GMc&fml
(lwo) il,
673-679
@ Longman Bfoup UK Ud lsg0
Na+-Dependent Ca2+ influx in bovine adrenal chromaffin cells P-S. LIU and L-S. KAO lnstiute of Biomedical Sciences, Academia Sin&?, Nankang, Taipei, Taiwan, Repubiic of China Abstrsct - Bovine adrenal chromaffln cells were loaded with Na+ via either acetylchollne receptor-assooiated Ion channels or voltage-sensltlve Na+ channels. There were lncreaws in [Ca*+ji, 45Ca2” uptake and catecholamine secretion in both types of Na+-louded cells relative to control cells in which Na+ loading had been prevented by hexamethonium and tetrodotoxln. respectfvely. These results show the presence of Na+dependent Ca2” inflw actlvlty In c~romkfln ceils which is probably media&d by the reverse &de of a Na”/C#’ exchanger. The essential role of Ca2+ in stimulus-secretion coupling in adrenal medullary chromaffin cells has been genemlly accepted [l]. The concentration of free &+ in the cytosol ([Ca2+]j) of chromaffin cells is under strict regulation and the level of [Ca2flt controls the occurrence of exocytosis. At resting state, the [Ca2flt of chromaffin cells is maintained at Upon maximal acetylcholine around 0.1 pM. stimulation, the [Ca2Tl increases to 5 to 10 pM which then triggers exocytosis [24]. The mechanisms responsible for [Ca2+Il regulation in many systems have been extensively studied. It has been suggested in other systems that Na’/Ca2’ exchange is one of the pthways in the plasma membrane regulating [Ca +]l [e.g., 5-71. The presence of Na+/Ca2+ exchange activity in the plasma membrane vesicles isolated from adrenal medulla and the intact chromaffin cells has been demonstrated [8, 91. In this study, we have taken advantage of the fact that Na+/Ca2+ exchange functions in both directions across the membrane, i.e. external Na+ exchanges with internal Ca2’ or internal Na+ exchanges with external Ca2+, depending on the concemrations of both ions on
each side of the membrane [lo, 111. Here, we demonstrate that the reverse mode of Na+/Ca2i exchange occurs in cultured bovine chromaffin cells after cytosolic Na+ concentration is increased via the acetylcholine receptor-associated ion channels and the volta e-sensitive Na+ channels. The effects on [Ca2Tt, 0‘Ca2’ influx and catecholamine secretion were examined.
Materials and Methods G.emicals
Collagenase (type I) was purchased from the Worthington Biochemical Corporation (NJ, USA). Deoxyribonuclease I, carbachol, vemtridine, ouabain, verapamil, hexamethonium, tetrodotoxin were obtained from Sigma Chemical Co. (MO, USA). [?Hj-Norepinephrine wm4sroy+New England Nuclear (MA, USA) and Ca was from Amersham (Amersham, UK). Sucrose and other salts were obtained from Merck (Darmstadt, FRG). Furaacetoxymethyl ester was obtained from
573
574
Molecular Probes (OR, USA) and ionomycln was from Calbiochem (CA, USA). Isolationof bovine a&em1 medullarycells
Bovine adrenal medullary cells were isolated by a minor modification of a previously described method 1121. Adrenal glands were first perfused via the adrenal vein 3 to 5 times using a syringe containing perfusion solution (145 mM NaCl, 5.4 mM KCl, 1.0 mM NaHzPO4 11.2 mM glucose, 100 units/ml penicillin G, 40 &ml gentamycin, and 15 mM HBPBS, pH 7.4) over a period of 30 min at room temperature. The glands were then perfused 1 to 3 times with a collagenase solution (0.2% collagenase and 0.002% deoxyribonuclease I in perfusion solution). After perfusion, the medulla was separated from the cortex and cut into small pieces. Minced medulla was then further digested twice, -each time for 30 min with collagenaae solution at 37’C. The isolated cells were filtered through 250 pm nylon mesh and collected by centrifugation (2,300 g) at room temperature. The isolated chrome cells were either used fresh for [Ca’+jt measurements or cultured in 24-well culture plates (5 x lo5 cells/well) for measuring 45Ca2+ uptake or in %-well plates (2 x 10’ cells/well) for secretion measurements.
CELLcALcIuM
included in the Cati-free Na+ loading solution to block the respective channels. After loading, the buffer was replaced by a Na+-free sucrose or N-methyl-D+camine (NMG) buffer (NaCl in the loading buffer was isotonically replaced by suctose or NMG with an osmolarity of 305 * 5 mGsmol/kg) containing 0.3 mM verapamil, 0.1 mM ouabain and 1 mM hexamethonium to establish a Na+ gradient across the cell membrane. A control experiment without any drug treatment in which chromaffln cells were placed in Na+-free sucrose or NMG solution after a 10 min incubation in Ca2’-free Na+ loading solution was also included. Catecholunaine secrettin
Cultured cells were loaded with [?I]-norepinephrine as described by Kilpatrlck et al. [13]. The [?Ij-norepinephrine-loaded chromaffin cells were loaded with Na+ as described above. At designated times after Na+-loading, supematant was removed and a solution of 0.1% Triton X-100 and 2 mM EGTA was added to the pellet. Radioactivlties of the supernatant and pellet were counted and used to calculate the percentage of the total radioactivity of the cells which was secreted. Results are expressed as mean * SEM of at least three determinations in each of three experiments using different batches of cells.
sodium loading calcium uptake
Sodium was introduced into chromaffin cells through either the acetylcholine receptor-associated ion channels or the voltage-sensitive Na+ channels. Briefly, freshly isolated or cultured chromaffin cells were incubated with either (a) 0.3 mM carbachol and 0.1 mM ouabain to open nicotinic receptor-associated ion channels or (b) 0.1 mM ouabain veratridine and 0.1 mM for voltage-sensitive Na+ channels, in a Ca’+-free Na+ loading solution (150 mM NaCl, 5 mM KCl, 2.2 mM CaClz, 1 mM MgCls 5 mM glucose, 10 mM HEPES, pH 7.4) for 10 min at room temperature. In one of the control experiments (with antagonists), either (a) 1 mM hexamethonium and 0.3 mM verapamil in addition to 0.3 mM carbachol and 0.1 mM ouabain or (b) 2 t&I tetrodotoxin in addition to 0.1 mM veratridine and 0.1 mM ouabain were
45Ca2+uptake was measured as previously described with minor modifications [14]. Cultured chromaffin cells were first washed six times with Krebs/HBPES solution (140 mM NaCl, 4.7 mM KCl, 1.2 mM KH904, 2.5 mM CaClz, 1.2 mM MgSO4, 11 mM glucose, 15 mM HEPES, pH 7.4) for 1 h a\;oog temperature. After Na+-loading, uptake of Ca was initiated by replacing Na+-loading sol$tioF+with Na+-free sucrose buffer containing Ca (4 @i/ml, 10-40 mCi/mg Ca). At designated times, 45Ca2+ uptake was stopped by washing the cells every 10 s for 1 min with a modified Ca2’-free Krebs/HEPBS solution containing 3.7 mM MgClz, 2 mM LaClh and 2 mM EGTA at pH 7.0 (prepared immediately before use). The cells were then lysed with 0.1% Triton X-100 solution and the
CXROh4AFFlNCELW Na+-DEPENDENTCa2+INFLUX
575
Table 1 Effect of Na+-loading oo the [h2+ji in chrotio
cells
Chromaffio cells received various treatments as described in the legends of Figure IA (A) and Wgwe 2A (B): awe Na+-loaded cells; curve B, control cells (t aotagoaist@; auve
A,
C, control cells (without any drug). ‘lk initial rates were
calculated from the cbaoges in [Ca2Ti within tbe first 30 s after the adis were a&d into the Na+-free solution by linear regresioo analysis. Values are meao t SEM of the number of experiments given in parentheses. s P ~0.02; bP
(lwo) il,
673-679
@ Longman Bfoup UK Ud lsg0
Na+-Dependent Ca2+ influx in bovine adrenal chromaffin cells P-S. LIU and L-S. KAO lnstiute of Biomedical Sciences, Academia Sin&?, Nankang, Taipei, Taiwan, Repubiic of China Abstrsct - Bovine adrenal chromaffln cells were loaded with Na+ via either acetylchollne receptor-assooiated Ion channels or voltage-sensltlve Na+ channels. There were lncreaws in [Ca*+ji, 45Ca2” uptake and catecholamine secretion in both types of Na+-louded cells relative to control cells in which Na+ loading had been prevented by hexamethonium and tetrodotoxln. respectfvely. These results show the presence of Na+dependent Ca2” inflw actlvlty In c~romkfln ceils which is probably media&d by the reverse &de of a Na”/C#’ exchanger. The essential role of Ca2+ in stimulus-secretion coupling in adrenal medullary chromaffin cells has been genemlly accepted [l]. The concentration of free &+ in the cytosol ([Ca2+]j) of chromaffin cells is under strict regulation and the level of [Ca2flt controls the occurrence of exocytosis. At resting state, the [Ca2flt of chromaffin cells is maintained at Upon maximal acetylcholine around 0.1 pM. stimulation, the [Ca2Tl increases to 5 to 10 pM which then triggers exocytosis [24]. The mechanisms responsible for [Ca2+Il regulation in many systems have been extensively studied. It has been suggested in other systems that Na’/Ca2’ exchange is one of the pthways in the plasma membrane regulating [Ca +]l [e.g., 5-71. The presence of Na+/Ca2+ exchange activity in the plasma membrane vesicles isolated from adrenal medulla and the intact chromaffin cells has been demonstrated [8, 91. In this study, we have taken advantage of the fact that Na+/Ca2+ exchange functions in both directions across the membrane, i.e. external Na+ exchanges with internal Ca2’ or internal Na+ exchanges with external Ca2+, depending on the concemrations of both ions on
each side of the membrane [lo, 111. Here, we demonstrate that the reverse mode of Na+/Ca2i exchange occurs in cultured bovine chromaffin cells after cytosolic Na+ concentration is increased via the acetylcholine receptor-associated ion channels and the volta e-sensitive Na+ channels. The effects on [Ca2Tt, 0‘Ca2’ influx and catecholamine secretion were examined.
Materials and Methods G.emicals
Collagenase (type I) was purchased from the Worthington Biochemical Corporation (NJ, USA). Deoxyribonuclease I, carbachol, vemtridine, ouabain, verapamil, hexamethonium, tetrodotoxin were obtained from Sigma Chemical Co. (MO, USA). [?Hj-Norepinephrine wm4sroy+New England Nuclear (MA, USA) and Ca was from Amersham (Amersham, UK). Sucrose and other salts were obtained from Merck (Darmstadt, FRG). Furaacetoxymethyl ester was obtained from
573
574
Molecular Probes (OR, USA) and ionomycln was from Calbiochem (CA, USA). Isolationof bovine a&em1 medullarycells
Bovine adrenal medullary cells were isolated by a minor modification of a previously described method 1121. Adrenal glands were first perfused via the adrenal vein 3 to 5 times using a syringe containing perfusion solution (145 mM NaCl, 5.4 mM KCl, 1.0 mM NaHzPO4 11.2 mM glucose, 100 units/ml penicillin G, 40 &ml gentamycin, and 15 mM HBPBS, pH 7.4) over a period of 30 min at room temperature. The glands were then perfused 1 to 3 times with a collagenase solution (0.2% collagenase and 0.002% deoxyribonuclease I in perfusion solution). After perfusion, the medulla was separated from the cortex and cut into small pieces. Minced medulla was then further digested twice, -each time for 30 min with collagenaae solution at 37’C. The isolated cells were filtered through 250 pm nylon mesh and collected by centrifugation (2,300 g) at room temperature. The isolated chrome cells were either used fresh for [Ca’+jt measurements or cultured in 24-well culture plates (5 x lo5 cells/well) for measuring 45Ca2+ uptake or in %-well plates (2 x 10’ cells/well) for secretion measurements.
CELLcALcIuM
included in the Cati-free Na+ loading solution to block the respective channels. After loading, the buffer was replaced by a Na+-free sucrose or N-methyl-D+camine (NMG) buffer (NaCl in the loading buffer was isotonically replaced by suctose or NMG with an osmolarity of 305 * 5 mGsmol/kg) containing 0.3 mM verapamil, 0.1 mM ouabain and 1 mM hexamethonium to establish a Na+ gradient across the cell membrane. A control experiment without any drug treatment in which chromaffln cells were placed in Na+-free sucrose or NMG solution after a 10 min incubation in Ca2’-free Na+ loading solution was also included. Catecholunaine secrettin
Cultured cells were loaded with [?I]-norepinephrine as described by Kilpatrlck et al. [13]. The [?Ij-norepinephrine-loaded chromaffin cells were loaded with Na+ as described above. At designated times after Na+-loading, supematant was removed and a solution of 0.1% Triton X-100 and 2 mM EGTA was added to the pellet. Radioactivlties of the supernatant and pellet were counted and used to calculate the percentage of the total radioactivity of the cells which was secreted. Results are expressed as mean * SEM of at least three determinations in each of three experiments using different batches of cells.
sodium loading calcium uptake
Sodium was introduced into chromaffin cells through either the acetylcholine receptor-associated ion channels or the voltage-sensitive Na+ channels. Briefly, freshly isolated or cultured chromaffin cells were incubated with either (a) 0.3 mM carbachol and 0.1 mM ouabain to open nicotinic receptor-associated ion channels or (b) 0.1 mM ouabain veratridine and 0.1 mM for voltage-sensitive Na+ channels, in a Ca’+-free Na+ loading solution (150 mM NaCl, 5 mM KCl, 2.2 mM CaClz, 1 mM MgCls 5 mM glucose, 10 mM HEPES, pH 7.4) for 10 min at room temperature. In one of the control experiments (with antagonists), either (a) 1 mM hexamethonium and 0.3 mM verapamil in addition to 0.3 mM carbachol and 0.1 mM ouabain or (b) 2 t&I tetrodotoxin in addition to 0.1 mM veratridine and 0.1 mM ouabain were
45Ca2+uptake was measured as previously described with minor modifications [14]. Cultured chromaffin cells were first washed six times with Krebs/HBPES solution (140 mM NaCl, 4.7 mM KCl, 1.2 mM KH904, 2.5 mM CaClz, 1.2 mM MgSO4, 11 mM glucose, 15 mM HEPES, pH 7.4) for 1 h a\;oog temperature. After Na+-loading, uptake of Ca was initiated by replacing Na+-loading sol$tioF+with Na+-free sucrose buffer containing Ca (4 @i/ml, 10-40 mCi/mg Ca). At designated times, 45Ca2+ uptake was stopped by washing the cells every 10 s for 1 min with a modified Ca2’-free Krebs/HEPBS solution containing 3.7 mM MgClz, 2 mM LaClh and 2 mM EGTA at pH 7.0 (prepared immediately before use). The cells were then lysed with 0.1% Triton X-100 solution and the
CXROh4AFFlNCELW Na+-DEPENDENTCa2+INFLUX
575
Table 1 Effect of Na+-loading oo the [h2+ji in chrotio
cells
Chromaffio cells received various treatments as described in the legends of Figure IA (A) and Wgwe 2A (B): awe Na+-loaded cells; curve B, control cells (t aotagoaist@; auve
A,
C, control cells (without any drug). ‘lk initial rates were
calculated from the cbaoges in [Ca2Ti within tbe first 30 s after the adis were a&d into the Na+-free solution by linear regresioo analysis. Values are meao t SEM of the number of experiments given in parentheses. s P ~0.02; bP
