisphosphates in AR4-2J cells.
Data from several cell types have indicated that activation of hormone receptors promotes the metabolism of inositol 1,3,4,5,6-pentakisphosphate (IP5) to inositol 3,4,5,6-tetrakisphosphate ((3,4,5,6)IP4). However, to date, metabolism of IP5 by cell-free preparations has resulted in the formation of only inositol 1,4,5,6-tetrakisphosphate ((1,4,5,6)IP4). Thus, the metabolic relationships of IP5 with various inositol tetrakisphosphate (IP4) isomers have been investigated in both intact cells and cell homogenates of the rat pancreatoma cell line, AR4-2J. The steady-state concentration of IP5 was estimated to be 65 microM, while the combined concentration of (3,4,5,6)IP4 and (1,4,5,6)IP4 was approximately 1.0 microM. AR4-2J cell homogenates converted (1,3,4,6)IP4, (3,4,5,6)IP4, and (1,4,5,6)IP4 to IP5. (1,4,5,6)IP4 previously has not been demonstrated to be a precursor of IP5. To alter steady-state levels of inositol phosphates that were maintained by phosphorylation-dephosphorylation cycles, intact cells were treated with 10 microM antimycin A which reduced ATP levels by > 90% within 10 min. Following 2 h of treatment with antimycin A, there was a 6-fold increase in both (3,4,5,6)IP4 and (1,4,5,6)IP4, presumably derived from IP5. Experiments with cell-free systems determined that IP5 was dephosphorylated to (1,4,5,6)IP4 by a predominantly particulate Mg(2+)-independent, Li(+)-insensitive IP5 3-phosphatase. However, in the presence of 5 mM MgATP, IP5 also was metabolized to (3,4,5,6)IP4. Therefore, our data demonstrate novel and complex relationships between IP5, (3,4,5,6)IP4, and (1,4,5,6)IP4.
Functional homogeneity of the non-mitochondrial Ca2+ pool in intact mouse lacrimal acinar cells.
In the absence of extracellular Ca2+, treatment of mouse lacrimal acinar cells with maximal concentrations of methacholine released Ca2+ from intracellular stores. No additional Ca2+ was mobilized by subsequent application of the intracellular Ca(2+)-ATPase inhibitor, thapsigargin, the stable inositol 1,4,5-trisphosphate ((1,4,5)IP3) analog, inositol 2,4,5-trisphosphate ((2,4,5)IP3) (by microinjection), or the Ca2+ ionophore, ionomycin. However, following prolonged activation of cells by methacholine in the presence of extracellular Ca2+, Ca2+ accumulated into a pool which was released by ionomycin but not by thapsigargin. This latter accumulation was blocked by prior microinjection of ruthenium red, indicating that it represents mitochondrial uptake. In saponin-permeabilized lacrimal cells, two Ca(2+)-sequestering pools were detected: (i) a ruthenium red-sensitive, thapsigargin-insensitive pool, presumed to be the mitochondria; and (ii) a ruthenium red-insensitive, thapsigargin-sensitive pool. Only the thapsigargin-sensitive pool accumulated Ca2+ at concentrations similar to those in unstimulated cells. The thapsigargin-sensitive Ca2+ pool was sensitive to (1,4,5)IP3; however, in contrast to findings in intact cells, only 44% of this pool was releasable by (1,4,5)IP3 or (2,4,5)IP3. These data indicate that, in intact lacrimal acinar cells, all exchangeable (ionomycin-sensitive) Ca2+ residues in a pool which responds homogeneously to agonists, (1,4,5)IP3, and thapsigargin. Prolonged elevation of [Ca2+]i results in Ca2+ accumulation into a second, ruthenium red-sensitive pool, presumably mitochondria. Finally, permeabilization of the cells fragments the non-mitochondrial pool, resulting in two pools, one sensitive and one insensitive to (1,4,5)IP3.
Sustained Ca2+ signaling in mouse lacrimal acinar cells due to photolysis of "caged" glycerophosphoryl-myo-inositol 4,5-bisphosphate.
In saponin-permeabilized mouse lacrimal acinar cells, glycerophosphoryl-myo-inositol 4,5-bisphosphate (GPIP2) activated the release of sequestered Ca2+ to the same extent as inositol 1,4,5-trisphosphate ((1,4,5)IP3) but with a potency about 1/10 that of (1,4,5)IP3. In lacrimal gland homogenates, [3H]GPIP2 was metabolized to two compounds which upon anion exchange high performance liquid chromatography eluted at positions indicating that they were [3H]GPIP and [3H]GPIP3. The rate of metabolism of [3H]GPIP2 was much slower than that of [3H](1,4,5)IP3, and its rate of phosphorylation was less than 1% of that of [3H] (1,4,5)IP3. In intact lacrimal cells, photolysis of a microinjected "caged" derivative of GPIP2, 1-(alpha-glycerophosphoryl)-myo-inositol 4,5-bisphosphate P4(5)-1-(2-nitrophenyl)ethyl ester, resulted in sustained activation of Ca2+ signaling; i.e. intracellular Ca2+ release followed by increased entry of Ca2+ across the plasma membrane. These findings indicate that caged GPIP2 should provide a useful tool for producing photolytically initiated, sustained activation of intracellular (1,4,5)IP3 receptors. They also provide strong support for the idea that sustained Ca2+ signaling can be achieved in lacrimal acinar cells by activation of intracellular receptors for (1,4,5)IP3 in the absence of stimulated production of inositol 1,3,4,5-tetrakisphosphate.
Actions of vasopressin and the Ca(2+)-ATPase inhibitor, thapsigargin, on Ca2+ signaling in hepatocytes.
When hepatocytes were loaded with fura-2 by incubation with the acetoxymethyl ester (fura-2/AM), addition of Mn2+ resulted in a rapid quench of a fraction of cellular fura-2 fluorescence. Addition of vasopressin caused a second, rapid quench of cellular fura-2, whereas the addition of thapsigargin had no effect. When hepatocytes were loaded by microinjection of fura-2 acid, addition of Mn2+ caused a slower, sustained rate of quench, and both vasopressin and thapsigargin increased this rate of quench. When Mn2+ was removed from the medium of fura-2/AM-loaded cells after preincubation with Mn2+, vasopressin still caused quench of cellular fura-2. In contrast, neither vasopressin nor thapsigargin increased fura-2 quench when Mn2+ was removed from fura-2-injected cells. When fura-2/AM-loaded cells were permeabilized with saponin, only a fraction of the cell-associated fura-2 was quenched by addition of Mn2+. A second fraction was then quenched by addition of inositol 1,4,5-trisphosphate. These results indicate that in hepatocytes loaded with the acetoxymethyl ester of fura-2, the increased quench of cellular fura-2 seen with phospholipase C-linked agonists is not due to effects of the agonist on Mn2+ entry across the plasma membrane, but rather is due to agonist activation of Mn2+ penetration into an intracellular organelle, presumably through inositol 1,4,5-trisphosphate-regulated channels. Thus, it appears that compartmentalization of fura-2 accounts for previously reported anomalies in Ca2+ signaling in hepatocytes, such as the apparent failure of Ca(2+)-ATPase inhibition to increase divalent cation entry, as well as the apparent ability of phospholipase C-linked agonists to stimulate efflux of Ca2+.
Mechanisms of activated Ca2+ entry in the rat pancreatoma cell line, AR4-2J.
The characteristics of Ca2+ entry activated by surface receptor agonists and membrane depolarization were studied in the rat pancreatoma cell line, AR4-2J. Ca2+ mobilization activated by substance P, bombesin, or muscarinic receptor stimulation was found to involve both Ca2+ release and entry. In addition, depolarization of the surface membrane of AR4-2J cells with elevated concentrations of K+ activated Ca2+ entry. Ca2+ entry induced by membrane depolarization was inhibited by the L-channel antagonist, nimodipine, while that due to surface receptor agonists was not inhibited by this agent. The microsomal Ca(2+)-ATPase inhibitor, thapsigargin, caused both depletion of the agonist-sensitive intracellular Ca2+ pool and sustained Ca2+ influx indistinguishable from that produced by bombesin or methacholine. These results confirm that, unlike the pancreatic acinar cells from which they are presumably derived, AR4-2J cells express voltage-sensitive, dihydropyridine-inhibitable Ca2+ channels. However, in contrast to previous reports with this cell line, in the AR4-2J cells in use in our laboratory, and under our experimental conditions, surface receptor agonists (including substance P) do not cause Ca2+ influx through voltage-sensitive Ca2+ channels. Instead, we conclude that agonist-activated Ca2+ mobilization is initiated by (1,4,5)IP3-mediated intracellular Ca2+ release and that Ca2+ influx is regulated primarily, if not exclusively, by the state of depletion of the (1,4,5)IP3-sensitive intracellular Ca2+ pool.