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Calcium influx in cultured carotid body cells is stimulated by acetylcholine and hypoxia
140
Brain Research, 347 (1985) 140-143 Elsevier
BRE 21148
Calcium influx in cultured carotid body cells is stimulated by acetylcholine and hypoxia F. PIETRUSCHKA Max-Planck-lnstitut fiir Systemphysiologie, 4600 Dortmund (F.R. G. )
(Accepted June l lth, 1985) Key words: carotid body - - tissue culture - - calcium influx - - acetylcholine - - hypoxia
Calcium influx was measured in cultured carotid body and glioma cells. In carotid body cells stimulation with acetylcholine (ACh, 10 5 mol/l) increased the calcium influx to 135% of control values after 1 min and to 163% after 30 min. With a reduction of the pO 2 to nearly zero calcium influx increased to 170% of control values. In glioma cells there was only a slight or no increase. This sensitivity of carotid body cells is discussed in relation to their function in chemoreception.
The carotid body is an arterial c h e m o r e c e p t o r situated on the carotid bifurcation. It consists of catecholamine-enriched chief cells, which are enveloped in processes of glial-like sustentacular cells. The carotid body is sensitive to hypoxia, hypercapnia and acidosis. U n d e r hypoxia transmitter-like catecholamines and acetylcholine ( A C h ) are released 7,8, and c h e m o r e c e p t o r discharge is increased 5. Until now, it is not clear whether the c h e m o r e c e p t o r is part of the carotid body cells or of the adjacent nerve endings. Previous studieslS have shown that tile fine structure of sustentacular and chief cells, especially the dense-cored vesicles of the latter, is preserved in culture. As sustentacular cells are similar to Schwann cells and belong to glial elements, we carried out our experiments not only with cultures of carotid body cells, but also with glioma cells cultured under the same conditions. This should thus enable us to determine whether the reaction of carotid body cells to the given stimuli is a specific one, and may also provide information as to which cell type may be responsible for it. It is well established 19 that calcium plays a role in stimulus secretion coupling in endocrine cells; e.g. G r 6 n b l a d et al. 9 have shown an increase in intracellular calcium-induced exocytotic m e m b r a n e profiles in the chief cells of the rat carotid body, and Starlinger 21
has found a Ca 2+ activated A T P a s e in the cat carotid body. M e a s u r e m e n t of the calcium influx in carotid body cells should enable the investigation as to which stimulus has a direct effect on the cells. Carotid body cells were p r e p a r e d from rabbit embryos. The cells were dissociated by trypsin/collagenase and plated in multiwell plastic culture dishes for 2 days in D u i b e c c o ' s minimum essential m e d i u m ( D M E M ) + 10% foetal calf serum (fCS) at 37 °C and 10% CO2 in air. The a m o u n t of cells varied from about 1 x 104 to 5 × 104 cells per culture dish. G l i o m a cells were derived from the rat glioma cell line C 6 (passage n u m b e r 90-100). They were cultivated at similar densities and under the same conditions. F o r comparison with literature all values of calcium uptake are calculated for 1 x 106 cells. 45CAC12 ( 4 - 5 0 Ci/g calcium) was purchased from New England Nuclear (Boston, M A ) , A C h chloride from Sigma (St. Louis, M O ) . Multiwell culture dishes were obtained from Costar (Cambridge, M A ) , Nunc (Roskilde, D e n m a r k ) and Falcon ( O x n a r d ,
CA). 45Ca-uptake experiments were p e r f o r m e d as described by Barnes and M a n d e P . Cells were washed and p r e i n c u b a t e d in H E P E S - b u f f e r e d saline (the incubation m e d i u m containing in retool/l: NaCl 136, KC1 5.4, MgC12 1.4, CaCI 2 1.2, NaH2PO 4 1.0, glu-
Correspondence: F. Pietruschka, Max-Planck-Institut ffir Systemphysiologie, Rheinlanddamm 201, 4600 Dortmund 1, F.R.G. 0006-8993/85/$03,30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
141 cose 10, HEPES 20; pH 7.4). After one hour of preincubation on a rocker platform at 37 °C, the medium was changed to an incubation medium (as above) containing 45CAC12(0,8 ktCi in 200 kd). Uptake experiments lasted from 1 min to 3 h. ACh was dissolved and added to the labeled medium immediately before the beginning of incubation. Hypoxia was set up in an atmos bag, an inflatable polyethylene chamber, filled with either N 2 or air (for controls). After preincubation, culture dishes were transferred to an exsiccator in the bag. When the bag was filled with gas, the dishes were attached to a shaker and the medium was changed to the labeled incubation medium. Experiments were terminated by cooling the culture dish on an ice-cold metal plate and washing 4 times with icecold stop solution (similar to the incubation medium, but NaC1 concentration raised to 150 mmol/l). Because of the small amount of cells per culture dish we could not divide them for analyzing both protein and ~sCa content. Therefore, cells were detached by 200 ~1 trypsin/collagenase and 20 gl of the cell suspension were taken for cell counting under a microscope (magnification x 150). This method of cell counting showed reproducible results (error < 10%). Diges-
tion of the cells was performed with 200 gl of 0,5 N NaOH and 45Ca2+ labeling was determined in a scintillation counter (Beckmann, Fullerton, CA). Extracellular calcium was measured with cooled cell cultures following rapid incubation in labeled cool medium1< Furthermore, calcium bound to the plastic of the culture dish without cells was determined. Both blanks were subtracted from the experimental values. As in young animals carotid bodies show only a small part of connective tissue 15, we prepared carotid body cells from embryos and cultured them for merely two days to minimize growth of non-specific cells. During this time no mitosis of sustentacular cells or fibroblasts was observed. A short-term culture of carotid body cells shows mainly epithelial cells which extend some processes (Fig. 1). They could be recognized as chief cells by their characteristic catecholamine fluorescence 16 and their fine structure1< Among the epitheloid there are small elongated cells, which prove to be sustentacular cells in electron microscopyl< The exchangeable pool of calcium, measured in carotid body cells under steady state conditions from
Fig. 1. A 2-day-oldculture of carotid body cells shows mainly epitheloid cells which extend some processes ( x 209).
142 TABLE II
6!
.6
5-
-5
°4-
Effect o f hypoxia on Ca 2+ uptake
Values for Ca 2+ uptake are expressed as in Table I.
"4>
--""
3- /i/"
-3_
Carotid body cells Glioma cells
Normoxia
Hypoxia
n
1.63 _+0.29 0.88 _+0.11
2.78 _+(/.58 0.82 _+0.13
14 9
Fig. 2. Ca 2÷ uptake under steady-state conditions into carotid body (squares) and glioma cells (circles). Continuous lines show Ca 2+ uptake per cell number (means _+ S.E.M.), dotted lines show Ca2+ uptake based on cell volume.
showed different behavior. In glomus cells there is a m a r k e d stimulation of the calcium influx after a 30rain incubation in hypoxic medium. Influx increased significantly (P < 0.02) to 170% of control values (Table II). Rat glioma cells treated in the same way as carotid body cells did not show a c o m p a r a b l e difference in Ca 2+ influx under hypoxic and normoxic
1 rain to 3 h, was c o m p a r e d with that of rat glioma cells. Fig. 1 shows that the exchange is more rapid in glomus cells and that the amount of exchangeable calcium is also higher. In carotid body cells we find values of 3 nmol + 0.86/106 cells, c o m p a r e d to 2.3 nmol + 0.23/106 in glioma cells. Based on the cell volume this difference is even greater, as glioma cells are 1.8 times bigger than glomus cells (based on the spherical form of the cells m e a s u r e d after enzymatic isolation). In relation to cell volume the pool of exchangeable calcium exceeds more than twofold that of glioma cells (Fig. 2). The effect of neurotransmitters on Ca 2+ uptake was examined with A C h (10-5 mol/1), which is known to cause catecholamine release in adrenal medulla cells 20. A d d i t i o n of A C h stimulated 45Ca2+ uptake in glomus cells (Table I). This increase a m o u n t e d to 135% after 1 rain and to 163% after 30 min and is significant (P < 0.05) using the Wilcoxon-test 22. With rat glioma cells stimulation of Ca 2+ uptake by A C h was lower and not significant. With reduction of pO2 nearly to zero the cells also
conditions. Ca 2+ uptake in carotid body cells was measured to clarify the process by which c h e m o r e c e p t i o n occurs. The amount of exchangeable calcium is higher than in glioma cells, possibly because the dense-cored vesicles and clear vesicles of the chief cells take part in calcium uptake, as shown by G r 6 n b l a d et al.l° with ultracytochemistical techniques. Like other secretory cells 12-14 carotid body cells should show increased calcium uptake with a d e q u a t e stimuli. As A C h receptor sites could be localized in the m e m b r a n e s of one type of the carotid body chief cells 4.6, we used this neurotransmitter for stimulation. The data obtained are in good agreement with that for adrenal medulla cells. Increase of Ca 2+ uptake is significant after one minute, but it is higher after 30 rain with continued presence of hormone. In rat glioma cells, which are hyperpolarized with A C h (ref. 11), we find a much smaller increase in Ca 2+ uptake under the same conditions. (The increase of variability with incubation time is p r o b a b l y caused by more calcium binding to the culture dishes.) Should the carotid body cells be chemosensitive to
3'o
6'o
9;
~o
15'o
~o o t Imin)
TABLE I Effect o f acetylcholine (10 -s tool/l) on Ca 2+ uptake (nmol Ca2+/lO° cells)
The amount of cells varied from about 1 x 104 to 5 × 104 cells per culture dish. Values of calcium uptake are calculated for 1 × 10~cells. Means + S.E.M. are shown; n = number of experiments. Incubation 1 min
Carotid body cells Glioma cells
Incubation 30 min
Control
ACh
n
Control
A Ch
n
0.78 ± 0.12 0.41 ± 0.05
1.05 _+0.24 0.52 _+0.06
11 12
2.2 ± 0.65 1.4 _ 0.19
3.6 _+ 1.4 1.65 +_0.3
7 9
143 h y p o x i a , this stimulus should cause an i n c r e a s e in cal-
cellular calcium d e c r e a s e d in carotid b o d i e s d u r i n g
c i u m u p t a k e similar to that f o u n d with A C h . T h i s was
h y p o x i a . In a d d i t i o n , p r e v i o u s e l e c t r o p h y s i o l o g i c a l
c o n f i r m e d in o u r e x p e r i m e n t s . S e v e r e h y p o x i a for 30
studies on c u l t u r e d c a r o t i d b o d y cells 2 s h o w e d that
min increases calcium u p t a k e to 170% in c o n t r o l
c h r o n i c h y p o x i a for two days c a u s e d h y p e r p o l a r i z a -
values. ( T h e s e v a l u e s are slightly l o w e r t h a n in the
tion of their m e m b r a n e p o t e n t i a l . T h e d e s c r i b e d ex-
o t h e r set of e x p e r i m e n t s , p e r h a p s b e c a u s e of t h e dif-
p e r i m e n t s with 45Ca 2+ conclusively d e m o n s t r a t e that
f e r e n t e q u i p m e n t used.) A s g l i o m a cells do not s h o w
h y p o x i a increases the calcium u p t a k e in carotid b o d y
a c o m p a r a b l e i n f l u e n c e of h y p o x i a on calcium influx,
cells. T h e way d e c r e a s i n g p O 2 triggers the c a l c i u m in-
this s e e m s to be a p r o c e s s specific to c a r o t i d b o d y
flux and s u b s e q u e n t l y the d o p a m i n e s e c r e t i o n , re-
cells, w h e r e e l e v a t e d calcium influx m a y i n d u c e cate-
mains to be clarified in f u r t h e r e x p e r i m e n t s .
c h o l a m i n e secretion. This is s u p p o r t e d by e x p e r i m e n t s of F i d o n e et al.8 with chronically d e n e r v a t e d
I wish to t h a n k Mrs. A n e t t e L a n g e r a k for e x c e l l e n t
rabbit carotid b o d i e s , which react to h y p o x i a with do-
technical assistance and Dr. H. A c k e r for helpful
p a m i n e s e c r e t i o n . Similarly, A c k e r 1 f o u n d that extra-
criticism.
1 Acker, H., The meaning of tissue pO 2 and local blood flow for the chemoreceptive process of the carotid body, Fed. Proc. Fed. Am. Soc. Exp. Biol., 39 (1980) 2641-2647. 2 Acker, H. and Pietruschka, F., Membrane potential of cultured carotid body glomus cells under normoxia and hypoxia, Brain Research, 311 (1984) 148-151. 3 Barnes, E.M. and Mandel, P., Calcium transport by primary cultured neuronal and glial cells from chick embryo brain, J. Neurochem., 36 (1981) 82-85. 4 Chen, I. and Yates, R.D., Two types of glomus cell in the rat carotid body as revealed by a-bungarotoxin binding, J. Neurocytol., 13 (1984) 281-302. 5 Delpiano, M.A. and Acker, H., Relationship between tissue pO e and chemoreceptor activity of the carotid body in vitro, Brain Research, 195 (1980) 85-93. 6 Dinger, B., Gonzalez, C., Yoshizaki, K. and Fidone, S., Alpha-bungarotoxin binding in cat carotid body, Brain Research, 205 (1981) 187-193. 7 Eyzaguirre, C., Koyano, H. and Taylor, J.R., Presence of acetylcho[ine and transmitter release from carotid body chemoreceptors, J. Physiol. (London), 178 (1965) 463-476. 8 Fidone, S., Gonzalez, C. and Yoshizaki, K., Effects of low oxygen on the release of dopamine from the rabbit carotid body in vitro, J. Physiol. (London), 333 (1982) 93-110. 9 Gr6nblad, M., Akerman, K.E.Q. and Erfink6, O., Induction of exocytosis from glomus cells by incubation of the carotid body of the rat with calcium and ionophore A23187, Anat. Rec., 195 (1979) 387-396. 10 Gr6nblad, M., Akerman, K.E.O. and Er~ink6, O., Electron-dense precipitates in glomus cells of rat carotid body after fixation in glutaraldehyde and pyroantimonate-osmium tetroxide mixture as possible indicators of calcium localization, ('ell Tissue Res.. 217 ( 1981 ) 93-104. 11 Hamprecht, B., Kemper, W. and Amano, T., Electrical response of glioma cells to acetylcholine, Brain Research, 101 (1975) 129 135.
12 Holz, R.W., Senter, R.A. and Frye, R.A., Relationship between Ca 2+ uptake and catecholamine secretion in primary dissociated cultures of adrenal medulla, J. Neurochem., 39 (1982) 635-646. 13 Kilpatrick, D.L., Slepetis, R.J., Corcoran, J.J. and Kirshner, N., Calcium uptake and catecholamine secretion by cultured bovine adrenal medulla cells, J. Neurochem., 38 (1982) 427-435. 14 Kondo, S. and Schulz, 1., Calcium ion uptake in isolated pancreas cells induced by secretagogues, Biochim. Biophys. Acta, 419 (19761 76-92. 15 Nouhouayi, Y., N6gulesco, I., Coujard, R. and March, N., Structure du corpuscule carotidien du rat male Wistar au cours du vieillissement, Z. Mikrosk. Anat. Forsch. (Leipzig), 95 (1981) 293-303. 16 Pietruschka, F., Cytochemical demonstration of catecholamines in cells of the carotid body in primary tissue culture, Cell Tissue Res., 151 (1974) 317-321. 17 Pietruschka, F. and Langerak, A., Calcium influx in cultured glomus and glioma cells. In D.J. Pallot (Ed.), The Peripheral Arterial Chemoreceptors, University Press, Oxford, 1984, pp. 67-74. 18 Pietruschka, F. and Sch~ifer, D., Fine structure of chemosensitive cells (glomus caroticum) in tissue culture, Cell Tissue Res., 168 (1976) 55-63. 19 Rasmussen, H. and Barrett, P.Q., Calcium messenger system: an integrated view, Physiol. Rev., 64 (1984) 938-984. 20 Schneider, A.S., Cline, H.T., Rosenheck, K. and Sonenberg, M., Stimulus-secretion coupling in isolated adrenal chromaffin cells: calcium channel activation and possible role of cytoskeletal elements, J. Neurochem., 37 (19811 567-575. 21 Starlinger, H., ATPases of the cat carotid body and of the neighbouring ganglia, Z. Naturjbrsch.. 37 C (19821 532-539. 22 Wilcoxon, F. In L. Sachs (Ed.), Angewandte Statistik, Springer. Berlin, 1978, pp. 230-246.
Brain Research, 347 (1985) 140-143 Elsevier
BRE 21148
Calcium influx in cultured carotid body cells is stimulated by acetylcholine and hypoxia F. PIETRUSCHKA Max-Planck-lnstitut fiir Systemphysiologie, 4600 Dortmund (F.R. G. )
(Accepted June l lth, 1985) Key words: carotid body - - tissue culture - - calcium influx - - acetylcholine - - hypoxia
Calcium influx was measured in cultured carotid body and glioma cells. In carotid body cells stimulation with acetylcholine (ACh, 10 5 mol/l) increased the calcium influx to 135% of control values after 1 min and to 163% after 30 min. With a reduction of the pO 2 to nearly zero calcium influx increased to 170% of control values. In glioma cells there was only a slight or no increase. This sensitivity of carotid body cells is discussed in relation to their function in chemoreception.
The carotid body is an arterial c h e m o r e c e p t o r situated on the carotid bifurcation. It consists of catecholamine-enriched chief cells, which are enveloped in processes of glial-like sustentacular cells. The carotid body is sensitive to hypoxia, hypercapnia and acidosis. U n d e r hypoxia transmitter-like catecholamines and acetylcholine ( A C h ) are released 7,8, and c h e m o r e c e p t o r discharge is increased 5. Until now, it is not clear whether the c h e m o r e c e p t o r is part of the carotid body cells or of the adjacent nerve endings. Previous studieslS have shown that tile fine structure of sustentacular and chief cells, especially the dense-cored vesicles of the latter, is preserved in culture. As sustentacular cells are similar to Schwann cells and belong to glial elements, we carried out our experiments not only with cultures of carotid body cells, but also with glioma cells cultured under the same conditions. This should thus enable us to determine whether the reaction of carotid body cells to the given stimuli is a specific one, and may also provide information as to which cell type may be responsible for it. It is well established 19 that calcium plays a role in stimulus secretion coupling in endocrine cells; e.g. G r 6 n b l a d et al. 9 have shown an increase in intracellular calcium-induced exocytotic m e m b r a n e profiles in the chief cells of the rat carotid body, and Starlinger 21
has found a Ca 2+ activated A T P a s e in the cat carotid body. M e a s u r e m e n t of the calcium influx in carotid body cells should enable the investigation as to which stimulus has a direct effect on the cells. Carotid body cells were p r e p a r e d from rabbit embryos. The cells were dissociated by trypsin/collagenase and plated in multiwell plastic culture dishes for 2 days in D u i b e c c o ' s minimum essential m e d i u m ( D M E M ) + 10% foetal calf serum (fCS) at 37 °C and 10% CO2 in air. The a m o u n t of cells varied from about 1 x 104 to 5 × 104 cells per culture dish. G l i o m a cells were derived from the rat glioma cell line C 6 (passage n u m b e r 90-100). They were cultivated at similar densities and under the same conditions. F o r comparison with literature all values of calcium uptake are calculated for 1 x 106 cells. 45CAC12 ( 4 - 5 0 Ci/g calcium) was purchased from New England Nuclear (Boston, M A ) , A C h chloride from Sigma (St. Louis, M O ) . Multiwell culture dishes were obtained from Costar (Cambridge, M A ) , Nunc (Roskilde, D e n m a r k ) and Falcon ( O x n a r d ,
CA). 45Ca-uptake experiments were p e r f o r m e d as described by Barnes and M a n d e P . Cells were washed and p r e i n c u b a t e d in H E P E S - b u f f e r e d saline (the incubation m e d i u m containing in retool/l: NaCl 136, KC1 5.4, MgC12 1.4, CaCI 2 1.2, NaH2PO 4 1.0, glu-
Correspondence: F. Pietruschka, Max-Planck-Institut ffir Systemphysiologie, Rheinlanddamm 201, 4600 Dortmund 1, F.R.G. 0006-8993/85/$03,30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
141 cose 10, HEPES 20; pH 7.4). After one hour of preincubation on a rocker platform at 37 °C, the medium was changed to an incubation medium (as above) containing 45CAC12(0,8 ktCi in 200 kd). Uptake experiments lasted from 1 min to 3 h. ACh was dissolved and added to the labeled medium immediately before the beginning of incubation. Hypoxia was set up in an atmos bag, an inflatable polyethylene chamber, filled with either N 2 or air (for controls). After preincubation, culture dishes were transferred to an exsiccator in the bag. When the bag was filled with gas, the dishes were attached to a shaker and the medium was changed to the labeled incubation medium. Experiments were terminated by cooling the culture dish on an ice-cold metal plate and washing 4 times with icecold stop solution (similar to the incubation medium, but NaC1 concentration raised to 150 mmol/l). Because of the small amount of cells per culture dish we could not divide them for analyzing both protein and ~sCa content. Therefore, cells were detached by 200 ~1 trypsin/collagenase and 20 gl of the cell suspension were taken for cell counting under a microscope (magnification x 150). This method of cell counting showed reproducible results (error < 10%). Diges-
tion of the cells was performed with 200 gl of 0,5 N NaOH and 45Ca2+ labeling was determined in a scintillation counter (Beckmann, Fullerton, CA). Extracellular calcium was measured with cooled cell cultures following rapid incubation in labeled cool medium1< Furthermore, calcium bound to the plastic of the culture dish without cells was determined. Both blanks were subtracted from the experimental values. As in young animals carotid bodies show only a small part of connective tissue 15, we prepared carotid body cells from embryos and cultured them for merely two days to minimize growth of non-specific cells. During this time no mitosis of sustentacular cells or fibroblasts was observed. A short-term culture of carotid body cells shows mainly epithelial cells which extend some processes (Fig. 1). They could be recognized as chief cells by their characteristic catecholamine fluorescence 16 and their fine structure1< Among the epitheloid there are small elongated cells, which prove to be sustentacular cells in electron microscopyl< The exchangeable pool of calcium, measured in carotid body cells under steady state conditions from
Fig. 1. A 2-day-oldculture of carotid body cells shows mainly epitheloid cells which extend some processes ( x 209).
142 TABLE II
6!
.6
5-
-5
°4-
Effect o f hypoxia on Ca 2+ uptake
Values for Ca 2+ uptake are expressed as in Table I.
"4>
--""
3- /i/"
-3_
Carotid body cells Glioma cells
Normoxia
Hypoxia
n
1.63 _+0.29 0.88 _+0.11
2.78 _+(/.58 0.82 _+0.13
14 9
Fig. 2. Ca 2÷ uptake under steady-state conditions into carotid body (squares) and glioma cells (circles). Continuous lines show Ca 2+ uptake per cell number (means _+ S.E.M.), dotted lines show Ca2+ uptake based on cell volume.
showed different behavior. In glomus cells there is a m a r k e d stimulation of the calcium influx after a 30rain incubation in hypoxic medium. Influx increased significantly (P < 0.02) to 170% of control values (Table II). Rat glioma cells treated in the same way as carotid body cells did not show a c o m p a r a b l e difference in Ca 2+ influx under hypoxic and normoxic
1 rain to 3 h, was c o m p a r e d with that of rat glioma cells. Fig. 1 shows that the exchange is more rapid in glomus cells and that the amount of exchangeable calcium is also higher. In carotid body cells we find values of 3 nmol + 0.86/106 cells, c o m p a r e d to 2.3 nmol + 0.23/106 in glioma cells. Based on the cell volume this difference is even greater, as glioma cells are 1.8 times bigger than glomus cells (based on the spherical form of the cells m e a s u r e d after enzymatic isolation). In relation to cell volume the pool of exchangeable calcium exceeds more than twofold that of glioma cells (Fig. 2). The effect of neurotransmitters on Ca 2+ uptake was examined with A C h (10-5 mol/1), which is known to cause catecholamine release in adrenal medulla cells 20. A d d i t i o n of A C h stimulated 45Ca2+ uptake in glomus cells (Table I). This increase a m o u n t e d to 135% after 1 rain and to 163% after 30 min and is significant (P < 0.05) using the Wilcoxon-test 22. With rat glioma cells stimulation of Ca 2+ uptake by A C h was lower and not significant. With reduction of pO2 nearly to zero the cells also
conditions. Ca 2+ uptake in carotid body cells was measured to clarify the process by which c h e m o r e c e p t i o n occurs. The amount of exchangeable calcium is higher than in glioma cells, possibly because the dense-cored vesicles and clear vesicles of the chief cells take part in calcium uptake, as shown by G r 6 n b l a d et al.l° with ultracytochemistical techniques. Like other secretory cells 12-14 carotid body cells should show increased calcium uptake with a d e q u a t e stimuli. As A C h receptor sites could be localized in the m e m b r a n e s of one type of the carotid body chief cells 4.6, we used this neurotransmitter for stimulation. The data obtained are in good agreement with that for adrenal medulla cells. Increase of Ca 2+ uptake is significant after one minute, but it is higher after 30 rain with continued presence of hormone. In rat glioma cells, which are hyperpolarized with A C h (ref. 11), we find a much smaller increase in Ca 2+ uptake under the same conditions. (The increase of variability with incubation time is p r o b a b l y caused by more calcium binding to the culture dishes.) Should the carotid body cells be chemosensitive to
3'o
6'o
9;
~o
15'o
~o o t Imin)
TABLE I Effect o f acetylcholine (10 -s tool/l) on Ca 2+ uptake (nmol Ca2+/lO° cells)
The amount of cells varied from about 1 x 104 to 5 × 104 cells per culture dish. Values of calcium uptake are calculated for 1 × 10~cells. Means + S.E.M. are shown; n = number of experiments. Incubation 1 min
Carotid body cells Glioma cells
Incubation 30 min
Control
ACh
n
Control
A Ch
n
0.78 ± 0.12 0.41 ± 0.05
1.05 _+0.24 0.52 _+0.06
11 12
2.2 ± 0.65 1.4 _ 0.19
3.6 _+ 1.4 1.65 +_0.3
7 9
143 h y p o x i a , this stimulus should cause an i n c r e a s e in cal-
cellular calcium d e c r e a s e d in carotid b o d i e s d u r i n g
c i u m u p t a k e similar to that f o u n d with A C h . T h i s was
h y p o x i a . In a d d i t i o n , p r e v i o u s e l e c t r o p h y s i o l o g i c a l
c o n f i r m e d in o u r e x p e r i m e n t s . S e v e r e h y p o x i a for 30
studies on c u l t u r e d c a r o t i d b o d y cells 2 s h o w e d that
min increases calcium u p t a k e to 170% in c o n t r o l
c h r o n i c h y p o x i a for two days c a u s e d h y p e r p o l a r i z a -
values. ( T h e s e v a l u e s are slightly l o w e r t h a n in the
tion of their m e m b r a n e p o t e n t i a l . T h e d e s c r i b e d ex-
o t h e r set of e x p e r i m e n t s , p e r h a p s b e c a u s e of t h e dif-
p e r i m e n t s with 45Ca 2+ conclusively d e m o n s t r a t e that
f e r e n t e q u i p m e n t used.) A s g l i o m a cells do not s h o w
h y p o x i a increases the calcium u p t a k e in carotid b o d y
a c o m p a r a b l e i n f l u e n c e of h y p o x i a on calcium influx,
cells. T h e way d e c r e a s i n g p O 2 triggers the c a l c i u m in-
this s e e m s to be a p r o c e s s specific to c a r o t i d b o d y
flux and s u b s e q u e n t l y the d o p a m i n e s e c r e t i o n , re-
cells, w h e r e e l e v a t e d calcium influx m a y i n d u c e cate-
mains to be clarified in f u r t h e r e x p e r i m e n t s .
c h o l a m i n e secretion. This is s u p p o r t e d by e x p e r i m e n t s of F i d o n e et al.8 with chronically d e n e r v a t e d
I wish to t h a n k Mrs. A n e t t e L a n g e r a k for e x c e l l e n t
rabbit carotid b o d i e s , which react to h y p o x i a with do-
technical assistance and Dr. H. A c k e r for helpful
p a m i n e s e c r e t i o n . Similarly, A c k e r 1 f o u n d that extra-
criticism.
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