The Inhibition of Capacitative Calcium Entry Due to ATP Depletion but Not Due to Glucosamine Is Reversed by Staurosporine
1999; Elsevier BV; Volume: 274; Issue: 29 Linguagem: Inglês
10.1074/jbc.274.29.20165
ISSN1083-351X
AutoresSudha Vemuri, Richard B. Marchase,
Tópico(s)Pancreatic function and diabetes
ResumoThe capacitative Ca2+ entry pathway in J774 macrophages is rapidly inhibited by the amino sugar glucosamine. This pathway is also inhibited by treatments such as 2-deoxy-d-glucose (2dGlc) or glucose deprivation that inhibit glycolysis and lead to significant decreases in cellular ATP and other trinucleotides. We sought to determine whether glucosamine's effect on capacitative Ca2+ entry was also due to ATP depletion, as has been suggested recently for its link to insulin resistance. In contrast to brief treatments with 2dGlc, there was no significant decrease in ATP following exposure to glucosamine. In addition, the 2dGlc-mediated inhibition of capacitative Ca2+ influx was reversed by staurosporine, a microbial alkaloid that inhibits a broad range of protein kinases. Staurosporine was also able to reverse the inhibition of capacitative Ca2+ entry seen following other treatments that decreased cellular ATP levels, including cytochalasin B and iodoacetic acid. Other inhibitors of protein kinase C, including bisindolylmaleimide, K252a, H-7, and calphostin C, were unable to mimic this effect of staurosporine. However, the inhibition of capacitative Ca2+influx in the presence of glucosamine was not reversed by staurosporine. These data indicate that the inhibitory action on capacitative Ca2+ entry of glucosamine is distinct from that caused by ATP depletion. The capacitative Ca2+ entry pathway in J774 macrophages is rapidly inhibited by the amino sugar glucosamine. This pathway is also inhibited by treatments such as 2-deoxy-d-glucose (2dGlc) or glucose deprivation that inhibit glycolysis and lead to significant decreases in cellular ATP and other trinucleotides. We sought to determine whether glucosamine's effect on capacitative Ca2+ entry was also due to ATP depletion, as has been suggested recently for its link to insulin resistance. In contrast to brief treatments with 2dGlc, there was no significant decrease in ATP following exposure to glucosamine. In addition, the 2dGlc-mediated inhibition of capacitative Ca2+ influx was reversed by staurosporine, a microbial alkaloid that inhibits a broad range of protein kinases. Staurosporine was also able to reverse the inhibition of capacitative Ca2+ entry seen following other treatments that decreased cellular ATP levels, including cytochalasin B and iodoacetic acid. Other inhibitors of protein kinase C, including bisindolylmaleimide, K252a, H-7, and calphostin C, were unable to mimic this effect of staurosporine. However, the inhibition of capacitative Ca2+influx in the presence of glucosamine was not reversed by staurosporine. These data indicate that the inhibitory action on capacitative Ca2+ entry of glucosamine is distinct from that caused by ATP depletion. The amino sugar glucosamine has been shown to have a variety of effects on cell and animal physiology. Numerous reports dating from over 40 years ago (1Quastel J.H. Cantero A. Nature. 1953; 171: 252-254Crossref PubMed Scopus (97) Google Scholar, 2Friedman S.J. Skehan P. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1172-1176Crossref PubMed Scopus (60) Google Scholar) document that dietary glucosamine is selectively toxic to some experimentally induced tumors in rodents. In addition, Marshall et al. (3Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar) determined that exogenous glucosamine induced insulin resistance in cultured adipocytes in a manner similar to that caused by hyperglycemia but at a 40-fold lower concentration than that required for glucose. They also showed that inhibition of glucose flux through the hexosamine biosynthetic pathway prevented hyperglycemia-induced insulin resistance from developing. These results have been extended to show that insulin resistance develops in animals infused with glucosamine (4Rossetti L. Hawkins M. Chen W. Gindi J. Barzilai N. J. Clin. Invest. 1995; 96: 132-140Crossref PubMed Scopus (243) Google Scholar, 5Kim Y.-B. Zhu J.-S. Zierath J.R. Shen H.-Q. Baron A.D. Kahn B.B. Diabetes. 1999; 48: 310-320Crossref PubMed Scopus (83) Google Scholar) or in cells (6Crook E.D. Daniels M.C. Smith T.M. McClain D.A. Diabetes. 1993; 42: 1289-1296Crossref PubMed Google Scholar) and animals (7Hebert Jr., L.F. Daniels M.C. Zhou J. Crook E.D. Turner R.L. Simmons S.T. Neidigh J.L. Zhu J.S. Baron A.D. McClain D.A. J. Clin. Invest. 1996; 98: 930-936Crossref PubMed Scopus (282) Google Scholar) that overexpress the rate-limiting enzyme in the hexosamine biosynthetic pathway, glutamine:fructose-6-phosphate amidotransferase. Glucosamine treatment has also been shown to elicit the expression of transforming growth factor α (8Daniels M.C. Kansal P. Smith T.M. Paterson A.J. 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Forsch. 1998; 48: 469-474PubMed Google Scholar, 14Barclay T.S. Tsourounis C. McCart G.M. Ann. Pharmacother. 1998; 32: 574-579Crossref PubMed Scopus (98) Google Scholar) as a treatment for osteoarthritis. It has been suggested that glucosamine's efficacy is due to a stimulation in the synthesis of glycosaminoglycans and other glycoconjugates (12Theodosakis J. Adderly B. Fox B. The Arthritis Cure. St. Martin's Press, New York1997Google Scholar,14Barclay T.S. Tsourounis C. McCart G.M. Ann. Pharmacother. 1998; 32: 574-579Crossref PubMed Scopus (98) Google Scholar), although experimental data supporting this suggestion are limited. Here, we present data that suggest an alternative mechanism to explain at least some of glucosamine's effects. In macrophages and other nonexcitable cells, Ca2+ influx across the plasma membrane is triggered by the depletion of Ca2+ from intracellular, inositol 1,4,5-trisphosphate-sensitive stores. The formation of inositol 1,4,5-trisphosphate is not a prerequisite for this influx to be initiated, as release of Ca2+ from these stores by other means, including an inhibition of the endoplasmic reticulum Ca2+-ATPase, can cause this influx pathway to be activated (15Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3010) Google Scholar). Thus influx has been termed store-operated (16Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2272) Google Scholar) or capacitative Ca2+ entry (17Putney Jr., J.W. Bird G.S.J. Endocr. Rev. 1993; 14: 610-631Crossref PubMed Scopus (486) Google Scholar). The mechanisms regulating the capacitative Ca2+ entry pathway are unknown. It is at present uncertain as to whether information regarding the concentration of Ca2+ sequestered in the endoplasmic reticulum is communicated via a direct physical coupling (18Petersen C.C.H. Berridge M.J. J. Biol. 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A. 1999; 96: 121-126Crossref PubMed Scopus (103) Google Scholar). In addition, several protein kinases (18Petersen C.C.H. Berridge M.J. J. Biol. Chem. 1994; 269: 32246-32253Abstract Full Text PDF PubMed Google Scholar, 26Shibata K. Morita K. Kitayama S. Okamoto H. Dohi T. Biochem. Pharmacol. 1996; 52: 167-171Crossref PubMed Scopus (17) Google Scholar, 27Morley P. Chakravarthy B.R. Mealing G.A.R. Tsang B.K. Whitfield J.F. Eur. J. Endocrinol. 1996; 134: 734-750Crossref Scopus (11) Google Scholar) have been proposed as playing regulatory and antagonistic roles in the capacitative Ca2+ entry pathway. A facilitating role for certain protein phosphorylation events is suggested by the findings that depletion of ATP in intact cells inhibits capacitative Ca2+ entry (28Gamberucci A. Innocenti B. Fulceri R. Banhegyi G. Guinti R. Pozzan T. Benedetti A. J. Biol. Chem. 1994; 269: 23597-23602Abstract Full Text PDF PubMed Google Scholar, 29Marriott I. Mason M.J. Am. J. Physiol. 1995; 269: C766-C774Crossref PubMed Google Scholar, 30Darbha S. Marchase R.B. Cell Calcium. 1996; 20: 361-371Crossref PubMed Scopus (9) Google Scholar), and that okadaic acid, a phosphatase inhibitor, augments it (31Randriamampita C. Tsien R.Y. J. Biol. Chem. 1995; 270: 29-32Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 32Thomas D. Hanley M.R. J. Biol. Chem. 1995; 270: 6429-6432Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). On the other hand, in whole cell patch clamp experiments ATP is not necessary for the activation of the current underlying capacitative Ca2+entry, and in fact the presence of ATP leads to its inhibition (33Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (155) Google Scholar). Also, activators of protein kinase C have repeatedly been found to regulate this process negatively (18Petersen C.C.H. Berridge M.J. J. Biol. Chem. 1994; 269: 32246-32253Abstract Full Text PDF PubMed Google Scholar, 26Shibata K. Morita K. Kitayama S. Okamoto H. Dohi T. Biochem. Pharmacol. 1996; 52: 167-171Crossref PubMed Scopus (17) Google Scholar, 27Morley P. Chakravarthy B.R. Mealing G.A.R. Tsang B.K. Whitfield J.F. Eur. J. Endocrinol. 1996; 134: 734-750Crossref Scopus (11) Google Scholar). Staurosporine, an inhibitor of several protein kinases, including protein kinase C (34Meggio F. Donella Deana A. Ruzzene M. Brunati A.M. Cesaro L. Guerra B. Meyer T. Mett H. Fabbro D. Furet P. Dobrawalska G. Pinna L.A. Eur. J. Biochem. 1995; 234: 317-322Crossref PubMed Scopus (251) Google Scholar), has in several reports been shown to augment capacitative Ca2+ entry (35Liu X. O'Connell A. Ambudkar I.S. J. Biol. Chem. 1998; 273: 33295-33304Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 36Tojyo Y. Tanimura A. Matsumoto Y. Jpn. J. Pharmacol. 1995; 69: 381-389Crossref PubMed Scopus (12) Google Scholar, 37Petersen C.C.H. Berridge M.J. Biochem. J. 1995; 307: 663-668Crossref PubMed Scopus (54) Google Scholar, 38Mene P. Pugliese F. Cinotti G.A. J. Am. Soc. Nephrol. 1996; 7: 983-990PubMed Google Scholar, 39Ahnadi C.E. Payet M.D. Dupuis G. Cell Calcium. 1996; 19: 509-520Crossref PubMed Scopus (17) Google Scholar). This suggests that different protein kinases may be active at different steps in the regulation of this pathway and that the balance among antagonistic regulatory reactions dependent on protein phosphorylation may dictate the time dependence and/or steady-state level of Ca2+ influx. Here, these issues were investigated in cells in which ATP had been depleted with several inhibitors of glycolysis, including 2-deoxyglucose (2dGlc). 1The abbreviations 2dGlc2-deoxy-d-glucoseHBSHepes-buffered saline solutionGTPγSguanosine 5′-3-O-(thio)triphosphateTgthapsigarginstpstaurosporine Following ATP depletion capacitative Ca2+ entry is inhibited, as might be expected if an activating kinase were operating less than optimally. However, in the presence of staurosporine a balance in regulation appears to be re-achieved such that capacitative Ca2+ entry is substantially restored. 2-deoxy-d-glucose Hepes-buffered saline solution guanosine 5′-3-O-(thio)triphosphate thapsigargin staurosporine In the course of these investigations monosaccharides other than 2dGlc were also examined for their effects on capacitative Ca2+entry. Surprisingly, short incubations with glucosamine and mannosamine were found to selectively inhibit this pathway via a mechanism that was not accompanied by a loss of ATP and could not be reversed by staurosporine. This inhibition may be significant to the utilization of glucosamine as a treatment for osteoarthritis (14Barclay T.S. Tsourounis C. McCart G.M. Ann. Pharmacother. 1998; 32: 574-579Crossref PubMed Scopus (98) Google Scholar), since capacitative Ca2+ entry is critical to the transcriptional regulation of cytokines in immune cells (40Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar, 41Dolmetsch R.E. Lewis R.S. Goodenow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1564) Google Scholar). J774 cells (American Type Culture Collection) were cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.) and 1% penicillin/streptomycin. Hepes-buffered saline solution (HBS) comprised 125 mm NaCl, 5 mm KCl, 1 mm MgSO4, 1 mm KH2PO4, 10 mm NaHCO3, 1 mmCaCl2, 20 mm Hepes, pH 7.3. J774 cells were washed in serum-free Dulbecco's modified Eagle's medium and resuspended in Dulbecco's modified Eagle's medium containing 1 mg/ml bovine serum albumin and 2.5 mm probenecid. Probenecid was added to decrease the leakage of Fura-2 (42DiVirgilio F. Meyer B.S. Greenberg S. Silverstein S.C. J. Cell Biol. 1988; 106: 657-666Crossref PubMed Scopus (146) Google Scholar). After a 30-min incubation at 37 °C, the cells were centrifuged, washed, and resuspended in HBS. Fluorescence measurements were performed in a fluorescence spectrophotometer (Photon Technologies Inc.) with the cells suspended in a cuvette in a temperature-controlled chamber (37 °C) equipped with a magnetic stirrer. The fluorescence intensity was measured at 510 nm with excitation wavelengths of 340 and 380 nm. [Ca2+]i was calculated as described by Grynkiewicz et al. (43Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar): [Ca2+]i =K d × [R −R min]/[R max −R] ×S f2/S b2, whereK d is the Fura-2 dissociation constant for Ca2+ (224 nm), R is the ratio of the intensities at 340 nm and 380 nm, and R min andR max are the R values at 0 and saturating levels of Ca2+, respectively.S f2/S b2 is the ratio of the intensities at 380 nm excitation underR min and R max conditions. In presentations in which traces overlap, base-line values ranged between 70 and 120 nm with no significant differences being seen among the various conditions. Cellular ATP levels were determined using an ATP assay kit (Calbiochem) based on firefly luciferase-catalyzed oxidation of d-luciferin. The emitted light was quantitated by luminometry. Cells (106/ml) were incubated in HBS containing the indicated nutrients or inhibitors for 4 min at 37 °C. The cells were then centrifuged and extracted with 1m HCl04 at 4 °C. The extracts were neutralized with 5 m K2CO3. A 10-μl aliquot of the extract was added to 400 μl of HBS buffer, and the reaction was initiated by addition of the luciferase. The addition of thapsigargin to Fura-2-loaded J774 cells leads to a sustained elevation of [Ca2+]i. This irreversible inhibitor of the endoplasmic reticulum Ca2+-ATPase causes depletion of intracellular Ca2+ stores and thereby activates the capacitative influx of Ca2+ (15Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3010) Google Scholar). The elevated plateau value of [Ca2+]i is ∼100 nm higher than the starting base line in J774 macrophages under our standard assay conditions (Fig. 1 A), reflecting the new balance between Ca2+-extruding mechanisms active at the plasma membrane and the ongoing capacitative influx of Ca2+. As we reported previously (30Darbha S. Marchase R.B. Cell Calcium. 1996; 20: 361-371Crossref PubMed Scopus (9) Google Scholar), a 4-min pretreatment with 2dGlc inhibits the capacitative Ca2+ influx pathway in these cells (Fig. 1 E). We speculated that local changes in ATP levels caused by 2dGlc could lead to a selective decrease in the activity of a protein kinase that was necessary for a sustained capacitative Ca2+ entry, thus altering the equilibrium between antagonistic kinases controlling this response. Staurosporine is a microbial alkaloid that was initially described as an inhibitor of protein kinase C but has since been shown to be a broad range inhibitor of protein kinase activity (34Meggio F. Donella Deana A. Ruzzene M. Brunati A.M. Cesaro L. Guerra B. Meyer T. Mett H. Fabbro D. Furet P. Dobrawalska G. Pinna L.A. Eur. J. Biochem. 1995; 234: 317-322Crossref PubMed Scopus (251) Google Scholar). Staurosporine augments capacitative Ca2+ influx in rat parotid acinar (36Tojyo Y. Tanimura A. Matsumoto Y. Jpn. J. Pharmacol. 1995; 69: 381-389Crossref PubMed Scopus (12) Google Scholar) and mesangial cells (38Mene P. Pugliese F. Cinotti G.A. J. Am. Soc. Nephrol. 1996; 7: 983-990PubMed Google Scholar) and modulates Ca2+ responses in Jurkat T lymphocytes (39Ahnadi C.E. Payet M.D. Dupuis G. Cell Calcium. 1996; 19: 509-520Crossref PubMed Scopus (17) Google Scholar). Also, in Xenopus oocytes the t 12of inhibition of capacitative Ca2+ entry by GTPγS was found to be increased by staurosporine (37Petersen C.C.H. Berridge M.J. Biochem. J. 1995; 307: 663-668Crossref PubMed Scopus (54) Google Scholar). Most recently, a staurosporine-sensitive kinase was shown to be critical to the Ca2+-dependent down-regulation of capacitative Ca2+ entry in human submandibular gland cells (35Liu X. O'Connell A. Ambudkar I.S. J. Biol. Chem. 1998; 273: 33295-33304Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The effect of staurosporine on capacitative Ca2+ influx in J774 macrophages was therefore tested both with and without 2dGlc pretreatment. We found that a 5-min incubation with staurosporine, either prior to or after 2dGlc addition, resulted in approximately an 80% reversal of the inhibition seen in the presence of 2dGlc alone (Fig. 1, Cand D). As can be seen by comparing Fig. 1, A with E, the initial peak height following the addition of thapsigargin, in addition to the capacitative plateau, is lower in the presence of 2dGlc. This is due not only to the inhibition of capacitative Ca2+ influx by 2dGlc, but to the ability of 2dGlc to partially deplete intracellular, thapsigargin-sensitive Ca2+ stores (30Darbha S. Marchase R.B. Cell Calcium. 1996; 20: 361-371Crossref PubMed Scopus (9) Google Scholar). Upon the addition of staurosporine, the 2dGlc-mediated inhibition of capacitative Ca2+ influx is relieved, although the release of Ca2+ from intracellular stores is not reversed (Fig. 1and data not shown). Unlike the finding in rat parotid acinar cells (44Tojyo Y. Tanimura A. Matsui S. Matsumoto Y. Sugiya H. Furuyama S. Am. J. Physiol. 1991; 260: C194-C200Crossref PubMed Google Scholar), staurosporine did not affect the capacitative Ca2+entry pathway in untreated J774 cells (Fig. 1 B), even at concentrations of up to 150 nm. This suggests that the effect of staurosporine is not due to an inhibition of the plasma membrane Ca2+ATPase or the opening of a different Ca2+ channel, but rather to a reversal of the inhibition of capacitative Ca2+ entry seen with 2dGlc. The inhibition of capacitative Ca2+ influx by 2dGlc can also be demonstrated by its addition after the pathway has been activated. In Fig. 2 Fura-2-loaded cells were treated with thapsigargin, and once a plateau value for [Ca2+]i was established, 2dGlc was added. Within ∼1 min of its addition [Ca2+]i decreased. The subsequent addition of 40 nm staurosporine caused [Ca2+]i to return to the initial, higher plateau value. Following the recovery of Ca2+ influx by staurosporine, treatment with SKF 96365, an inhibitor of Ca2+ influx via the capacitative entry pathway (45Merritt J.E. Armstrong W.P. Benham C.D. Hallam T.J. Jacob R. Jaxa-Chamiec A. Leigh B.K. McCarthy S.A. Biochem. J. 1990; 271: 515-522Crossref PubMed Scopus (704) Google Scholar), caused [Ca2+]i to return to base line. This, like the finding that staurosporine itself caused no increase over the normal capacitative Ca2+ plateau (Fig. 1), supports the premise that the effect of staurosporine on Ca2+ influx is attributable to capacitative Ca2+ entry. In order to determine whether the effect of staurosporine is limited to reversing only 2dGlc-mediated inhibition of capacitative Ca2+ entry, we tried alternate ways of inhibiting glycolysis. The addition of iodoacetic acid also caused a decrease in Ca2+ influx that was reversed by the subsequent addition of staurosporine (Fig.3 A). Addition of cytochalasin B, an inhibitor of glucose transport (46Salter D.W. Weber M.J. J. Biol. Chem. 1979; 254: 3554-3561Abstract Full Text PDF PubMed Google Scholar), also caused a decrease in Ca2+ influx similar to that seen upon the addition of 2dGlc (Fig. 3 B). The subsequent addition of staurosporine restored the influx to normal. Dihydrocytochalasin B, which has a similar effect as cytochalasin B on the cytoskeleton but does not interfere with glucose transport (47Atlas S.J. Magargal W.W. Lin S. J. Recept. Res. 1980; 1: 113-135Crossref PubMed Scopus (5) Google Scholar), had no effect on capacitative Ca2+influx (data not shown). This is in agreement with our previous results indicating that glucose deprivation inhibits the influx of Ca2+ via the capacitative entry pathway (30Darbha S. Marchase R.B. Cell Calcium. 1996; 20: 361-371Crossref PubMed Scopus (9) Google Scholar). Thus, it appears that staurosporine is able to reverse compromised capacitative Ca2+ entry that is accompanied by and presumably due to a decrease in cellular ATP levels. An excess of glucose can overcome the 2dGlc-mediated inhibition in capacitative Ca2+ influx. As shown in Fig. 3 C, an inhibition of capacitative Ca2+ entry can also be achieved by the addition of 5 mm 2dGlc to cells suspended in medium containing 1 mm glucose. Upon the addition of excess glucose (10 mm), the inhibition is reversed. As expected, the reversal in this case is accompanied by an increase in cellular ATP levels (data not shown). We next sought to determine the effects of a 4-min incubation with monosaccharides other than 2dGlc on the activation of capacitative Ca2+ influx by thapsigargin. As shown in Fig. 4, galactose, mannose,N-acetylglucosamine, and 3-O-methylglucose were without effect. However, glucosamine and mannosamine caused nearly complete inhibition in the ongoing elevation of Ca2+, indicating an absence of capacitative Ca2+ influx. The inhibition in capacitative Ca2+ entry by these amino sugars can also be demonstrated in a Ca2+ add-back experiment. Fura-2-loaded J774 cells were suspended in a nominally Ca2+-free buffer. Glucosamine was added 5 min prior to the addition of thapsigargin. Following release from intracellular stores, Ca2+ was added to the medium so that the entry of Ca2+ via the capacitative Ca2+ pathway could be observed in isolation (Fig. 5). Glucosamine caused little change in the release of intracellular Ca2+ from stores by thapsigargin. However, the capacitative entry of Ca2+ was clearly inhibited. We asked if staurosporine could reverse the inhibition of the capacitative Ca2+ influx seen in the presence of the amino sugars. As shown in Fig. 6, staurosporine was unable to reverse the inhibition in capacitative Ca2+influx that is brought about by the action of glucosamine or mannosamine. Since glucosamine's link to insulin resistance has been attributed to ATP depletion (11Hresko R.C. Heimberg H. Chi M.M.-Y. Mueckler M. J. Biol. Chem. 1998; 273: 20658-20668Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), we asked if, like 2Glc, short term glucosamine treatments sufficient to inhibit capacitative Ca2+ entry would lead to drops in cellular ATP levels. A 4-min treatment in the presence of the amino sugars did not decrease ATP levels to an extent that was statistically significant. This is in contrast to the much more substantial decreases obtained with 2dGlc (Fig. 7). The effect of staurosporine on cellular ATP levels was also investigated. Staurosporine had no effect on ATP levels in the presence or absence of 2dGlc. Thus, staurosporine is not reversing the 2dGlc-mediated inhibition by countering the effect of 2dGlc on intracellular ATP levels. Based on previous reports on the involvement of protein kinases in the capacitative pathway for Ca2+ entry, the most likely target for staurosporine's action is protein kinase C (35Liu X. O'Connell A. Ambudkar I.S. J. Biol. Chem. 1998; 273: 33295-33304Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 37Petersen C.C.H. Berridge M.J. Biochem. J. 1995; 307: 663-668Crossref PubMed Scopus (54) Google Scholar). To determine whether staurosporine was having its effects via inhibition of this serine/threonine kinase, the effects of alternate protein kinase C inhibitors were examined. K252a, bisindolylmaleimide, calphostin C, and H-7 were all unable to mimic the effect of staurosporine (Fig. 8). A dose response with staurosporine (Fig. 9) also revealed that its effect is not likely to be due to protein kinase C inhibition, since the IC50 for protein kinase C inhibition has been reported to be ∼5 nm (34Meggio F. Donella Deana A. Ruzzene M. Brunati A.M. Cesaro L. Guerra B. Meyer T. Mett H. Fabbro D. Furet P. Dobrawalska G. Pinna L.A. Eur. J. Biochem. 1995; 234: 317-322Crossref PubMed Scopus (251) Google Scholar), whereas a concentration of ∼18 nm is half-maximal for the reversal of the inhibition of capacitative Ca2+ influx in our experiments.Figure 9Dose-dependent effect of staurosporine on capacitative Ca2+ influx. Tg was added to Fura-2-loaded cells suspended in HBS containing 5 mm glucose and 5 mm pyruvate, and a stable plateau of [Ca2+]i was achieved. At each of the staurosporine concentrations used, the new plateau value of Ca2+ was then measured following the addition of nothing further or stp alone (A); the latter is depicted as a percent of the former; 25 mm 2dGlc or 25 mm2dGlc and then 4 nm staurosporine (B); 25 mm 2dGlc or 40 nm stp and then 2dGlc (C). InB and C the percent reversal of the 2dGlc-mediated inhibition seen as a result of staurosporine treatment is depicted. Each determination is the mean of three independent experiments.View Large Image Figure ViewerDownload (PPT) Other experiments were performed to determine whether activators of protein kinase C might cause a decrease in capacitative Ca2+ entry similar to that seen with 2dGlc. Phorbol myristate acetate, an activator of protein kinase C, did not have any effects on the capacitative influx pathway in these cells in the presence or absence of 2dGlc. Phorbol myristate acetate has been shown previously to inhibit the Ca2+ response to IgG in these cells (42DiVirgilio F. Meyer B.S. Greenberg S. Silverstein S.C. J. Cell Biol. 1988; 106: 657-666Crossref PubMed Scopus (146) Google Scholar). The finding that in our experiments phorbol myristate acetate was again able to suppress the response to IgG excludes the possibility that under the conditions/concentrations used the phorbol ester was ineffective in activating protein kinase C in our cells (data not shown). We had shown previously an inhibition of capacitative Ca2+ influx within 4 min of 2dGlc treatment or glucose deprivation in J774 macrophages (30Darbha S. Marchase R.B. Cell Calcium. 1996; 20: 361-371Crossref PubMed Scopus (9) Google Scholar). Here, we found that staurosporine, a microbial alkaloid that inhibits a broad range of protein kinases, is able to reverse the 2dGlc-mediated inhibition of the capacitative Ca2+ influx pathway. The decreases in influx following other means of inhibiting glycolysis were also reversed by staurosporine. However, the identity of staurosporine's target remains unclear, as protein kinase C does not appear to be the object of its action. We also found that the amino sugars glucosamine and mannosamine inhibited capacitative Ca2+ influx. The inhibitory action of these amino sugars appears to be distinct from that of 2dGlc. This conclusion is based in part on our observation that in the presence of either of these sugars there was no significant decrease in ATP levels. In addition, all treatments investigated that led to an inhibition of the capacitative Ca2+ pathway that was accompanied by a decrease in cellular ATP were reversed by the addition of staurosporine. In contrast, the inhibitory effects of glucosamine and mannosamine were not reversed by this kinase inhibitor. The mechanism by which staurosporine restores the block in capacitative Ca2+ entry caused by depletion of ATP is still undetermined. It is likely that protein kinases play regulatory and possibly counter-balancing roles in controlling the magnitude of capacitative Ca2+ influx. It is possible that an activating kinase is preferentially inhibited when ATP levels fall, allowing an inhibitory kinase with a lower K m for ATP to dominate. This could be responsible for the inhibition seen with 2dGlc and other glycolytic inhibitors. In such a model, staurosporine is proposed to selectively inhibit the proposed inhibitory kinase so as to allow the system to re-achieve a balance permissive for capacitative Ca2+ influx. A related possibility is that a staurosporine-sensitive inhibitory kinase with a lowK m for ATP is kept in check by phosphorylation. When ATP falls, it becomes dephosphorylated and in turn phosphorylates and thereby inhibits an element important for capacitative Ca2+entry. In evaluating such models it should be noted that an activating kinase does not appear to be essential for the initiation of capacitative Ca2+ entry, at least in patch clamp experiments. When such experiments are carried out with no ATP in the pipette solution capacitative Ca2+ entry activates within minutes of whole cell break-in (33Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (155) Google Scholar). The relationship between this mechanism of activation and the necessity for ATP in whole cell experiments observed both by us (30Darbha S. Marchase R.B. Cell Calcium. 1996; 20: 361-371Crossref PubMed Scopus (9) Google Scholar) and, for instance, Gamberucci et al. (28Gamberucci A. Innocenti B. Fulceri R. Banhegyi G. Guinti R. Pozzan T. Benedetti A. J. Biol. Chem. 1994; 269: 23597-23602Abstract Full Text PDF PubMed Google Scholar) is still unclear. It should also be noted that a decrease in GTP cannot be experimentally dissociated from a decrease in ATP (33Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (155) Google Scholar). Thus, the inhibitory effects observed with 2dGlc could be due to decreases in a trinucleotide other than ATP. Previously, staurosporine had been shown in a variety of cell types to augment capacitative Ca2+ influx (36Tojyo Y. Tanimura A. Matsumoto Y. Jpn. J. Pharmacol. 1995; 69: 381-389Crossref PubMed Scopus (12) Google Scholar, 37Petersen C.C.H. Berridge M.J. Biochem. J. 1995; 307: 663-668Crossref PubMed Scopus (54) Google Scholar, 38Mene P. Pugliese F. Cinotti G.A. J. Am. Soc. Nephrol. 1996; 7: 983-990PubMed Google Scholar, 39Ahnadi C.E. Payet M.D. Dupuis G. Cell Calcium. 1996; 19: 509-520Crossref PubMed Scopus (17) Google Scholar). In several of these experiments staurosporine's effect targeted a kinase other than protein kinase C, since other inhibitors of this enzyme were, as seen here, ineffective. The most parsimonious explanation for the data presented here is that staurosporine's effect is on a protein kinase that is capable of inactivating capacitative Ca2+ entry and that is relatively resistant to the initial decreases in intracellular ATP brought about by glycolytic inhibitors. The assertion that glucosamine can affect biological processes via a mechanism independent of ATP depletion contrasts with arguments recently put forth by Hresko et al. (11Hresko R.C. Heimberg H. Chi M.M.-Y. Mueckler M. J. Biol. Chem. 1998; 273: 20658-20668Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). They reported that glucosamine treatment of 3T3-L1 adipocytes dramatically decreased cellular ATP and that subsequently this prevented normal levels of insulin-stimulated protein phosphorylation from occurring. Furthermore, they suggested that this was the mechanism responsible for the insulin resistance caused by glucosamine in these cells. They went on to suggest that many of the other biological effects of glucosamine were likely to be due to ATP depletion. However, in the data presented here and in a related study by Bounelis et al. 2P. Bounelis, Z. Su, E. A. Johnson, H. McFerrin, W. Bennett, J. E. Blalock, and R. B. Marchase, submitted for publication. we did not observe significant decreases in ATP in response to short term glucosamine treatments. The difference between our findings and those reported by Hresko et al. (11Hresko R.C. Heimberg H. Chi M.M.-Y. Mueckler M. J. Biol. Chem. 1998; 273: 20658-20668Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) would appear to be due to the provision of alternate fuels from which ATP can be generated. In our experiments 5 mm glucose and 5 mm pyruvate were present along with glucosamine. In the experiments reported by Hresko et al. (11Hresko R.C. Heimberg H. Chi M.M.-Y. Mueckler M. J. Biol. Chem. 1998; 273: 20658-20668Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) the cells were starved for glucose for several hours prior to the addition of glucosamine. This no doubt exacerbated ATP depletion and would appear to be a significant procedural difference relative to the experiments reported here. Under conditions in which glucose is present along with glucosamine, the data presented here and elsewhere2 suggest that a non-ATP-dependent inhibition of capacitative Ca2+ entry must be considered as a mechanism by which glucosamine affects cell physiology. In addition, a recent publication by Kim et al. (5Kim Y.-B. Zhu J.-S. Zierath J.R. Shen H.-Q. Baron A.D. Kahn B.B. Diabetes. 1999; 48: 310-320Crossref PubMed Scopus (83) Google Scholar) showed following glucosamine treatment no inhibition of insulin-stimulated phosphorylation of the insulin receptor or IRS-1. The authors concluded that the inhibition caused by glucosamine was at a step distal to these early phosphorylation events. In a related study by Bounelis et al.,2 we have determined that short term glucosamine inhibits capacitative Ca2+ entry in Jurkat T lymphocytes, RBL-2 cells, and BHK-21 cells. In those experiments, the influx and metabolism of glucosamine was followed utilizing [3H]glucosamine. We observed an increase in intracellular levels of glucosamine, glucosamine-6-P, and UDP-GlcNAc. The most likely candidate for mediating the effect of extracellular glucosamine on capacitative Ca2+ entry is the initial intracellular metabolite of glucosamine, glucosamine 6-phosphate. Whole-cell patch clamp experiments in RBL-2 cells determined that glucosamine 6-phosphate, but not other intracellular metabolites of glucosamine, inhibited the trans-plasma membrane currentI CRAC, the Ca2+release activated Ca2+current regulated by Ca2+ store depletion and responsible for capacitative Ca2+ entry in those cells. We propose that this metabolite is also responsible for the inhibition seen here in J774 cells. Increased flux through the glucosamine pathway that could lead to the accumulation of intracellular glucosamine metabolites has been reported to be a response to hyperglycemia (49Buse M.G. Robinson K.A. Gettys T.W. McMahon E.G. Gulve E.A. Am. J. Physiol. 1997; 272: E1080-E1088PubMed Google Scholar). In addition, such increases may occur in response to dietary glucosamine, which is currently being widely used as an alternative treatment for osteoarthritis (12Theodosakis J. Adderly B. Fox B. The Arthritis Cure. St. Martin's Press, New York1997Google Scholar). An inhibition in capacitative Ca2+ entry due to excessive hexosamine biosynthesis could affect a number of physiological processes, including several important to the capacity to combat infections and the onset of inflammation. For instance, increases in [Ca2+]i have been shown to accompany phagocytosis in macrophages (42DiVirgilio F. Meyer B.S. Greenberg S. Silverstein S.C. J. Cell Biol. 1988; 106: 657-666Crossref PubMed Scopus (146) Google Scholar) and to be necessary for phagosome/lysosome fusion in neutrophils (50Jaconi M.E. Lew D.P. Carpentier J.L. Magnusson K.E. Sjogren M. Stendahl O. J. Cell Biol. 1990; 110: 1555-1564Crossref PubMed Scopus (177) Google Scholar). However, phagosome/lysosome fusion in macrophages appears to be a Ca2+-independent event (51Zimmerli S. Majeed M. Gustavsson M. Stendahl O. Sanan D.A. Ernst J.D. J. Cell Biol. 1996; 132: 49-61Crossref PubMed Scopus (91) Google Scholar). In addition, capacitative Ca2+ entry is important to certain aspects of gene regulation. The best described examples of this involve transcription mediated by members of the nuclear factor of activated T cells family. These transcription factors require capacitative Ca2+ entry in order to provide for the ongoing activation of calcineurin and the subsequent sustained nuclear localization of nuclear factor of activated T cells family members (40Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). This transcription factor family is critical to production of a number of cytokines that greatly influence both the selection of peripheral T cell populations and the function of other cells more directly involved in combating infections. For instance, granulocyte-macrophage colony-stimulating factor is dependent upon an nuclear factor of activated T cells family member for its transcriptional control (40Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). Such alterations in cytokine environment could be expected to have effects on host defense against a variety of infectious agents. Because glucosamine metabolites increase during hyperglycemia (49Buse M.G. Robinson K.A. Gettys T.W. McMahon E.G. Gulve E.A. Am. J. Physiol. 1997; 272: E1080-E1088PubMed Google Scholar), the inhibition of capacitative Ca2+ entry seen here may be relevant to the inability of diabetic individuals to effectively combat infections. For instance, Rayfield et al. (52Rayfield E.J. Ault M.J. Keusch G.T. Brothers M.J. Nechemias C. Smith H. Am. J. Med. 1982; 72: 439-450Abstract Full Text PDF PubMed Scopus (530) Google Scholar) found a striking correlation between the prevalence of infection and mean plasma glucose levels in diabetic outpatients. Consistent with these data is a more recent report by Zerr et al. (48Zerr K.J. Furnary A.P. Grunkemeier G.L. Bookin S. Kanhere V. Starr A. Ann. Thorac. Surg. 1997; 63: 356-361Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar) in which infection rates increased with blood glucose in patients recovering from chest surgery. Those individuals with the highest blood glucose levels suffered from rates of infection more than 15 times those seen in a control population. Interestingly, implementation of a glucose control protocol led to a 40% drop in infection rate among the diabetic population. Last, glucosamine is currently being widely used as an alternative treatment for osteoarthritis (12Theodosakis J. Adderly B. Fox B. The Arthritis Cure. St. Martin's Press, New York1997Google Scholar). The data presented here suggest that, rather than acting to enhance glycosaminoglycan synthesis, a change in cytokine profiles and subsequent decreases in inflammatory immune responses could underlie the putative effectiveness of such treatments. We thank Sherry Crittenden for expert secretarial assistance and Pam Bounelis, Ph.D. for her insightful critique and assistance with graphics.
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