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46 1
Rinard, G. A . , and Jensen, A. 1981. Preparation of homonesensitive airway smooth muscle adenylate cyclase from dissociated canine trachealis cells. Biochim. Biophys. Acta, 678: 287 -2 12. Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Am. N.Y. Acad. Sci. 51: 660. Takagi, K., a d Takayanagi, I. 8958. Chemico-phamcological studies on antispasmodic action XV. Non-specific antispasmodic action on trachea. Chern. P b m . Bull. (Tokyo), 6: 786-720. Tayanagi, I., and Koike, K. 1991. Minireview: Effects of ageing on postsynaptic a,-adrenoceptor mechanisms in rat aorta. Gene Pharmcol. 22: 211-218. Tkayanagi, I., Koike, K., md Himta, T. 1986. Interactions of muscarinic drugs with their receptors in single cells guinea-pig taenia caecum. J. Pham. Phamcol. 38: 476-478. Takayanagi, I., Maeda, O . , and Koike, K. 1987. Postsynaptic a,-adrenoceptor mechanisms in rat vas deferens and ageing. J. Pham. Phamcol. 39: 754-757. Tdcayanagi, I., Shinhi, M.,and Yamasawa, K. 1989. Effects of ageing on a,-adrenoceptor mechanisms and inhibitory effect of diltimem on noradremaline maximum response in isolated rat aortic preparation. Can. B. Physiol. Pharmcol. 67: 1398- 1402. Takayaanagi, I., Kawano, K.-I., and Koilce, K. 1990. Effect of aging on the response of guinea pig trachea to isoprenaline. Bpn. J. Phamacol. 53: 359-366. Tsujimoto, G., Lee, C.-H., and H o h a n , B. B. 1986. Age-related decreases in beta-adrenergic receptor-mediated vascular smooth muscle relaxation. J. Phamacol. Exp. Ther. 239: 41 1-415.
Davis, C., Conslly, M. E., and Greenace, J. K. 1980.8-Ackenoceptors in human lung, bronchus and lymphocytes. Br. J. Clin. P b m c o l . 10: 425 -432. Duncan, D. B. 1955. Multiple range and multiple Ftest. Biometries, 3: 1-24. Duncan, P. G., Brink, C., and Douglas, J. S. 1982. fl-Receptorsdurk g aging in respiratory tissues. Eur. J. Phamacol. 78: 45 -52. Ooldie, G. R.,Papadimitriou, J. M., Paterson, J. W., Rigby, P. J . , Self, H. M., and Spina, D. 1986. Influence of the epithelium on the responsiveness of guinea-pig isolatd trachea to contractile and relaxant agonists. Br- 9. Phamacol. 87: 5 - 14. Himyarna, T., Tahyamgi, I., Nhgoshi, F., and Hiram, K. 1988. Epithelium selectively controls hypersensitization of the response of smooth muscle to leukotriene D, by endogenous prosknoid(s) in guinea-pig trachea. Naunyn-Schmideberg's Arch. Phamcol. 337: 296-300. Johs, A., and Riehl, R. M. 1982. A simple method for preparing single cell suspensions of heart and smooth muscle for radiorwep. Methods, 7: 153- 159. tor labeling studies. 9. Pha Comparison of ,6-adrenoceptors KoiEre, K., and Takayanagi, I. in single smooth muscle cells and isolated smmth muscle preparations from the guinea pig he& caecum. Arch. Int. Phamacodyn. %er. 296: 184-191. Mita, M.,and Uchida, M. K. 1988. Muswrinic receptor binding and CaZ+influx in the all-or-none o acetylcholine of isolated smooth muscle cells. Eur. J. 1. 151: 9- 17. Momox, K., and Gomi, Y. 1978. Studies on isolated smooth muscle cells. IV. Isolation and acetylcholine contraction of single smooth muscle cells from kenia coli of guinea-pig. J. Bhamacobio-Dyn. It: 184-198e
Differing significance of Na+ -Ca2+ exchange in the regulation of cytosolic CaZ+ in rat exocrine gland acini and cardiac myocytes THOMASWeHURLEY~~ WILLIAM E. DALE, AND MICHAEL J. ROVETTO Depa~aneentsof ChiM Heallbe a d PhysioEogyDUniversity of Missouri School of Medicine, Columbia, MO 65212, U.S.A.
Received July 12, 1991 H~RLEY, T* W , DALE,W. E., and R O V B T ~M. , J. 1992. Differing significance of Na+ -Ca2+ exchange in the regulation of cytosolic Ca2+ in rat exocrine gland acini and cardiac myocytes. Can. J. Physiol. Phamcol. 70: 461 -465. In order to compare the importance of Na+ -@a2+ exchange in the regulation of cytosolic Ca2+ concentration (Ca"), acini obtained from rat pancreas and submandibular glands as well as cardiac myocytes were loaded with Na+ by inhibition of Na+-K+ AThse activity then loaded with hra-2. In the exocrine tissues, incubation in K+-free buffer or with ouabain had no substantid effect on resting Ca:' or on the changes in Ca" following exposure to carbachol as compared with acini incubated under control conditions. In contrast, rat cardiac myocytes, treated identically, showed marked changes in Ca:+ under resting and stimulated conditions as compared with controls. We conclude that the Nag-Ca2+ exchange systems of rat pancreatic and submndihlar gland acini contribute little to the overall regulation of Ca'+ at rest during cholinergic stimulation. Key words : submandibular glands, pancreatic acini, salivary glands, hra-2, cytosolic Ca2+,Na+ -Caw exchange. --
HURLEY,T. W., DALE,W. E., et R O V E ~M. ~, J. 1992. Differing significance of Na+-Ca2+ exchange in the regulation of cytosolie Ca2+ in rat exocrine gland aciri and cardiac myocyas. Can. J. Physiol. Phamcol. 70 : 461 -465. Afin d'6valuer l ' i m p m c e de 19&hangeNa+ -Ca2' dans la rkgulation de la concentration de Ca2+cytosolique (Caf +), des acini de pancreas, de glandes sous-maxillaires et des myocytes cardiaques de rats ont d'abord kt6 chargks avec du Na+ par 19inhibitionde I'activit6 de Na+-K+ AThse, puis avec du hra-2. Dans les tissus exocrines, l'incubation dam un
"uthor for correspondence at the following address: Dep Drive, Columbia, MO 65212, U.S.A. Printed in Canada I Imprim6 au Canada
nt of Child Health, University of Missouri School of Medicine, 1 Hospital
CAN. B. PHYSIBL. PHAFSvfACOL. VOL. 70, I992
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tampon sans K+ ou avec de l'ouabai'ne n'a pas eu dkffet substantiel sur le Ca'+ au repos, ni sur les variations Be @a'+, aprks une expsition au carbachol, csmparativement anx acini incub6s dans des conditions t6moins. A 190pposC,les myocytes cardiaques de rats trait& de manike indentique ont montr6 des variations marquhs de Ca'+ dans des conditions de stimubat b n et de r e p s , comparativement a m conditions tkmoins. Nsus concluons que les syst2mes d'khange Na9 -Ca2+ des acini sous-mxillaires et pancrbatiques des rats influencent p u la rkgulation globale du @a;+ au repos et durant une stimulation cholinergique. Mots ck&s : glandes som-mxillaires, achi pancr&tiques, glandes salivaires, hra-2, cytosoltique, Cchange Na+-$la2+. [Traduit par h r&&ction]
Introduction Many differentiated cells respond to stimuli with an increase in cytosolic Ca2+ levels (Ca:+), which is due to release of C$+ from stores within the cell and (or) to influx sf Ca2+ from outside the ell merridge md Hmine 1989). Overdl control of CaTf may be achieved in part by uptake of Ca2+ by cellular organelles but dso requires that Ca2+ eventually be expelled from the cell. Two important mechanisms by which C$+ may be lost from the cell are an ATP-dependent C$+ pump (Rega md Garrhan 1986) and a Na+-Ca2+ exchanger (Reeves 1990). The former directly couples the hydrolysis of ATP to &la2+movement against its electrochemical potential gradient; the latter couples the movement of Na+ down its electrochemical potential gradient to the movement of @a2+in the opposite direction and does not expend ATP directly. This exchange of Na+ and Ca2+ does, however, rely on a Na+ concentration gradient that, in most cells, is maintained by the Na+-K+ AThse of the plasma membrane. A Na+ -Caw exchanger has k e n identified in many cell types, but evaluations of its significance in the regulation of CaT+ are less co on. In cardiac myocytes (Men et d. 1989), retinal rod cells (Koch md Stryer 1988), and rend tubules (Snowdowne and Borle 1985) Na+-Ca2+ exchange has been reported to be a major regulator of moment-tomoment changes in Caf . Indeed, inhibition of the Na+ -K+ A T h e of cardiac myocytes, which increases @a;+ by lowering the Na+ gradient and reducing Na+ -Ca2+ exchange, is %laoughtto be among the mechanisms by which cardiac glycosides exert their positive inotropic effect. Evidence for a Na -Ca2 exchange system has been reported in plasm membrane preparations of pancreatic and salivary gland acini (Schulz 1980; Takuma et d. 1985), but evaluations of their significance in the regulation of CaT+ at rest and during stimulation are few. Muallem et al. (1988) observed at, in pancreatic acini, Ca2+ efflux, resting Cat+, and changes in &la:+ during stimulation were unaffected by direct manipulation of the Na9 gradient across the cell membrane. They concluded that the Na+-CaZi exchanger was relatively unipnpomwt cornpad with ATP-dependent Ca2 pumping in mediating Ca" efflux. Though convincing, their report dso noted hat the cations used to replace Na+ in manipulating extracellular Na+ concentrations were not inert but had appreciable effects on celldm Ca2+ handling and on acinar cell function. We have taken a different approach in evaluating the role of Na+ -Ca2+ exchange in the overdl handing of Ga2+ in exocrine glmd acini. Rather than manipulating Na6 gradients by substituting other extracellular cations, we have exposed acinar suspnsisns to conditions that inhibit Na+-K+ AFhse activity, seducing the Na' gradient across the plasma membrane md thereby reducing any ~ a -+ ca2+ exchange activity. Acid were then loaded with the acetoxymethyl ester of hua-2, and Ca:' was measured at rest and during maximal +
+
+
+
stimulation. To the extent that Na9-C$+ exchange contributes to regulation of resting or stimulated Ca;+, Na+ -K9 ATWse inhibition should result in increased Caf+ compared with cells not subjected to such a manipulation. h addition, we have compared the effects of %la+-K+ ATkse inhibition in pancreatic and submandibular gland acini with those induced in rat cardiac myocytes treated similarly. Our results show that, while Na+ -K+ AThse inhibition results in substantial changes in @a2+handing in cardiac myocytea, such inhibition has little effect on Ca;+ at rest and during stimulation in either pancreatic or submandibdar gland acini. We conclude that, under conditions of this study, the Na+ -Ca2+ exchange system of rat pancreatic md submandibular gland acini csntributes little to the overall regulation of C++ at rest and during cholinergic stimulation.
Methods Acinar preparations were obtained by digestion s f the minced glands with collagenase and hyaluronidase as described elsewhere for the pancreas (Hurley and Brinck 1990) and submndibular glands (Hurley and Ryan 1988). Rat cardiac myocytes were obtained as previously described (GeisbuMer et al. 1987). Cell suspensions were incubated with the acetoxymethyl ester s f hra-2 (4-6 pM) for 45 -60 min at 39°C. Most measurements of Ca;+ were obtained within 2 k of the end of loading. For dye loading and to measure Ca;+, cells were suspend& in control buffer of the following compsition (d): HEPES (pH 7.41, 25; NaCl, 125; KC1, 4; glucose, 2.5; CaCl,, 2.5; bovine serum albumin, 0. 1 % w/v; and amino acids, 1% v k . After loading, cells were washed twice to remove extracdlular dye and stored in buffer at room temperature. Aliqusts of loaded cells were washed once more before being placed in the sample chamber of a dual excitation beam spectrofluoromekr. Ca:+ was measured by the ratio method as described by Bydciewicz et d. (1985) with excitation at 380 and 340 m at 1.0-s intervals and eontinuous recording of emission intensity at 505 m. A dissociation constant for the Eaara-2 -Ca2+ complex of 224 nM was used to cdculate Ca;+ (Grynkewicz et daB. 1985). Fluorescence emission maxima were obtained by lysing cells with digitonin in the presence of a saturating Ca" concentration; a sixfold excess of BGTA (final pH > 8.0) was then added to obtain the minimum fluorescence emission intensity. Pancreatic and submndibular gland acini were stimuhtai with a supramximl concentration of carbachsl(48 pM);cardiac cells were depolaked by the addition of KCl to a final concentration of 30 mM. Two methods were used to inhibit Na+ -K9 AThse activity: (dl cells were incubated 45 -60 min in buffer in which K+ was replaced with an equimoltar comentratisn of NaCl and (ii) cells were incubated 45 -60 HBPiPP in control buffer containing 2.5 mh4 ouabain, a concentration shown, in preliminary experiments, to cause maximum rduc$ion of ATPase activity. The results shown were d l obtained using aliqots of the same tissue preparations and are representative of results obtained in at least four preparations of each tissue type. Artifacts introduced into the recordings when the sample chamber was opened to add reagents Rave been removed to improve the clarity sf presentation.
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Time (s)
Time (s) FIG. 1. Rat pancreatic asinar CaPc at rest and during stimulation with 48 pM carbiichol. (a) Acini incubated in control buffer. (b) Acini incubated in K+-free buffer. (e) Acini incubated in control buffer containing 2.5 mM ouabain.
FIG. 2. Rat submandibular gland acinar Ca" at rest and during stimulation with 40 p M cabaehol. (a) Acini incubated in control buffer. (b) Acini incubated in K+-free buffer. (c) Acini incubated in control buffer containing 2.5 rnM ouabain.
Results The effects of inhibiting Na+ -K+ AThse activity on Ca:+ in pancreatic acini are shown in Fig. 1. Figure la shows changes in Ca:+ in acini incubated in control buffer during exposure to 40 p M cmbachol at about 15 s. The resting Caf+ of 150-200 nM is immediately elevated upon exposure to the agsnist and then fdls to a steady-state value near 300 nM, which is maintained as long as carbachol occupies its receptor (Hoarley md Brinck 1998). Figure 1b shows carbachol-induced changes in Caf+ in pancreatic acini incubated in Kc-free buffer. The resting and stimulated levels of Ca:+ are not significantly different from those measured in acini exposed to control buffer. Likewise, the changes in Ca:+ in pancreatic acini expsed to ouabdw (Fig. 1c) are not different from those measured under control conditions. In submndibular gland acini not expsed to NaC-K* ATPase inhibition (Fig. 2n) resting Ca:+ (100 nM) rises several fold during exposure to cmbachol (at about 38 s) md soon fdls to a steady-state value two to three times the resting Ca:+ where it remains as long as the muscarinic receptor remains occupied @rinck a d PIaarley 1989). Incubation of the acid in KQ-free buffer results ody in a slightly prolonged initial elevation of Ca:+ (Fig. 2b). Resting Ca;+ and the steady-state Ca:+ during carbachol exposure do not differ markedly from control levels. Incubation with o w b h is dso followed by a minimally prolonged initial elevation in Ca:+ in response to carbachol (Fig. 2e). However, this change is neither pronounced nor prolonged9 and within a minute
Ca:+reaches Levels virtually identical to those measured in control acini . Figure 3a shows the C q + of cardiac tnyocytes incubated under control conditions: a resting level of a b u t 75 wM undergoes a modest but persistent increase upon expsare to 30 mE&% KC1 at a b u t 30 s. Incubation in Kc-free buffer results in a increase in resting Ca:* to more than 100 nM (Pig. 3b), but there is little change compmed with controls in the response sf &la:+ to depolarization. (It shodd be pointed out hat depolarization with 30 m M KC1 vitiates the test condition of K+-free incubation.) After incubation with ouabain, the i m e diate change in CaZ+ in response to depolarization (Pig. 3c) is little changed but, thereafter, Ca;+ steadily increases rather than reaching a stable plateau. After some minutes, C g + reaches levels sufficient to induce contraction that is apparent microscopidly when these nomally elongated cells become spherical (data not shown).
These studies were undertaken to assess the role of Na+ -Ca2 exchange in regulating Ca;' in different tissues that depend on changes in Ca;+ for their functions. Inhibition of Na+ -K+ AThse activity is followed by major changes in overdl calcium handling in cardiac mymytes. These changes are reflected in the marked elevations in Ca?+ during exposure to KC1 compared with the changes in ~ a f in + myocytes n d e x p s d to Na+-K+ AThse inhibition. This suggests h i t the Na+ -Ca2+ exchanger in cardiac myocytes is a +
+
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Time (s) FIG. 3. Rat cardiac pn~wyte at rest and during depolarization with KCl. (a) Myocytes incubated in control buffer. (b) Myocytes incubated in M+-free buffer. (c) Myocytes incubated in control buffer containing 2.5 mM ouabain.
major regulator of CafC at rest and during stimulation, a conclusion consistent with a large body of evidence from many laboratories (Allen et d. 1989). More important for this report, these results establish that the conditions used to assess the role of Nae -Ca2+ exchange are sufficient to induce and reved derangements in C$+ handling in a tissue in which the Na+ -CaD exchanger is known to be functionally important. This comparative approach to the assessment of physiologicd relevance is especially usehl, since no specific inhibitor is available that selectively reduces Naf -C$ * exchange activity. In addition, comparing the effects on Caf+ of directly inhibiting Nae -Ke AThse activity avoids the difficulties of substituting extracellular Na+ with other anions that may themselves deer cellular functions, as Mudlem et diH. (1988) have pointed out. In contrast with its effects in cardiac myocytes, Na+ -K+ AThse inhibition had no significant effect compared with unheated cells om resting or stimulated Ca:+ in pancreatic or submmdibular gland acini. The differences in response between exocrine gland acini and the cardiac myocytes were not due to differences in the stimuli used to evoke the responses, since expsure of acini from either g l a d to 30 mM KC1 produced no changes in Caf+ (data not shown). This arized by Petersen and is consistent with reports, su Gallacher (11988), that exocrine glmd achi lack voltagedependent Gas influx pathways and with earlier reports of ours (Hurley m d Ryan 1988; Hudey and Brinck 1998) that blockers of deplarization-dependent, L-type channels are without effect on carbachol-induced changes in Ca?+ in acini
from either the pancreas or submandibular gland. These results suggest that Ca2+ does not enter pancreatic or submandibular gland acini via voltage-sensitive influx pathways and that the ligand-operated influx pathways that are present in both types of acini (Hurley and Brinck 4998; Brine% and Hurley 1989) have little stmcturd similarity to the %-type Ca" channels of excitable cells. Some studies have suggested a link between the cellular handling of cytosoIic Ca2+ and Na+ in salivary gland aeini. Wa&on et d, (1981) and Martinez et id. ( 1990) showed that exposure of mouse parotid acini and rat submandibular gland acini to monensin stimulates amylase release (from parotid cells) and Na+ uptake (in both cell types). These observations are consistent with the presence of a Na+ - Ca2+ exchange system in those cells. As those authors point out, however, rnonensin &so stimulates Na+ -H+ countertransport so that its effects may be due, at least in part, to the dkdinization of the cell in the presence of monensin. The effects of andoride and hrosemide reported by Martinez et d. (1990) may be due, in part, to the inhibition of Na+-H+ exchange by the former and to the latter's M i bition of Na+ -K+ -2Cl- co-transport. That co-transpofier may be stimulated by increased extracellular K + secondary to Caw-activated K + efflux and could also contribute to the accumulation of Na+ by acini exposed to Ca2+-mobilizing agents. Of course our results do not rule out the presence of a Na+ -Ca2+ exchmger in rat pancreatic or submandibular g l a d acini. They bear, instead, on its apparent contribution to the regulation of Caf+ under our experimental conditions. It is clear that, under those conditions, the Na+ -Ca2+ exchanger contributes little to the ovemll regulation of Ca:+ at rest and during stimulation.
Acknowledgements This work was supported by grants from the National Institutes of Health. The assistance of Mrs. Shirley Haden is deeply appreciated. Allen, %. J. A., Noble, D., and Reuter, H. (Editors). 1989. Sodium -calcium exchange. Oxford University Press, Oxford ,
U.K. Bemidge, M.b., and Irvine, W. F. 1989. Hnositol phosphates and cell signalling. Nature (London), 341: % 97 -285. Brinek, R. W., and Hurley, T. W. 1989. Regulation sf eytosolic Ca2' in resting and stimulated rat submandibular salivary gland acini. Arch. Ord BioB. 34: 91'7-922. CkisbuMer, T. P., Johnson9DsA mand p Rovetts, M. 3. 1987. Cardkc myocyte guanssine transport and metabolism. Am. J. Physiol. 253: C645 -C65 1. Srynkiewicz, G . , Poenie, M., and Tsien, 8. Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. 1. Biol. CIrern. 260: 340-3450. Hurley, T. W., and Brinck, R.W. 1998. Regulating transient and sustained changes of cytosolic @a2+in rat pancreatic acini. Am. J. Physiol. 25%: C54 -C61. Hurley, T. W., and Ryan, M. P. 1988. The control s f cytosolic Caw concentration: studies of high affinity Ca2+ transport in perrnabirized spcini of rat submmdibdar glands. Arch. Ord Biol. 33: 793 - 800. Koch, K.-W., and Stryer, L. 1988. Highly cooperative feedback control of retinal md panylate qclase by calcium ions. Nature (London), 3%: 64-66. Martinez, J. R., Camden, J . , and Baker, S. 1990. Effects of acetylcholine and monensin on 2Na uptake and cytosolic Ca2' in rat submandibular salivary cells. Arch. Ord Biol. 35: 359 -364.
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Mullem, S . , Beeker, T o , and Psandol, S. J. 1988. Role of Na+dCa2' exchange and the plasma membrane Ca2' pump in homowe-mdiated C 3 + efflux from pancreatic acini. J. Membr. Biol. 102: 153- 152. Petersen, 8.H., and Gallacher, D. V. 1988. Electrsphysiology sf pancreatic and salivary acinar cells. Amu. Rev. Phyaisl. 50: 65 - 80. Reeves, J. P. 1990. Hwtracdlular calcium regulation. Edited by F. Brsnner. Alan R. Eiss, New York. pp. 305-348. Rega, A. F., and Garrhan, P. S. 1986. The calcium pump of plasma membranes. CRC Press, Boca Raton, Fla. Schula, I. 1980. Messenger role of calcium in function of pancreatic acinar cells. Am. J. Physiol. 239: G335 -G347.
Snowdowne, K. W., and Borle, A. B. 1985. Effects s f low extracdlalar sodium on eytosolic ionized calcium. Na+ -Ca2+ exchange as a major calcium influx pathway in kidney cells. J. Biol. Chem. 268: 14 998 - 15 007. T a h m a , T., Kuyatt, B. L., and Baum, B.J. 1985. Calcium transport mechanisms in basolaterd plasma membrane-enriched vesicles from rat parotid g l a d . Biochem. 3. 227: 239 -245. Watson, E. L., Farrham, C. J., Friedman, S., and Farham, W. 8981. Effects s f rnonensin on amylase release from mouse parotid acini. Am. J. Physiol. 240: C189-C192.
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46 1
Rinard, G. A . , and Jensen, A. 1981. Preparation of homonesensitive airway smooth muscle adenylate cyclase from dissociated canine trachealis cells. Biochim. Biophys. Acta, 678: 287 -2 12. Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Am. N.Y. Acad. Sci. 51: 660. Takagi, K., a d Takayanagi, I. 8958. Chemico-phamcological studies on antispasmodic action XV. Non-specific antispasmodic action on trachea. Chern. P b m . Bull. (Tokyo), 6: 786-720. Tayanagi, I., and Koike, K. 1991. Minireview: Effects of ageing on postsynaptic a,-adrenoceptor mechanisms in rat aorta. Gene Pharmcol. 22: 211-218. Tkayanagi, I., Koike, K., md Himta, T. 1986. Interactions of muscarinic drugs with their receptors in single cells guinea-pig taenia caecum. J. Pham. Phamcol. 38: 476-478. Takayanagi, I., Maeda, O . , and Koike, K. 1987. Postsynaptic a,-adrenoceptor mechanisms in rat vas deferens and ageing. J. Pham. Phamcol. 39: 754-757. Tdcayanagi, I., Shinhi, M.,and Yamasawa, K. 1989. Effects of ageing on a,-adrenoceptor mechanisms and inhibitory effect of diltimem on noradremaline maximum response in isolated rat aortic preparation. Can. B. Physiol. Pharmcol. 67: 1398- 1402. Takayaanagi, I., Kawano, K.-I., and Koilce, K. 1990. Effect of aging on the response of guinea pig trachea to isoprenaline. Bpn. J. Phamacol. 53: 359-366. Tsujimoto, G., Lee, C.-H., and H o h a n , B. B. 1986. Age-related decreases in beta-adrenergic receptor-mediated vascular smooth muscle relaxation. J. Phamacol. Exp. Ther. 239: 41 1-415.
Davis, C., Conslly, M. E., and Greenace, J. K. 1980.8-Ackenoceptors in human lung, bronchus and lymphocytes. Br. J. Clin. P b m c o l . 10: 425 -432. Duncan, D. B. 1955. Multiple range and multiple Ftest. Biometries, 3: 1-24. Duncan, P. G., Brink, C., and Douglas, J. S. 1982. fl-Receptorsdurk g aging in respiratory tissues. Eur. J. Phamacol. 78: 45 -52. Ooldie, G. R.,Papadimitriou, J. M., Paterson, J. W., Rigby, P. J . , Self, H. M., and Spina, D. 1986. Influence of the epithelium on the responsiveness of guinea-pig isolatd trachea to contractile and relaxant agonists. Br- 9. Phamacol. 87: 5 - 14. Himyarna, T., Tahyamgi, I., Nhgoshi, F., and Hiram, K. 1988. Epithelium selectively controls hypersensitization of the response of smooth muscle to leukotriene D, by endogenous prosknoid(s) in guinea-pig trachea. Naunyn-Schmideberg's Arch. Phamcol. 337: 296-300. Johs, A., and Riehl, R. M. 1982. A simple method for preparing single cell suspensions of heart and smooth muscle for radiorwep. Methods, 7: 153- 159. tor labeling studies. 9. Pha Comparison of ,6-adrenoceptors KoiEre, K., and Takayanagi, I. in single smooth muscle cells and isolated smmth muscle preparations from the guinea pig he& caecum. Arch. Int. Phamacodyn. %er. 296: 184-191. Mita, M.,and Uchida, M. K. 1988. Muswrinic receptor binding and CaZ+influx in the all-or-none o acetylcholine of isolated smooth muscle cells. Eur. J. 1. 151: 9- 17. Momox, K., and Gomi, Y. 1978. Studies on isolated smooth muscle cells. IV. Isolation and acetylcholine contraction of single smooth muscle cells from kenia coli of guinea-pig. J. Bhamacobio-Dyn. It: 184-198e
Differing significance of Na+ -Ca2+ exchange in the regulation of cytosolic CaZ+ in rat exocrine gland acini and cardiac myocytes THOMASWeHURLEY~~ WILLIAM E. DALE, AND MICHAEL J. ROVETTO Depa~aneentsof ChiM Heallbe a d PhysioEogyDUniversity of Missouri School of Medicine, Columbia, MO 65212, U.S.A.
Received July 12, 1991 H~RLEY, T* W , DALE,W. E., and R O V B T ~M. , J. 1992. Differing significance of Na+ -Ca2+ exchange in the regulation of cytosolic Ca2+ in rat exocrine gland acini and cardiac myocytes. Can. J. Physiol. Phamcol. 70: 461 -465. In order to compare the importance of Na+ -@a2+ exchange in the regulation of cytosolic Ca2+ concentration (Ca"), acini obtained from rat pancreas and submandibular glands as well as cardiac myocytes were loaded with Na+ by inhibition of Na+-K+ AThse activity then loaded with hra-2. In the exocrine tissues, incubation in K+-free buffer or with ouabain had no substantid effect on resting Ca:' or on the changes in Ca" following exposure to carbachol as compared with acini incubated under control conditions. In contrast, rat cardiac myocytes, treated identically, showed marked changes in Ca:+ under resting and stimulated conditions as compared with controls. We conclude that the Nag-Ca2+ exchange systems of rat pancreatic and submndihlar gland acini contribute little to the overall regulation of Ca'+ at rest during cholinergic stimulation. Key words : submandibular glands, pancreatic acini, salivary glands, hra-2, cytosolic Ca2+,Na+ -Caw exchange. --
HURLEY,T. W., DALE,W. E., et R O V E ~M. ~, J. 1992. Differing significance of Na+-Ca2+ exchange in the regulation of cytosolie Ca2+ in rat exocrine gland aciri and cardiac myocyas. Can. J. Physiol. Phamcol. 70 : 461 -465. Afin d'6valuer l ' i m p m c e de 19&hangeNa+ -Ca2' dans la rkgulation de la concentration de Ca2+cytosolique (Caf +), des acini de pancreas, de glandes sous-maxillaires et des myocytes cardiaques de rats ont d'abord kt6 chargks avec du Na+ par 19inhibitionde I'activit6 de Na+-K+ AThse, puis avec du hra-2. Dans les tissus exocrines, l'incubation dam un
"uthor for correspondence at the following address: Dep Drive, Columbia, MO 65212, U.S.A. Printed in Canada I Imprim6 au Canada
nt of Child Health, University of Missouri School of Medicine, 1 Hospital
CAN. B. PHYSIBL. PHAFSvfACOL. VOL. 70, I992
462
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tampon sans K+ ou avec de l'ouabai'ne n'a pas eu dkffet substantiel sur le Ca'+ au repos, ni sur les variations Be @a'+, aprks une expsition au carbachol, csmparativement anx acini incub6s dans des conditions t6moins. A 190pposC,les myocytes cardiaques de rats trait& de manike indentique ont montr6 des variations marquhs de Ca'+ dans des conditions de stimubat b n et de r e p s , comparativement a m conditions tkmoins. Nsus concluons que les syst2mes d'khange Na9 -Ca2+ des acini sous-mxillaires et pancrbatiques des rats influencent p u la rkgulation globale du @a;+ au repos et durant une stimulation cholinergique. Mots ck&s : glandes som-mxillaires, achi pancr&tiques, glandes salivaires, hra-2, cytosoltique, Cchange Na+-$la2+. [Traduit par h r&&ction]
Introduction Many differentiated cells respond to stimuli with an increase in cytosolic Ca2+ levels (Ca:+), which is due to release of C$+ from stores within the cell and (or) to influx sf Ca2+ from outside the ell merridge md Hmine 1989). Overdl control of CaTf may be achieved in part by uptake of Ca2+ by cellular organelles but dso requires that Ca2+ eventually be expelled from the cell. Two important mechanisms by which C$+ may be lost from the cell are an ATP-dependent C$+ pump (Rega md Garrhan 1986) and a Na+-Ca2+ exchanger (Reeves 1990). The former directly couples the hydrolysis of ATP to &la2+movement against its electrochemical potential gradient; the latter couples the movement of Na+ down its electrochemical potential gradient to the movement of @a2+in the opposite direction and does not expend ATP directly. This exchange of Na+ and Ca2+ does, however, rely on a Na+ concentration gradient that, in most cells, is maintained by the Na+-K+ AThse of the plasma membrane. A Na+ -Caw exchanger has k e n identified in many cell types, but evaluations of its significance in the regulation of CaT+ are less co on. In cardiac myocytes (Men et d. 1989), retinal rod cells (Koch md Stryer 1988), and rend tubules (Snowdowne and Borle 1985) Na+-Ca2+ exchange has been reported to be a major regulator of moment-tomoment changes in Caf . Indeed, inhibition of the Na+ -K+ A T h e of cardiac myocytes, which increases @a;+ by lowering the Na+ gradient and reducing Na+ -Ca2+ exchange, is %laoughtto be among the mechanisms by which cardiac glycosides exert their positive inotropic effect. Evidence for a Na -Ca2 exchange system has been reported in plasm membrane preparations of pancreatic and salivary gland acini (Schulz 1980; Takuma et d. 1985), but evaluations of their significance in the regulation of CaT+ at rest and during stimulation are few. Muallem et al. (1988) observed at, in pancreatic acini, Ca2+ efflux, resting Cat+, and changes in &la:+ during stimulation were unaffected by direct manipulation of the Na9 gradient across the cell membrane. They concluded that the Na+-CaZi exchanger was relatively unipnpomwt cornpad with ATP-dependent Ca2 pumping in mediating Ca" efflux. Though convincing, their report dso noted hat the cations used to replace Na+ in manipulating extracellular Na+ concentrations were not inert but had appreciable effects on celldm Ca2+ handling and on acinar cell function. We have taken a different approach in evaluating the role of Na+ -Ca2+ exchange in the overdl handing of Ga2+ in exocrine glmd acini. Rather than manipulating Na6 gradients by substituting other extracellular cations, we have exposed acinar suspnsisns to conditions that inhibit Na+-K+ AFhse activity, seducing the Na' gradient across the plasma membrane md thereby reducing any ~ a -+ ca2+ exchange activity. Acid were then loaded with the acetoxymethyl ester of hua-2, and Ca:' was measured at rest and during maximal +
+
+
+
stimulation. To the extent that Na9-C$+ exchange contributes to regulation of resting or stimulated Ca;+, Na+ -K9 ATWse inhibition should result in increased Caf+ compared with cells not subjected to such a manipulation. h addition, we have compared the effects of %la+-K+ ATkse inhibition in pancreatic and submandibular gland acini with those induced in rat cardiac myocytes treated similarly. Our results show that, while Na+ -K+ AThse inhibition results in substantial changes in @a2+handing in cardiac myocytea, such inhibition has little effect on Ca;+ at rest and during stimulation in either pancreatic or submandibdar gland acini. We conclude that, under conditions of this study, the Na+ -Ca2+ exchange system of rat pancreatic md submandibular gland acini csntributes little to the overall regulation of C++ at rest and during cholinergic stimulation.
Methods Acinar preparations were obtained by digestion s f the minced glands with collagenase and hyaluronidase as described elsewhere for the pancreas (Hurley and Brinck 1990) and submndibular glands (Hurley and Ryan 1988). Rat cardiac myocytes were obtained as previously described (GeisbuMer et al. 1987). Cell suspensions were incubated with the acetoxymethyl ester s f hra-2 (4-6 pM) for 45 -60 min at 39°C. Most measurements of Ca;+ were obtained within 2 k of the end of loading. For dye loading and to measure Ca;+, cells were suspend& in control buffer of the following compsition (d): HEPES (pH 7.41, 25; NaCl, 125; KC1, 4; glucose, 2.5; CaCl,, 2.5; bovine serum albumin, 0. 1 % w/v; and amino acids, 1% v k . After loading, cells were washed twice to remove extracdlular dye and stored in buffer at room temperature. Aliqusts of loaded cells were washed once more before being placed in the sample chamber of a dual excitation beam spectrofluoromekr. Ca:+ was measured by the ratio method as described by Bydciewicz et d. (1985) with excitation at 380 and 340 m at 1.0-s intervals and eontinuous recording of emission intensity at 505 m. A dissociation constant for the Eaara-2 -Ca2+ complex of 224 nM was used to cdculate Ca;+ (Grynkewicz et daB. 1985). Fluorescence emission maxima were obtained by lysing cells with digitonin in the presence of a saturating Ca" concentration; a sixfold excess of BGTA (final pH > 8.0) was then added to obtain the minimum fluorescence emission intensity. Pancreatic and submndibular gland acini were stimuhtai with a supramximl concentration of carbachsl(48 pM);cardiac cells were depolaked by the addition of KCl to a final concentration of 30 mM. Two methods were used to inhibit Na+ -K9 AThse activity: (dl cells were incubated 45 -60 min in buffer in which K+ was replaced with an equimoltar comentratisn of NaCl and (ii) cells were incubated 45 -60 HBPiPP in control buffer containing 2.5 mh4 ouabain, a concentration shown, in preliminary experiments, to cause maximum rduc$ion of ATPase activity. The results shown were d l obtained using aliqots of the same tissue preparations and are representative of results obtained in at least four preparations of each tissue type. Artifacts introduced into the recordings when the sample chamber was opened to add reagents Rave been removed to improve the clarity sf presentation.
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Time (s)
Time (s) FIG. 1. Rat pancreatic asinar CaPc at rest and during stimulation with 48 pM carbiichol. (a) Acini incubated in control buffer. (b) Acini incubated in K+-free buffer. (e) Acini incubated in control buffer containing 2.5 mM ouabain.
FIG. 2. Rat submandibular gland acinar Ca" at rest and during stimulation with 40 p M cabaehol. (a) Acini incubated in control buffer. (b) Acini incubated in K+-free buffer. (c) Acini incubated in control buffer containing 2.5 rnM ouabain.
Results The effects of inhibiting Na+ -K+ AThse activity on Ca:+ in pancreatic acini are shown in Fig. 1. Figure la shows changes in Ca:+ in acini incubated in control buffer during exposure to 40 p M cmbachol at about 15 s. The resting Caf+ of 150-200 nM is immediately elevated upon exposure to the agsnist and then fdls to a steady-state value near 300 nM, which is maintained as long as carbachol occupies its receptor (Hoarley md Brinck 1998). Figure 1b shows carbachol-induced changes in Caf+ in pancreatic acini incubated in Kc-free buffer. The resting and stimulated levels of Ca:+ are not significantly different from those measured in acini exposed to control buffer. Likewise, the changes in Ca:+ in pancreatic acini expsed to ouabdw (Fig. 1c) are not different from those measured under control conditions. In submndibular gland acini not expsed to NaC-K* ATPase inhibition (Fig. 2n) resting Ca:+ (100 nM) rises several fold during exposure to cmbachol (at about 38 s) md soon fdls to a steady-state value two to three times the resting Ca:+ where it remains as long as the muscarinic receptor remains occupied @rinck a d PIaarley 1989). Incubation of the acid in KQ-free buffer results ody in a slightly prolonged initial elevation of Ca:+ (Fig. 2b). Resting Ca;+ and the steady-state Ca:+ during carbachol exposure do not differ markedly from control levels. Incubation with o w b h is dso followed by a minimally prolonged initial elevation in Ca:+ in response to carbachol (Fig. 2e). However, this change is neither pronounced nor prolonged9 and within a minute
Ca:+reaches Levels virtually identical to those measured in control acini . Figure 3a shows the C q + of cardiac tnyocytes incubated under control conditions: a resting level of a b u t 75 wM undergoes a modest but persistent increase upon expsare to 30 mE&% KC1 at a b u t 30 s. Incubation in Kc-free buffer results in a increase in resting Ca:* to more than 100 nM (Pig. 3b), but there is little change compmed with controls in the response sf &la:+ to depolarization. (It shodd be pointed out hat depolarization with 30 m M KC1 vitiates the test condition of K+-free incubation.) After incubation with ouabain, the i m e diate change in CaZ+ in response to depolarization (Pig. 3c) is little changed but, thereafter, Ca;+ steadily increases rather than reaching a stable plateau. After some minutes, C g + reaches levels sufficient to induce contraction that is apparent microscopidly when these nomally elongated cells become spherical (data not shown).
These studies were undertaken to assess the role of Na+ -Ca2 exchange in regulating Ca;' in different tissues that depend on changes in Ca;+ for their functions. Inhibition of Na+ -K+ AThse activity is followed by major changes in overdl calcium handling in cardiac mymytes. These changes are reflected in the marked elevations in Ca?+ during exposure to KC1 compared with the changes in ~ a f in + myocytes n d e x p s d to Na+-K+ AThse inhibition. This suggests h i t the Na+ -Ca2+ exchanger in cardiac myocytes is a +
+
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Time (s) FIG. 3. Rat cardiac pn~wyte at rest and during depolarization with KCl. (a) Myocytes incubated in control buffer. (b) Myocytes incubated in M+-free buffer. (c) Myocytes incubated in control buffer containing 2.5 mM ouabain.
major regulator of CafC at rest and during stimulation, a conclusion consistent with a large body of evidence from many laboratories (Allen et d. 1989). More important for this report, these results establish that the conditions used to assess the role of Nae -Ca2+ exchange are sufficient to induce and reved derangements in C$+ handling in a tissue in which the Na+ -CaD exchanger is known to be functionally important. This comparative approach to the assessment of physiologicd relevance is especially usehl, since no specific inhibitor is available that selectively reduces Naf -C$ * exchange activity. In addition, comparing the effects on Caf+ of directly inhibiting Nae -Ke AThse activity avoids the difficulties of substituting extracellular Na+ with other anions that may themselves deer cellular functions, as Mudlem et diH. (1988) have pointed out. In contrast with its effects in cardiac myocytes, Na+ -K+ AThse inhibition had no significant effect compared with unheated cells om resting or stimulated Ca:+ in pancreatic or submmdibular gland acini. The differences in response between exocrine gland acini and the cardiac myocytes were not due to differences in the stimuli used to evoke the responses, since expsure of acini from either g l a d to 30 mM KC1 produced no changes in Caf+ (data not shown). This arized by Petersen and is consistent with reports, su Gallacher (11988), that exocrine glmd achi lack voltagedependent Gas influx pathways and with earlier reports of ours (Hurley m d Ryan 1988; Hudey and Brinck 1998) that blockers of deplarization-dependent, L-type channels are without effect on carbachol-induced changes in Ca?+ in acini
from either the pancreas or submandibular gland. These results suggest that Ca2+ does not enter pancreatic or submandibular gland acini via voltage-sensitive influx pathways and that the ligand-operated influx pathways that are present in both types of acini (Hurley and Brinck 4998; Brine% and Hurley 1989) have little stmcturd similarity to the %-type Ca" channels of excitable cells. Some studies have suggested a link between the cellular handling of cytosoIic Ca2+ and Na+ in salivary gland aeini. Wa&on et d, (1981) and Martinez et id. ( 1990) showed that exposure of mouse parotid acini and rat submandibular gland acini to monensin stimulates amylase release (from parotid cells) and Na+ uptake (in both cell types). These observations are consistent with the presence of a Na+ - Ca2+ exchange system in those cells. As those authors point out, however, rnonensin &so stimulates Na+ -H+ countertransport so that its effects may be due, at least in part, to the dkdinization of the cell in the presence of monensin. The effects of andoride and hrosemide reported by Martinez et d. (1990) may be due, in part, to the inhibition of Na+-H+ exchange by the former and to the latter's M i bition of Na+ -K+ -2Cl- co-transport. That co-transpofier may be stimulated by increased extracellular K + secondary to Caw-activated K + efflux and could also contribute to the accumulation of Na+ by acini exposed to Ca2+-mobilizing agents. Of course our results do not rule out the presence of a Na+ -Ca2+ exchmger in rat pancreatic or submandibular g l a d acini. They bear, instead, on its apparent contribution to the regulation of Caf+ under our experimental conditions. It is clear that, under those conditions, the Na+ -Ca2+ exchanger contributes little to the ovemll regulation of Ca:+ at rest and during stimulation.
Acknowledgements This work was supported by grants from the National Institutes of Health. The assistance of Mrs. Shirley Haden is deeply appreciated. Allen, %. J. A., Noble, D., and Reuter, H. (Editors). 1989. Sodium -calcium exchange. Oxford University Press, Oxford ,
U.K. Bemidge, M.b., and Irvine, W. F. 1989. Hnositol phosphates and cell signalling. Nature (London), 341: % 97 -285. Brinek, R. W., and Hurley, T. W. 1989. Regulation sf eytosolic Ca2' in resting and stimulated rat submandibular salivary gland acini. Arch. Ord BioB. 34: 91'7-922. CkisbuMer, T. P., Johnson9DsA mand p Rovetts, M. 3. 1987. Cardkc myocyte guanssine transport and metabolism. Am. J. Physiol. 253: C645 -C65 1. Srynkiewicz, G . , Poenie, M., and Tsien, 8. Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. 1. Biol. CIrern. 260: 340-3450. Hurley, T. W., and Brinck, R.W. 1998. Regulating transient and sustained changes of cytosolic @a2+in rat pancreatic acini. Am. J. Physiol. 25%: C54 -C61. Hurley, T. W., and Ryan, M. P. 1988. The control s f cytosolic Caw concentration: studies of high affinity Ca2+ transport in perrnabirized spcini of rat submmdibdar glands. Arch. Ord Biol. 33: 793 - 800. Koch, K.-W., and Stryer, L. 1988. Highly cooperative feedback control of retinal md panylate qclase by calcium ions. Nature (London), 3%: 64-66. Martinez, J. R., Camden, J . , and Baker, S. 1990. Effects of acetylcholine and monensin on 2Na uptake and cytosolic Ca2' in rat submandibular salivary cells. Arch. Ord Biol. 35: 359 -364.
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Mullem, S . , Beeker, T o , and Psandol, S. J. 1988. Role of Na+dCa2' exchange and the plasma membrane Ca2' pump in homowe-mdiated C 3 + efflux from pancreatic acini. J. Membr. Biol. 102: 153- 152. Petersen, 8.H., and Gallacher, D. V. 1988. Electrsphysiology sf pancreatic and salivary acinar cells. Amu. Rev. Phyaisl. 50: 65 - 80. Reeves, J. P. 1990. Hwtracdlular calcium regulation. Edited by F. Brsnner. Alan R. Eiss, New York. pp. 305-348. Rega, A. F., and Garrhan, P. S. 1986. The calcium pump of plasma membranes. CRC Press, Boca Raton, Fla. Schula, I. 1980. Messenger role of calcium in function of pancreatic acinar cells. Am. J. Physiol. 239: G335 -G347.
Snowdowne, K. W., and Borle, A. B. 1985. Effects s f low extracdlalar sodium on eytosolic ionized calcium. Na+ -Ca2+ exchange as a major calcium influx pathway in kidney cells. J. Biol. Chem. 268: 14 998 - 15 007. T a h m a , T., Kuyatt, B. L., and Baum, B.J. 1985. Calcium transport mechanisms in basolaterd plasma membrane-enriched vesicles from rat parotid g l a d . Biochem. 3. 227: 239 -245. Watson, E. L., Farrham, C. J., Friedman, S., and Farham, W. 8981. Effects s f rnonensin on amylase release from mouse parotid acini. Am. J. Physiol. 240: C189-C192.
