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Regulation by Purinergic Agonists of Zinc Uptake by Rat Submandibular Glands
J. Trace Elements Med. BioI. Vol. 9, pp. 94-101 (1995)
Regulation by Purinergic Agonists of Zinc Uptake by Rat Submandibular Glands J.P. DEHAYE Laboratoire de Biochimie generale et humaine, Institut de Pharmacie, Universite libre de Bruxelles B1050 - Bruxelles, Belgium (Received September 1994IFebruary 1995)
Summary
The zinc uptake in rat submandibular cells was measured using fura2 as a fluorescent probe. Basal zinc uptake was observed in a 100 flM - 1 mM concentration range. Carbachol and isoproterenol had no effect but ATp4- dose-dependently increased the basal zinc uptake (half-maximal concentration: 250 flM). The purinergic agonist shifted the concentration curve for zinc to the left by one order of magnitude. The response to ATP was not reprodu~ed by adenosine or ADP and was blocked by Coomassie blue. Calcium, nickel or lanthanum were inhibitors of zinc uptake, while the substitution of extracellular sodium by potassium or lithium increased the basal zinc uptake. We conclude that in submandibular cells zinc can permeate through the non-specific cation channel coupled to ATP-sensitive purinergic receptors. Keywords: fura2, calcium channels; ATP; carbamylcholine; non-selective cation channels; salivation. Abbreviations: cAMP, adenosine, 3',5' cyclic monophosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DTPA, diethylenetriamine pentaacetic acid.
Introduction
Zinc plays a major role in cell metabolism and division. It is a cofactor of many metalloenzymes and binds to proteins involved in the regulation of DNA synthesis (1). These proteins interact with DNA through so-called zinc fingers, secondary structures designed for the coordination of zinc to cysteine and histidine residues (2). These major functions of zinc probably account for the fact that zinc is one of the most abundant trace elements in the human body (3). The clinical picture of zinc deficiency includes loss of appetite, inducing a loss of body weight. This symptom has been related to the hypogeusia which is probably the most general complain of these patients (4). Since patients with xerostomia also generally have some taste imEairment (5) it was suggested that zinc binds to a salivary protein involved in taste. This then led to the characterization of gustins, a family of salivary proteins able to bind zinc (6). It has also been shown that zinc binds to 7S-nerve growth factor (7) and might possi I
To whom the correspondence should be addressed.
© 1995 by Gustav Fischer Verlag Stuttgart· Jena . New York
bly stabilize the structure of this pro-form of NGF in secretory granules. Considering the interaction between zinc and salivary proteins we decided to investigate the mechanisms of zinc uptake in salivary glands. Three secretagogues, acting via distinct pathways, were tested. The muscarinic agonist carbachol activates a polyphosphoinositide-specific phospholipase C and increases the intracellular concentration of inositol 1,4,5-trisphosphate and diglycerides. The inositol derivative binds to an intracellular receptor and mobilizes an intracellular pool of calcium. The subsequent increase in cytosolic calcium, coupled with the activation of protein kinase C by diglycerides triggers t
Zinc and submandibular glands
95
Materials and Methods
Fura2-AM was purchased from Molecular Probes (Eugene OR) and Coomassie blue from Aldrich (Milwaukee, WI). Zinc sulfate heptahydrate and lanthanum nitrate were purchased from Merck (Darmstadt, Germany), isoproterehol bitartrate, c~rbamylcholine, ATP, ADP, adenosine, digitonin and diethylenetriamine-pentaacetic acid (DTPA) from Sigma (St Louis, MO). All the other reagents were of analytical grade. Male Wistar rats (150-200 g) fed ad libitum on a standard chow (40 mg/kg of Zn) and with free access to water were used for these experiments. The animals were killed with ether and their submandibular glands were removed and dissected. A crude cellular suspension from the submandibular glands was prepared as previously described (12), with minor modifications (13). The glands were finely minced and the fragments were resuspended with 10 ml isotonic saline in a 15 ml tube. After 3 minutes at room temperature, the floating debris was aspirated. The fragments which had sedimented were resuspended in 10 ml digestion medium containing (mM): NaCI 96, KCI6, MgCI2 1, NaHl04 2.5, glucose 11, Na-pyruvate 5, Na-glutamate 5, Na-fumarate 5, HEPES 24.5 (pH=7.4 with NaOH), amino acid, mixture (without glutamate) 1 %, albumin 0.1 % (w/v) and 1.4 mg collagenase per 10 ml (collagenase P, 2.6 U/mg). The fragments were incubated in this medium for 20 minutes at 37°C under constant shaking (160 cycles/min). After the first 10 minutes, the fragments were aspirated 10 times through a 10 ml glass pipette. At the end of the next 10 minutes, tqey were aspirated 10 times through pipettes (10 ml, 5 ml, 2 ml and 1 ml) of decreasing diameters (from 2 to 0.5 mm). This crude suspension was washed four times with the digestion medium (in the absence of collagenase) by successive centrifugation at 500 g, removal of the supernatant and resuspension of the pellet in 10 ml fresh medium. After the last wash, the final pellet was resuspended in 5 ml fresh medium. This suspension was kept at 4°C until use. The uptake of zinc was measured using fura2 as the intracellular fluorescent probe. This dye is used as a probe to measure the intracellular free calcium concentration. The binding of calcium to the dye shifts the excitation spectrum to lower wavelength and the isosbestic point for calcium (the excitation wavelength for which the emitted light is not affected by the concentration of calcium) is 360 nm. The binding of zinc to fura2 also shifts the excitation spectrum of the dye but the isosbestic point is higher (375 nm) (ref 14 and Figure 1 of this paper). By using this excitation wavelength (360 nm) and 495 nm as the emission wavelength, it is thus possible to measure the relative variations in zinc concentration without any interference from variations in the calcium concentration.
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Excitation wavelenght (nm) Figure I. Excitation spectrum for fura2 in the absence and in the presence of zinc. A 10 mM free fura2 was prepared in 2 ml ofmedium containing (mM): NaCI 145, KCI 6, HEPES 10 and 100 mM EGTA. The sample was placed in the cuvette of the fluorimeter and the excitation wavelength was increased every second by 1 nm starting at 320 nm and ending at 450 nm. The emission wavelength was set at 495 nm (dotted line). At the end of the measurement, ZnCI, was added to the cuvette (I mM final concentration) and the excitation spectrum was recorded (full line). Results are from one experiment representative of 2 others.
To load the cells with fura2, 1 ml of the crude cellular suspension was mixed with 1 ml of a similar medium but containing 1% albumin and 0.5 mM CaCI2. The cells were incubated for 30 min at 25°C in the dark, in the presence of 2 mM fura2/AM and 0.02% pluronic acid. At the end of the incubation, 1 ml was removed and washed with 12 ml isotonic NaCI. The pellet was resuspended in 2 ml of medium containing (mM): NaCI 145, KCl 6, HEPES 10. The suspension was placed in the cuvette of a spectrofluorimeter (SLM Aminco Bowman 2, Urbana IN) and constantly stirred. The measurements were perfonned at room temperature. The excitation wavelength was switched every second from 345 nm to 360 nm. The emission wavelength was set at 495 nm. The fluorescence signals obtained at 345 nm and 360 nm excitation wavelength were separately recorded to distinguish between alterations in [Cali (reflected by an increase in the fluorescence at 345 nm excitation) and an increase in fluorescence due to zinc uptake (apparent at both excitation wavelengths). To compare quantitatively the rates of zinc-induced increase in fluorescence between different preparations, the initial fluorescence of all samples was amplified by the fluorimeter to a set initial fluorescence reading. This procedure allowed us to obtain similar basal rates on zinc uptake between different preparations from submandibular glands and different fura2 loading. After an equilibration period, the suspension was successively submitted to the tested agents and zinc was then added. In some traces, 1 mM DTPA was added to confirm that the
96
J. P. Dehaye
increased fluorescence signals were not due to dye leakage but truly to zinc uptake (data not shown). The files were transferred to Lotus 123 and the data were analyzed by nonlinear regression. The rate of fluorescence increase due to zinc uptake was estimated by measuring the slope of the curve 10 seconds after the addition of zinc, at a time when no interference due to zinc binding on extracellular fura2 could obscure the calculations. In some experiments, the fluorescence at 360 nm excitation slightly and steadily declined before the addition of zinc. This has been previously reported and is probably due to photobleaching of free fura2. Results were corrected for this decrease. The results were analyzed using Student's t-test.
Results
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2.1 Figure 2 shows the fluorescence at 360 nm excitation, :: in the presence of 100 11M zinc and of various secretago- C':I ;.. gues. Under basal conditions, the addition of zinc to the :s;.. 1.9 cellular suspension sharply increased the fluorescence for C':I '-' the first 10 seconds, but the emitted signal increased only c;; (j slightly over the next ten minutes (slope after 10 sec1.7 onds: 0.011 ± 0.001 arbitrary units (a.u.)/min). The cells 8
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Zinc and submandibular glands
proterenol did not interfere with the stimulatory effect of the purinergic agonist on zinc uptake and these cells responded to the purinergic stimulation. It can also be seen that the stimulatory effect of ATP appeared during the first seconds after the exposure to the purinergic agent. Figure 3 shows that the basal zinc uptake became apparent at metal concentrations higher than 100 IlM. At a 1 mM zinc concentration, this uptake was still increasing. The presence of ATP in the medium 5 minutes before the addition of zinc increased the sensitivity of the transporter to zinc: in the presence of the nucleotide, a significant zinc uptake was observed at a 30 IlM zinc concentration. The maximal response to ATP could not be estimated since a plateau was not reached in the dose-response curve. But the major effect of ATP seemed to be an increase in the affinity of the transporter to zinc, since in the presence of ATP the curve was shifted to the left by one order of magnitude. Figure 4 shows the response of submandibular cells preincubated for 5 minutes in the presence of increasing ATP concentrations. The maximal effect was observed at 1000 IlM ATP with a half-maximal effective concentration at 250 IlM. Purinergic receptors have been classified according to their sensitivity to adenosine and ATP analogs. As shown in Figure 5, adenosine and ADP at a 1 mM level had no significant effect on the uptake of zinc. Coomassie blue is a dye used to color proteins and which has been shown to block the response of parotid (15) and submandibular cells (16) to exogenous ATP. The response to ATP was completely blocked by the previous addition of 10 IlM
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Con enlralion of ATP (JIM) Figure 4: Effect of various concentrations of ATP on zinc uptake by rat submandibular glands Submandibular cells loaded with fura2 were incubated for 2 minutes under basal conditions. Various concentrations of ATP were then added and at 7 minutes, 100 11M zinc was added to the cuvette. Results are expressed as fluorescence variation (arbitrary units per minute) as a function of the concentration of ATP in the assay. Results are means ± s.e.m. of 4 experiments.
Coomassie blue to the cuvette. These results suggest that the response to ATP does not involve a PI receptor but rather a P 2 receptor of the P2z type. As shown in Figure 6, the zinc uptake was inhibited by cations. At time 0, the cells were resuspended in the assay medium in the absence or in the presence of either 1 mM calcium or 0.2 mM nickel (as a chloride salt) or lanthanum (as a nitrate salt). After 2 minutes, the cells were preincubated for 5 minutes in the presence of 300 IlM ATP and the subsequent zinc uptake was measured. Cal-
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Concentration of Zn (Log M) Figure 3: Effect of various extracellular concentrations of zinc on the uptake of this ion by rat submandibular glands. Submandibular glands loaded with fura2 were incubated for 2 minutes under basal conditions. The cells were incubated under the same condition for the rest of the assay (open circles) or were incubated in the presence of 300 11M ATP (closed triangles). At 7 minutes, various concentrations of zinc were added. Results are expressed as fluorescence variation (arbitrary units per minute) as a function of the concentration of zinc in the assay. Results are means ± s.e.m. of 4 experiments.
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Figure 5: Characterization of the purinergic receptors involved in the zinc uptake by rat submandibular glands. Submandibular cells loaded with fura2 were incubated for 2 minutes under basal conditions or in the presence of 10 11M Coomassie blue (last column). One millimolar ATP (first and last columns), ADP (second column) or adenosine (third column) were added to the cuvette. After 5 minutes in the presence ofthe purinergic agonist, 100 11M zinc was added. Results are expressed as fluorescence variation (arbitrary units per minute) as a function of the agonist. Results are means ± s.e.m. of 4 experiments.
98
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Figure 6: Effect of cations on zinc uptake by rat submandibular glands. Submandibular cells loaded with fura2 were resuspended in control medium or in the presence of I mM calcium, 0.2 mM nickel or lanthanum. At 2 minutes, 300 mM ATP was added to the cuvette. After 5 minutes in the presence of the purinergic agonist, 100 /!M zinc was added. Results are expressed as fluorescence variation (arbitrary units per minute) as a function of the agortist. Results are means ± s.e.m. of 4 experiments.
cium completely blocked the uptake of zinc. Nickel and lanthanum inhibited the zinc uptake by 45% and 65% respectively. These inhibitions were statistically significant (p
Discussion The synthesis of calcium-sensitive permeant fluorescent dyes has greatly facilitated studies of variations in the intracellular calCium concentration. But in the first description of these probes it was already reported that they were sensitive to metal ions (17). As a matter of fact, their sensitivity to these ions was sometimes much better than for calcium. Fura2, for instance, has a Kd for calci-
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Figure 7: Effect of the removal of extracellular sodium on zinc uptake by rat submandibular glands. Submandibular cells loaded with fura2 were resuspended in control medium or in the presence of 150 mM KCI or 5 mM KCI and 145 mM LiCI or in the presence of 5 mM KCI and of various concentrations of NaCl and LiC\. After 5 minutes, 100 mM zinc was added to the cuvette and the assay was run for 5 minutes. The cells were then exposed to 300 /!M ATP for the next 5 minutes. Only basal zinc uptake has been represented. Results are expressed as fluorescence variation (arbitrary units per minute) as a function of the salt condition. Results are means ± s.e.m. of 3 experiments.
urn in the 10.7 M range while its pK for zinc is 8.6-8.8. The property of dyes like fura2 to bind metal ions was used in 1989 by Merritt, Jacob and Hallam (18) to measure the uptake of manganese, an ion which can flow through calcium channels. This technique was designed to give an estimate of the opening of calcium channels. More recently, quin2 and fura2 were used to study the selectivity of calcium channels towards divalent cations (19-22). Fura2 was also used to measure cadmium movements in GH3 cells (23) and recently zinc movements in bovine liver nuclei (14). In this last study, the excitation spectrum of fura2 was tested in the absence and in the presence of zinc or calcium. Zinc had an effect similar to calcium, shifting the excitation wavelength to shorter values. But interestingly, the isosbestic wavelength was slightly higher than 370 nm, i.e. different from the isosbestic wavelength for calcium (360 nm). Similar results were observed in our system (see Figure 1). Using an excitation wavelength of 360 nm, it was thus possible to measure variations in the spectrum of fura2 which were due to zinc ions, independently of any effect of calcium. Besides by shifting the excitation wavelength regularly from 360 nm to 345 nm (an excitation wavelength at which the dye is sensitive to zinc and calcium), it was possible to measure the contribution of both calcium and zinc to the signal. For instance, we observed that carbachol did not modify the signal at 360 nm, but increased
Zinc and submandibular glands
the signal at 345 nm, allowing the conclusion that carbachol did not modify zinc uptake but still induced the mobilization of intracellular calcium pools. On the other hand, ATP did not affect the signal at 345 nm in the absnece of extracellular calcium but increased the signals at 345 and 360 nm in the presence of zinc, a reflection of zinc liptake. The validity of the method was assessed by addition of DTPA, a non-permeant chelator, to the medium. Since this did not modify the fluorescence observed at a 360 nm excitation wavelength it confirmed that the dye was intracellular. The high affinity of the dye (around 3 nM) for zinc could lead to saturation of the dye during stimulation. We observed, however, that the fluorescence increased from a basal value set at 1.7 arbitrary units to 2.3 a.u. in the presence of zinc and a maximal concentration of ATP. The addition of digitonin to the cells increased the fluorescence to values around 3.1 a.u. These results confirm that the intracellular concentration of free zinc is unable to saturate the fura2 and remains very low, in the nanomolar range, in spite of the fact that the total intracellular concentration of zinc is high. The discrepancy between these two estimates probably accounts for the large amount of zinc which is bound to proteins. , Our results show that the basal uptake of zinc is not sensitive to carbachoLrhis analog of acetylcholine has been reported to increase the intracellular free calcium concentration via mobilization of intracellular calcium pools and the activation of calcium uptake by a second messenger operated calcium channel (24). This channel is thus able to discriminate between zinc and calcium. Isoproterenol which increases the intracellplar concentration of cAMP, also has no effect on zinc uptake, suggesting that the transporter is not regulated by cAMP-dependent protein kinase. The only effective agonist was ATP. This purinergic agonist increased the zinc uptake within seconds. Since these measurements were performed in the absence of extracellular magnesium, one can assume that the true agonist was ATP 4-. The stimulatory effect of ATP was not reproduced by ADP or adenosine, but was blocked by Coomassie blue. These results suggest that the purinergic receptors involved in the uptake of zinc are similar to the receptors recently involved in the increase in intracellular calcium in rat submandibular glands (10, 11) or in rat parotid glands (25). The stimulatory effect of ATP was blocked by calcium or inhibitors of calcium channels such as nickel or lanthanum. These results confirm that the zinc uptake is mediated by a calcium channel activated by ATP. Soltoff, McMillian and Talamo (26) reported that in rat parotid glands ATP promoted the uptake of sodium and calcium and that the two ions competed for the same non-specific cation channel. The uptake of sodium also induced a depolarization of the plasma membrane. Similarly, we observed that, in submandibu-
99
lar glands, sodium and calcium compete for an uptake mechanism activated by ATP (16). Results shown in Figure 5 confirm that zinc behaved like calcium with respect to the ATP-activated calcium channels. Indeed, replacement of sodium by potassium increased the basal zinc uptake. This stimulatory effect of potassium was not related to the opening of a voltage-sensitive calcium channel in response to the depolarization induced by potassium (27) since it could also be reproduced by lithium. And since lithium (in contrast with potassium) can substitute for sodium at the sodium/proton exchanger (28), the stimulation induced by lithium is not secondary to any modification of intracellular pH which might have activated a zinc/proton exchanger. It thus appears that the so-called "non-selective" cation channel opened by purinergic agonists can in fact discriminate among cations: it is permeable to sodium, calcium or zinc but impermeable to potassium or lithium. These data are at variance with previous results obtained with lacrimal glands: it was reported that the ATP-sensitive cation channel could not discriminate between sodium and potassium (29). More recently it has been shown that a similar channel present in lymphocytes is permeable to sodium but not to potassium (30). It thus appears that the ATP-sensitive cation channels are heterogeneous, with their specificity varying with the tissue under consideration. This conclusion is in line with recent results of Nuttle and Dubyak (31) who showed that P 2Z receptors could be coupled to two distinct types of channels, which differed in the size of their pores. Several modalities for zinc uptake in cells have been described. In the intestinal cells, the process is mediated by a peptide carrier system (32). Zinc enters the renal proximal cells chelated to cysteine or histidine (33). In human red blood cells, zinc is taken up by two mechanisms. One involves the anion exchanger (34), while the second one occurs in the presence of thiocyanate or salicylate and consists in transport of a neutral complex with zinc (35). In giant neurons of snails (36) and in insect muscle membrane (37) zinc can permeate through voltage-sensitive calcium channels. In HL-60 cells, Demaurex et al. (22) reported that zinc depletion of intracellular calcium pools by inhibitors of Ca-ATPases increases the influx of divalent cations among which zinc. In hepatocytes, Crofts and Barritt (38) demonstrated the existence of a divalent cation inflow system permeable to zinc and activp.ted by vasopressin and angiotensin II. Our results suggest some interesting perspectives. First, since carbachol does not affect the uptake of zinc in salivary glands, the treatment of hypogeusia due to impairment of zinc secretion should benefit from the introduction to the therapeutic arsenal of an agonist able to stimulate the non-specific cation channels; it should be used in conjunction with a more classical sialagogue such
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J. P. Dehaye
as pilocarpine. Second, saliva contains trace elements (some of them toxic) other than zinc (39) and it is conceivable that these other ions enter the salivary glands via the ATP-sensitive non-specific cation channels. From a toxicological point of view, the activation of these channels should thus help to get rid of toxic metals. .In conclusion, the purinergic receptors of salivary glands (parotid and submandibular glands) are coupled to a class of non-selective cation channels. These channels are not only the trigger mechanism for the activation of salivary secretion but might also playa major role in the transcellular transport of charged molecules from the vascular compartment to the mouth.
Acknowledgements
This work was supported by grant n° 3.4558.92 from the National Fund for Scientific Research, Belgium. The author wishes to thank Professor J. NEVE for his helpful criticism during the preparation of this manuscript.
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volve analogous 26-kDa integral membrane phosphoproteins. Proc. Natl. Acad. Sci. 82, 3237-3241. 10. DEHAYE, J.P. (1993) ATP" increases the intracellular calcium concentration in rat submandibular glands. Gen. Pharmacol. 24, 1097-1100. 11. HURLEY, T.w., RYAN. M.P. AND SHOEMAKER, D.D. (1994) Mobilization of Ca"+ influx, but not of stored Ca"+, by extracellular ATP in rat submandibular gland acini. Arch. oral BioI. 39, 205-212. 12. DEHAYE, J.P. and TURNER, R.I. (1991) Isolation and characterization of rat submandibular intralobular ducts. Am. J. Physiol. 261, C490-C494. 13. GROSFILS, K., METIOUI, M., TIOULI, M. AND DEHAYE, J.P. (1993) Isolation of rat pancreatic acini with crude collagenase and permeabilization of these acini with streptolysin O. Res. Commun. Chern. Pathol. Pharmacol. 79, 99-115. 14. HECHTENBERG, S. and BEYERSMANN, D. (1993) Differential control of free calcium and free zinc levels in isolated bovine liver nuclei. Biochem. J. 289,757-760. 15. SOLTOFF, S.P., McMILLIAN, M.K. AND TALAMO, B.R. (1989) Coomassie brilliant blue G is a more potent antagonist of P, purinergic responses than reactive blue 2 (Cibacron blue 3GA) in rat parotid acinar cells. Biochem. biophys. Res. Commun. 165,1279-1285. 16. METIOUI, M., GROSFILS, K. AND DEHAYE, J.P. (1994) Interaction between thapsigargin and ATP" in the regulation of the intracellular calcium in rat submandibular glands. J. cell. Physiol. 161,243-248. 17. GRYNKIEWICZ, G., POENIE, M. AND TSIEN, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. bioI. Chern. 260,3440-3450. 18. MERRITT, J.E., JACOB, R. AND HALLAM, T.J. (1989) Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophil. J. bioI. Chern. 264, 1522-1527. 19. JACOB, R. (1990) Agonist-stimulated divalent cation entry into single cultured human umbilical vein endothelial cells. J. Physiol. (Lond) 421, 55-77. 20. KWAN, C.-H. AND PUTNEY, J.w.Jr. (1990) Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. J. bioI. Chern. 265, 678-684. 21. HIDE, M. AND BEAVEN, M.A. (1991) Calcium influx in a rat mast cell (RBL-2H3) line. J. bioI. Chern. 266,15221-15229. 22. DEMAUREX, N., LEW, D.P. AND KRAUSE, K.-H. (1992) Cyclapiazonic acid depletes intracellular Ca2+ stores and activates an influx pathway for divalent cations in HL-60 cells. J. bioI. Chern. 267, 2318-2324. 23. HINKLE, P.M., SHANSHALA, E.D. AND NELSON, E.I. (1992) Measurement of intracellular cadmium with fluorescent dyes. J. bioI. Chern. 267, 25553-25559. 24. METIOUI, M., GROSFILS, K. AND DEHAYE, J.P. (1994) Regulation by thapsigargin and carbachol of the intracellular calcium concentration in rat submandibular glands. Gen. Pharmacol. 25,1353-1359.
Zinc and submandibular glands 25. SOLTOFF, S.P., McMILLIAN, M.K., CRAGOE, E.I., CANTLEY, L.e. AND TALAMO, B.R. (1990) Effects of extracellular ATP on ion transport systems and [Ca'+], in rat parotid acinar cells. I. gen. Physiol. 95, 319-346. 26. SOLTOFF, S.P., McMILLIAN, M.K. AND TALAMO, B.R. (1992) ATP activates a cation-permeable pathway in rat parotid acinar cells. Am. I. Physiol. 262, C934-C940. 27. GALLACHER, D.V. AND MORRIS , A.P. (1987) The receptorregulated calcium influx in mouse submandibular acinar cells is sodium dependent: a patch-clamp study. J. Physiol. (Lond) 387, 119-130. 28. ELLIOTT, A.e., LAU, K.R. AND BROWN, P.D. (1991) The effects ofNa+ replacement on intracellular pH and [Ca'+] in rabbit salivary gland acinar cells. J. Physiol. (Lond) 444, 419-439. 29. SASAKI, T. AND GALLACHER, D.V. (1990) Extracellular ATP activates receptor-operated cation channels in mouse lacrimal acinar cells to promote calcium influx in the absence of phosphoinositide metabolism. FEBS Lett. 264, 130-134. 30. CHEN, I .R. , JAMIESON, G.P. AND WILEY, J.S. (1994) Extracellular ATP increases NH. + permeability in human lymphocytes by opening a P,z purinoceptor operated ion channel. Biochem. biophys. Res. Commun. 202,1511-1516. 31. NUTTLE, L.C. AND DUBYAK, G.R. (1994) Differential activation of cation channels and non-selective pores by macrophage P" purinergic receptors expressed in Xenopus oocytes. J. BioI. Chern. 266, 13988-13996. 32. TACNET, F., LAUTH.IER, F. AND RlPOCHE, P. (1993) Mechanisms of zinc transport into pig small intestine brush-border membrane vesicles. J. Physiol. (Lond) 465,57-72.
10 I
33. GACHOT, B., TAUC, M. AND POUJEOL, P. (1991) Zinc up~ take by proximal cells isolated from rabbit kidney: Effects of cysteine and histidine, Pflugers Arch. 419, 583-587. 34. TORRUBIA, J.O.A. AND GARAY, R. (1989) Evidence for a major route for zinc uptake in human red blood cells: [zinc(HCO,),Clj'influx through the [C!'/HCO;] anion exchanger. J. cell. PhYsiol. 138, 316-322. . 35. KALFAKAKOU, V. AND SIMONS , T.J.B. (1990) Anionic mechanisms of zinc uptake across the human red cell membrane. I . Physiol. (Lond) 421 , 485-497. 36. KAWA, K. (1979) Zinc-dependent action potentials in giant neurons of the snail, Euhadra quaestia. I . Membrane BioI. 49, 325-344. 37. FUKUDA, J. AND KAWA, K. (1977) Permeation of manganese, cadmium, zinc, and beryllium through calcium channels of an insect muscle membrane. Science 196,309-311. 38. CROFTS, I.N. and BARRITT, G.J. (1990) The liver cell plasma membrane Ca' + inflow systems exhibit a broad specificity for divalent metal ions. Biochem. I . 269, 579-587. 39.0LMEZ, I., GULOVALI, M.e., GORDON, G.E. and HENKIN, R.1. (1988) Trace elements in human parotid saliva. BioI. Trace Elem. Res. 17, 259-270.
Regulation by Purinergic Agonists of Zinc Uptake by Rat Submandibular Glands J.P. DEHAYE Laboratoire de Biochimie generale et humaine, Institut de Pharmacie, Universite libre de Bruxelles B1050 - Bruxelles, Belgium (Received September 1994IFebruary 1995)
Summary
The zinc uptake in rat submandibular cells was measured using fura2 as a fluorescent probe. Basal zinc uptake was observed in a 100 flM - 1 mM concentration range. Carbachol and isoproterenol had no effect but ATp4- dose-dependently increased the basal zinc uptake (half-maximal concentration: 250 flM). The purinergic agonist shifted the concentration curve for zinc to the left by one order of magnitude. The response to ATP was not reprodu~ed by adenosine or ADP and was blocked by Coomassie blue. Calcium, nickel or lanthanum were inhibitors of zinc uptake, while the substitution of extracellular sodium by potassium or lithium increased the basal zinc uptake. We conclude that in submandibular cells zinc can permeate through the non-specific cation channel coupled to ATP-sensitive purinergic receptors. Keywords: fura2, calcium channels; ATP; carbamylcholine; non-selective cation channels; salivation. Abbreviations: cAMP, adenosine, 3',5' cyclic monophosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DTPA, diethylenetriamine pentaacetic acid.
Introduction
Zinc plays a major role in cell metabolism and division. It is a cofactor of many metalloenzymes and binds to proteins involved in the regulation of DNA synthesis (1). These proteins interact with DNA through so-called zinc fingers, secondary structures designed for the coordination of zinc to cysteine and histidine residues (2). These major functions of zinc probably account for the fact that zinc is one of the most abundant trace elements in the human body (3). The clinical picture of zinc deficiency includes loss of appetite, inducing a loss of body weight. This symptom has been related to the hypogeusia which is probably the most general complain of these patients (4). Since patients with xerostomia also generally have some taste imEairment (5) it was suggested that zinc binds to a salivary protein involved in taste. This then led to the characterization of gustins, a family of salivary proteins able to bind zinc (6). It has also been shown that zinc binds to 7S-nerve growth factor (7) and might possi I
To whom the correspondence should be addressed.
© 1995 by Gustav Fischer Verlag Stuttgart· Jena . New York
bly stabilize the structure of this pro-form of NGF in secretory granules. Considering the interaction between zinc and salivary proteins we decided to investigate the mechanisms of zinc uptake in salivary glands. Three secretagogues, acting via distinct pathways, were tested. The muscarinic agonist carbachol activates a polyphosphoinositide-specific phospholipase C and increases the intracellular concentration of inositol 1,4,5-trisphosphate and diglycerides. The inositol derivative binds to an intracellular receptor and mobilizes an intracellular pool of calcium. The subsequent increase in cytosolic calcium, coupled with the activation of protein kinase C by diglycerides triggers t
Zinc and submandibular glands
95
Materials and Methods
Fura2-AM was purchased from Molecular Probes (Eugene OR) and Coomassie blue from Aldrich (Milwaukee, WI). Zinc sulfate heptahydrate and lanthanum nitrate were purchased from Merck (Darmstadt, Germany), isoproterehol bitartrate, c~rbamylcholine, ATP, ADP, adenosine, digitonin and diethylenetriamine-pentaacetic acid (DTPA) from Sigma (St Louis, MO). All the other reagents were of analytical grade. Male Wistar rats (150-200 g) fed ad libitum on a standard chow (40 mg/kg of Zn) and with free access to water were used for these experiments. The animals were killed with ether and their submandibular glands were removed and dissected. A crude cellular suspension from the submandibular glands was prepared as previously described (12), with minor modifications (13). The glands were finely minced and the fragments were resuspended with 10 ml isotonic saline in a 15 ml tube. After 3 minutes at room temperature, the floating debris was aspirated. The fragments which had sedimented were resuspended in 10 ml digestion medium containing (mM): NaCI 96, KCI6, MgCI2 1, NaHl04 2.5, glucose 11, Na-pyruvate 5, Na-glutamate 5, Na-fumarate 5, HEPES 24.5 (pH=7.4 with NaOH), amino acid, mixture (without glutamate) 1 %, albumin 0.1 % (w/v) and 1.4 mg collagenase per 10 ml (collagenase P, 2.6 U/mg). The fragments were incubated in this medium for 20 minutes at 37°C under constant shaking (160 cycles/min). After the first 10 minutes, the fragments were aspirated 10 times through a 10 ml glass pipette. At the end of the next 10 minutes, tqey were aspirated 10 times through pipettes (10 ml, 5 ml, 2 ml and 1 ml) of decreasing diameters (from 2 to 0.5 mm). This crude suspension was washed four times with the digestion medium (in the absence of collagenase) by successive centrifugation at 500 g, removal of the supernatant and resuspension of the pellet in 10 ml fresh medium. After the last wash, the final pellet was resuspended in 5 ml fresh medium. This suspension was kept at 4°C until use. The uptake of zinc was measured using fura2 as the intracellular fluorescent probe. This dye is used as a probe to measure the intracellular free calcium concentration. The binding of calcium to the dye shifts the excitation spectrum to lower wavelength and the isosbestic point for calcium (the excitation wavelength for which the emitted light is not affected by the concentration of calcium) is 360 nm. The binding of zinc to fura2 also shifts the excitation spectrum of the dye but the isosbestic point is higher (375 nm) (ref 14 and Figure 1 of this paper). By using this excitation wavelength (360 nm) and 495 nm as the emission wavelength, it is thus possible to measure the relative variations in zinc concentration without any interference from variations in the calcium concentration.
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Excitation wavelenght (nm) Figure I. Excitation spectrum for fura2 in the absence and in the presence of zinc. A 10 mM free fura2 was prepared in 2 ml ofmedium containing (mM): NaCI 145, KCI 6, HEPES 10 and 100 mM EGTA. The sample was placed in the cuvette of the fluorimeter and the excitation wavelength was increased every second by 1 nm starting at 320 nm and ending at 450 nm. The emission wavelength was set at 495 nm (dotted line). At the end of the measurement, ZnCI, was added to the cuvette (I mM final concentration) and the excitation spectrum was recorded (full line). Results are from one experiment representative of 2 others.
To load the cells with fura2, 1 ml of the crude cellular suspension was mixed with 1 ml of a similar medium but containing 1% albumin and 0.5 mM CaCI2. The cells were incubated for 30 min at 25°C in the dark, in the presence of 2 mM fura2/AM and 0.02% pluronic acid. At the end of the incubation, 1 ml was removed and washed with 12 ml isotonic NaCI. The pellet was resuspended in 2 ml of medium containing (mM): NaCI 145, KCl 6, HEPES 10. The suspension was placed in the cuvette of a spectrofluorimeter (SLM Aminco Bowman 2, Urbana IN) and constantly stirred. The measurements were perfonned at room temperature. The excitation wavelength was switched every second from 345 nm to 360 nm. The emission wavelength was set at 495 nm. The fluorescence signals obtained at 345 nm and 360 nm excitation wavelength were separately recorded to distinguish between alterations in [Cali (reflected by an increase in the fluorescence at 345 nm excitation) and an increase in fluorescence due to zinc uptake (apparent at both excitation wavelengths). To compare quantitatively the rates of zinc-induced increase in fluorescence between different preparations, the initial fluorescence of all samples was amplified by the fluorimeter to a set initial fluorescence reading. This procedure allowed us to obtain similar basal rates on zinc uptake between different preparations from submandibular glands and different fura2 loading. After an equilibration period, the suspension was successively submitted to the tested agents and zinc was then added. In some traces, 1 mM DTPA was added to confirm that the
96
J. P. Dehaye
increased fluorescence signals were not due to dye leakage but truly to zinc uptake (data not shown). The files were transferred to Lotus 123 and the data were analyzed by nonlinear regression. The rate of fluorescence increase due to zinc uptake was estimated by measuring the slope of the curve 10 seconds after the addition of zinc, at a time when no interference due to zinc binding on extracellular fura2 could obscure the calculations. In some experiments, the fluorescence at 360 nm excitation slightly and steadily declined before the addition of zinc. This has been previously reported and is probably due to photobleaching of free fura2. Results were corrected for this decrease. The results were analyzed using Student's t-test.
Results
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2.1 Figure 2 shows the fluorescence at 360 nm excitation, :: in the presence of 100 11M zinc and of various secretago- C':I ;.. gues. Under basal conditions, the addition of zinc to the :s;.. 1.9 cellular suspension sharply increased the fluorescence for C':I '-' the first 10 seconds, but the emitted signal increased only c;; (j slightly over the next ten minutes (slope after 10 sec1.7 onds: 0.011 ± 0.001 arbitrary units (a.u.)/min). The cells 8
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Zinc and submandibular glands
proterenol did not interfere with the stimulatory effect of the purinergic agonist on zinc uptake and these cells responded to the purinergic stimulation. It can also be seen that the stimulatory effect of ATP appeared during the first seconds after the exposure to the purinergic agent. Figure 3 shows that the basal zinc uptake became apparent at metal concentrations higher than 100 IlM. At a 1 mM zinc concentration, this uptake was still increasing. The presence of ATP in the medium 5 minutes before the addition of zinc increased the sensitivity of the transporter to zinc: in the presence of the nucleotide, a significant zinc uptake was observed at a 30 IlM zinc concentration. The maximal response to ATP could not be estimated since a plateau was not reached in the dose-response curve. But the major effect of ATP seemed to be an increase in the affinity of the transporter to zinc, since in the presence of ATP the curve was shifted to the left by one order of magnitude. Figure 4 shows the response of submandibular cells preincubated for 5 minutes in the presence of increasing ATP concentrations. The maximal effect was observed at 1000 IlM ATP with a half-maximal effective concentration at 250 IlM. Purinergic receptors have been classified according to their sensitivity to adenosine and ATP analogs. As shown in Figure 5, adenosine and ADP at a 1 mM level had no significant effect on the uptake of zinc. Coomassie blue is a dye used to color proteins and which has been shown to block the response of parotid (15) and submandibular cells (16) to exogenous ATP. The response to ATP was completely blocked by the previous addition of 10 IlM
97
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Coomassie blue to the cuvette. These results suggest that the response to ATP does not involve a PI receptor but rather a P 2 receptor of the P2z type. As shown in Figure 6, the zinc uptake was inhibited by cations. At time 0, the cells were resuspended in the assay medium in the absence or in the presence of either 1 mM calcium or 0.2 mM nickel (as a chloride salt) or lanthanum (as a nitrate salt). After 2 minutes, the cells were preincubated for 5 minutes in the presence of 300 IlM ATP and the subsequent zinc uptake was measured. Cal-
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Concentration of Zn (Log M) Figure 3: Effect of various extracellular concentrations of zinc on the uptake of this ion by rat submandibular glands. Submandibular glands loaded with fura2 were incubated for 2 minutes under basal conditions. The cells were incubated under the same condition for the rest of the assay (open circles) or were incubated in the presence of 300 11M ATP (closed triangles). At 7 minutes, various concentrations of zinc were added. Results are expressed as fluorescence variation (arbitrary units per minute) as a function of the concentration of zinc in the assay. Results are means ± s.e.m. of 4 experiments.
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Figure 5: Characterization of the purinergic receptors involved in the zinc uptake by rat submandibular glands. Submandibular cells loaded with fura2 were incubated for 2 minutes under basal conditions or in the presence of 10 11M Coomassie blue (last column). One millimolar ATP (first and last columns), ADP (second column) or adenosine (third column) were added to the cuvette. After 5 minutes in the presence ofthe purinergic agonist, 100 11M zinc was added. Results are expressed as fluorescence variation (arbitrary units per minute) as a function of the agonist. Results are means ± s.e.m. of 4 experiments.
98
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Figure 6: Effect of cations on zinc uptake by rat submandibular glands. Submandibular cells loaded with fura2 were resuspended in control medium or in the presence of I mM calcium, 0.2 mM nickel or lanthanum. At 2 minutes, 300 mM ATP was added to the cuvette. After 5 minutes in the presence of the purinergic agonist, 100 /!M zinc was added. Results are expressed as fluorescence variation (arbitrary units per minute) as a function of the agortist. Results are means ± s.e.m. of 4 experiments.
cium completely blocked the uptake of zinc. Nickel and lanthanum inhibited the zinc uptake by 45% and 65% respectively. These inhibitions were statistically significant (p
Discussion The synthesis of calcium-sensitive permeant fluorescent dyes has greatly facilitated studies of variations in the intracellular calCium concentration. But in the first description of these probes it was already reported that they were sensitive to metal ions (17). As a matter of fact, their sensitivity to these ions was sometimes much better than for calcium. Fura2, for instance, has a Kd for calci-
K+ 5 Li+ .
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Figure 7: Effect of the removal of extracellular sodium on zinc uptake by rat submandibular glands. Submandibular cells loaded with fura2 were resuspended in control medium or in the presence of 150 mM KCI or 5 mM KCI and 145 mM LiCI or in the presence of 5 mM KCI and of various concentrations of NaCl and LiC\. After 5 minutes, 100 mM zinc was added to the cuvette and the assay was run for 5 minutes. The cells were then exposed to 300 /!M ATP for the next 5 minutes. Only basal zinc uptake has been represented. Results are expressed as fluorescence variation (arbitrary units per minute) as a function of the salt condition. Results are means ± s.e.m. of 3 experiments.
urn in the 10.7 M range while its pK for zinc is 8.6-8.8. The property of dyes like fura2 to bind metal ions was used in 1989 by Merritt, Jacob and Hallam (18) to measure the uptake of manganese, an ion which can flow through calcium channels. This technique was designed to give an estimate of the opening of calcium channels. More recently, quin2 and fura2 were used to study the selectivity of calcium channels towards divalent cations (19-22). Fura2 was also used to measure cadmium movements in GH3 cells (23) and recently zinc movements in bovine liver nuclei (14). In this last study, the excitation spectrum of fura2 was tested in the absence and in the presence of zinc or calcium. Zinc had an effect similar to calcium, shifting the excitation wavelength to shorter values. But interestingly, the isosbestic wavelength was slightly higher than 370 nm, i.e. different from the isosbestic wavelength for calcium (360 nm). Similar results were observed in our system (see Figure 1). Using an excitation wavelength of 360 nm, it was thus possible to measure variations in the spectrum of fura2 which were due to zinc ions, independently of any effect of calcium. Besides by shifting the excitation wavelength regularly from 360 nm to 345 nm (an excitation wavelength at which the dye is sensitive to zinc and calcium), it was possible to measure the contribution of both calcium and zinc to the signal. For instance, we observed that carbachol did not modify the signal at 360 nm, but increased
Zinc and submandibular glands
the signal at 345 nm, allowing the conclusion that carbachol did not modify zinc uptake but still induced the mobilization of intracellular calcium pools. On the other hand, ATP did not affect the signal at 345 nm in the absnece of extracellular calcium but increased the signals at 345 and 360 nm in the presence of zinc, a reflection of zinc liptake. The validity of the method was assessed by addition of DTPA, a non-permeant chelator, to the medium. Since this did not modify the fluorescence observed at a 360 nm excitation wavelength it confirmed that the dye was intracellular. The high affinity of the dye (around 3 nM) for zinc could lead to saturation of the dye during stimulation. We observed, however, that the fluorescence increased from a basal value set at 1.7 arbitrary units to 2.3 a.u. in the presence of zinc and a maximal concentration of ATP. The addition of digitonin to the cells increased the fluorescence to values around 3.1 a.u. These results confirm that the intracellular concentration of free zinc is unable to saturate the fura2 and remains very low, in the nanomolar range, in spite of the fact that the total intracellular concentration of zinc is high. The discrepancy between these two estimates probably accounts for the large amount of zinc which is bound to proteins. , Our results show that the basal uptake of zinc is not sensitive to carbachoLrhis analog of acetylcholine has been reported to increase the intracellular free calcium concentration via mobilization of intracellular calcium pools and the activation of calcium uptake by a second messenger operated calcium channel (24). This channel is thus able to discriminate between zinc and calcium. Isoproterenol which increases the intracellplar concentration of cAMP, also has no effect on zinc uptake, suggesting that the transporter is not regulated by cAMP-dependent protein kinase. The only effective agonist was ATP. This purinergic agonist increased the zinc uptake within seconds. Since these measurements were performed in the absence of extracellular magnesium, one can assume that the true agonist was ATP 4-. The stimulatory effect of ATP was not reproduced by ADP or adenosine, but was blocked by Coomassie blue. These results suggest that the purinergic receptors involved in the uptake of zinc are similar to the receptors recently involved in the increase in intracellular calcium in rat submandibular glands (10, 11) or in rat parotid glands (25). The stimulatory effect of ATP was blocked by calcium or inhibitors of calcium channels such as nickel or lanthanum. These results confirm that the zinc uptake is mediated by a calcium channel activated by ATP. Soltoff, McMillian and Talamo (26) reported that in rat parotid glands ATP promoted the uptake of sodium and calcium and that the two ions competed for the same non-specific cation channel. The uptake of sodium also induced a depolarization of the plasma membrane. Similarly, we observed that, in submandibu-
99
lar glands, sodium and calcium compete for an uptake mechanism activated by ATP (16). Results shown in Figure 5 confirm that zinc behaved like calcium with respect to the ATP-activated calcium channels. Indeed, replacement of sodium by potassium increased the basal zinc uptake. This stimulatory effect of potassium was not related to the opening of a voltage-sensitive calcium channel in response to the depolarization induced by potassium (27) since it could also be reproduced by lithium. And since lithium (in contrast with potassium) can substitute for sodium at the sodium/proton exchanger (28), the stimulation induced by lithium is not secondary to any modification of intracellular pH which might have activated a zinc/proton exchanger. It thus appears that the so-called "non-selective" cation channel opened by purinergic agonists can in fact discriminate among cations: it is permeable to sodium, calcium or zinc but impermeable to potassium or lithium. These data are at variance with previous results obtained with lacrimal glands: it was reported that the ATP-sensitive cation channel could not discriminate between sodium and potassium (29). More recently it has been shown that a similar channel present in lymphocytes is permeable to sodium but not to potassium (30). It thus appears that the ATP-sensitive cation channels are heterogeneous, with their specificity varying with the tissue under consideration. This conclusion is in line with recent results of Nuttle and Dubyak (31) who showed that P 2Z receptors could be coupled to two distinct types of channels, which differed in the size of their pores. Several modalities for zinc uptake in cells have been described. In the intestinal cells, the process is mediated by a peptide carrier system (32). Zinc enters the renal proximal cells chelated to cysteine or histidine (33). In human red blood cells, zinc is taken up by two mechanisms. One involves the anion exchanger (34), while the second one occurs in the presence of thiocyanate or salicylate and consists in transport of a neutral complex with zinc (35). In giant neurons of snails (36) and in insect muscle membrane (37) zinc can permeate through voltage-sensitive calcium channels. In HL-60 cells, Demaurex et al. (22) reported that zinc depletion of intracellular calcium pools by inhibitors of Ca-ATPases increases the influx of divalent cations among which zinc. In hepatocytes, Crofts and Barritt (38) demonstrated the existence of a divalent cation inflow system permeable to zinc and activp.ted by vasopressin and angiotensin II. Our results suggest some interesting perspectives. First, since carbachol does not affect the uptake of zinc in salivary glands, the treatment of hypogeusia due to impairment of zinc secretion should benefit from the introduction to the therapeutic arsenal of an agonist able to stimulate the non-specific cation channels; it should be used in conjunction with a more classical sialagogue such
100
J. P. Dehaye
as pilocarpine. Second, saliva contains trace elements (some of them toxic) other than zinc (39) and it is conceivable that these other ions enter the salivary glands via the ATP-sensitive non-specific cation channels. From a toxicological point of view, the activation of these channels should thus help to get rid of toxic metals. .In conclusion, the purinergic receptors of salivary glands (parotid and submandibular glands) are coupled to a class of non-selective cation channels. These channels are not only the trigger mechanism for the activation of salivary secretion but might also playa major role in the transcellular transport of charged molecules from the vascular compartment to the mouth.
Acknowledgements
This work was supported by grant n° 3.4558.92 from the National Fund for Scientific Research, Belgium. The author wishes to thank Professor J. NEVE for his helpful criticism during the preparation of this manuscript.
References 1. BACK, C.J., SISTONEN, L., ENKVIST, M.O.K., HEIKKILA, J.E. AND AKERMAN, K.E.O. (1993) Ca2+ and zinc 2+ dependence of DNA synthesis in untransformed and in Ha-ras v ,I.12 expressing NIH 3T3 cells. Expl. Cell Res. 208, 303-310. 2. JOHNSON, P.F. AND McKNIGHT S.L. (1989) Eukaryotic transcriptional regulatory proteins. Annu. Rev. Biochem. 58, 799-839. 3. PRASAD, A.S. (1979) Clinical, biochemical and pharmacological role of zinc. Annu. Rev. Pharmacol. Toxicol. 20, 393-426. 4. HENKIN, R.I., PATTEN, B.M., RE, P. AND BRONZERT, D. (1975) A syndrome of acute zinc loss. Cerebellar dysfunction, mental changes, anorexia and taste and smell dysfunction. Arch. Neurol. 32, 745-751. 5. HENKIN, R.1. (1984) Zinc in taste function. BioI. Trace Elem. Res. 6,263-280. 6. SHATZMAN,A.R. AND HENKIN, R.1. (1981) Gustin concentration changes relative to salivary zinc and taste in humans. Proc. Natl. Acad. Sci. 78, 3867-3871. 7. FREDERICKSON, c.J., PEREZ-CLAUSELL, J. AND DANSCHER, G. (1987) Zinc-containing 7S-NGF complex. Evidence from zinc histochemistry for localization in salivary secretory granules. J. Histochem. Cytochem. 35, 579-583. 8. PUTNEY, J.w.Jr. (1976) Identification of cellular activation mechanisms associafed with salivary secretion. Ann Rev. Physiol. 48, 75-88. 9. QUISSELL, D.O., DEISHER, L.M. AND BARZEN, K.A. (1985) The rate-determining step in cAMP-mediated exocytosis in the rat parotid and submandibular glands appears to in-
volve analogous 26-kDa integral membrane phosphoproteins. Proc. Natl. Acad. Sci. 82, 3237-3241. 10. DEHAYE, J.P. (1993) ATP" increases the intracellular calcium concentration in rat submandibular glands. Gen. Pharmacol. 24, 1097-1100. 11. HURLEY, T.w., RYAN. M.P. AND SHOEMAKER, D.D. (1994) Mobilization of Ca"+ influx, but not of stored Ca"+, by extracellular ATP in rat submandibular gland acini. Arch. oral BioI. 39, 205-212. 12. DEHAYE, J.P. and TURNER, R.I. (1991) Isolation and characterization of rat submandibular intralobular ducts. Am. J. Physiol. 261, C490-C494. 13. GROSFILS, K., METIOUI, M., TIOULI, M. AND DEHAYE, J.P. (1993) Isolation of rat pancreatic acini with crude collagenase and permeabilization of these acini with streptolysin O. Res. Commun. Chern. Pathol. Pharmacol. 79, 99-115. 14. HECHTENBERG, S. and BEYERSMANN, D. (1993) Differential control of free calcium and free zinc levels in isolated bovine liver nuclei. Biochem. J. 289,757-760. 15. SOLTOFF, S.P., McMILLIAN, M.K. AND TALAMO, B.R. (1989) Coomassie brilliant blue G is a more potent antagonist of P, purinergic responses than reactive blue 2 (Cibacron blue 3GA) in rat parotid acinar cells. Biochem. biophys. Res. Commun. 165,1279-1285. 16. METIOUI, M., GROSFILS, K. AND DEHAYE, J.P. (1994) Interaction between thapsigargin and ATP" in the regulation of the intracellular calcium in rat submandibular glands. J. cell. Physiol. 161,243-248. 17. GRYNKIEWICZ, G., POENIE, M. AND TSIEN, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. bioI. Chern. 260,3440-3450. 18. MERRITT, J.E., JACOB, R. AND HALLAM, T.J. (1989) Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophil. J. bioI. Chern. 264, 1522-1527. 19. JACOB, R. (1990) Agonist-stimulated divalent cation entry into single cultured human umbilical vein endothelial cells. J. Physiol. (Lond) 421, 55-77. 20. KWAN, C.-H. AND PUTNEY, J.w.Jr. (1990) Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. J. bioI. Chern. 265, 678-684. 21. HIDE, M. AND BEAVEN, M.A. (1991) Calcium influx in a rat mast cell (RBL-2H3) line. J. bioI. Chern. 266,15221-15229. 22. DEMAUREX, N., LEW, D.P. AND KRAUSE, K.-H. (1992) Cyclapiazonic acid depletes intracellular Ca2+ stores and activates an influx pathway for divalent cations in HL-60 cells. J. bioI. Chern. 267, 2318-2324. 23. HINKLE, P.M., SHANSHALA, E.D. AND NELSON, E.I. (1992) Measurement of intracellular cadmium with fluorescent dyes. J. bioI. Chern. 267, 25553-25559. 24. METIOUI, M., GROSFILS, K. AND DEHAYE, J.P. (1994) Regulation by thapsigargin and carbachol of the intracellular calcium concentration in rat submandibular glands. Gen. Pharmacol. 25,1353-1359.
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