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Influence of the calcium ionophores A23187 and X537A on calcitonin secretion from the isolated perfused porcine thyroid
Molecular and Cellular Endocrinology. 45 (1986) 71-75 Elsevier Scientific Publishers Ireland, Ltd.
71
MCE 01450
Influence
of the calcium ionophores A23187 and X537A on calcitonin secretion from the isolated perfused porcine thyroid J. Thomas
Pento
Division of Pharmacodynamics and Toxicology, College of Pharmacy, Uniuersity of Oklahoma Health Sciences Center, Oklahoma City, OK 73190 (U.S.A.) (Received
Key words: calcitonin;
calcitonin
secretion;
17 July 1985; accepted
thyroid;
calcium
ionophores;
12 December
A23187;
1985)
X537A.
Summary The present study examined the influence of the calcium ionophores A23187 and X537A on the calcitonin (CT) secretory process. The isolated perfused porcine thyroid was used to evaluate ionophore effects on CT secretion and thyroid slices were used to measure 45Ca uptake. Both A23187 and X537A enhanced the rate of CT release from the perfused thyroid. A23187 at a concentration of 19 PM (10 pg/ml) produced a maximal increase in CT secretion of 325% above control levels. X537A at a concentration of 16 PM (10 pg/ml) produced a peak rise in CT release of approximately 2000% over control levels. The CT secretory response to A23187 was found to be completely calcium dependent; however, the secretory response to X537A was partially, but not completely dependent upon the presence of perfusate calcium. The results demonstrate that these calcium ionophores are very potent CT secretagogues which vary considerably in their calcium dependency.
Introduction Calcitonin acts on bone to lower the circulating levels of calcium (Hirsch and Munson, 1969). This endocrine system is regulated in a negative feedback manner primarily in response to the level of calcium in the circulation (Lee et al., 1969). It has been demonstrated that the calcium ion is the most potent among the alkaline earth calcium in stimulating CT secretions and appears to be the only cation that is involved in the physiological regulation of CT secretory system (Pento et al., 1974a; Cooper, 1975). Further, it has been demonAll correspondence should be addressed to: Dr. J. Thomas Pento, College of Pharmacy, P.O. Box 26901, Oklahoma City, OK 73190 (U.S.A.). 0303-7207/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
strated that both organic and inorganic calcium antagonists inhibit basal and secretagogue-induced CT secretion (Pento, 1977, 1980). Calcium ionophores are antimicrobial derivatives which form a reversible complex with calcium ions and facilitate calcium translocation across biological membranes. The calcium ionophores such as A23187 and X537A have been shown to enhance calcium transport in a number of biological systems including endocrine tissue (Pressman, 1973). Accordingly, the calcium-related secretagogue activity of these ionophores has been demonstrated in a number of calcium-dependent endocrine systems (Pressman, 1976). Sinde the calcium ion is known to be involved in the physiological regulation of calcitonin secretion, the present study was designed to examine Ltd.
12
the influence of the calcium ionophores A23185 and X53lA on the CT secretory process and to compare the secretagogue influence of these two calcium ionophores with differing degrees of calcium ion specificity (Pressman, 1973). This study was conducted using the in vitro whole-thyroid perfusion system which has been shown to mimic both quantitatively and qualitatively the in vivo CT secretory response to a variety of CT secretagogues in the pig (Pento, 1985a). Materials and methods Tissue preparation
Fresh pig thyroids were collected from mature pigs of mixed breeding at a local abattoir (Wilson and Co., Oklahoma City, OK) immediately after slaughter, and returned to the laboratory in an oxygenated Tris-balanced salt (TBS) solution that was chilled on ice. The TBS solution contained 20 mM Tris-HCl, 146 mM NaCl, 1 mM MgSO,, 4.75 mM KCl, 1.25 mM CaCl,, and 5.6 mM glucose, and was titrated to pH 7.4. Intact whole thyroids were cleaned of connective tissue in an ice bath containing the TBS solution. Perfusion
methodology
The thyroid artery was isolated and cannulated and the whole thyroids were perfused as previously described (Pento, 1985a). The thyroids were then placed in a water-jacketed tissue chamber maintained at 37°C. The thyroid tissue was perfused at a rate of 2.5 ml/min with the TBS solution, which was continuously oxygenated with 95% O/5’% CO, and maintained at 37°C. After a preexperimental equilibration perfusion period of 20-30 min, total effluent fractions were collected at 5-min intervals. The various experimental treatments were added to the perfusion media at the times indicated following the initiation of the sample collection period. The calcium ionophores were dissolved in dimethylsulfoxide (DMSO) and added to the TBS perfusion solution so that the final concentration of DMSO was 0.15%. The same concentration of DMSO was added to the control perfusion media. At the conclusion of each experiment, the thyroids were perfused for 10 min with a 10% solution of carbon ink to estimate of the extent of tissue perfusion. Thyroids that appeared
to be less than 75% perfused were removed from experimental consideration. Following collection, tissue effluent samples were immediately frozen at -20°C for a period of up to one month until subsequent CT assay. Calcitonin radioimmunoassay
The CT concentration of the thyroid effluent samples was determined by means of a specific porcine CT radioimmunoassay (Pento et al., 1973). Pure porcine CT (Lederle Laboratories, Lot 4688C-140A) was used as the assay standard and for labelling with high specific activity 13’1 (Union Carbide Corp.). Equilibrium incubations were conducted at 4°C for 6 days in a 50 mM Verona1 buffer containing 20 mM disodium EDTA and 0.5% bovine serum albumin. Bound and free fractions were separated by talc adsorption. The individual bound and free fractions were counted on an autogamma counter (Beckman, Model G 300). Counting times were automatically adjusted to obtain a counting error of less than 1%. Data analysis
The radioimmunoassay data were processed by computer (IBM Systems/370) using an algorism that has been described (Pento et al., 1974b) and expressed as ng CT/ml effluent. Because of the variation in baseline effluent CT concentration between individual thyroids, the data is presented as percent change from baseline CT concentrations. The baseline CT concentration in these experiments ranged from 3 to 30 ng/ml. In this study a non-directional hypothesis was tested using a t-test for correlated data in comparing treatment-induced changes in CT levels during the treatment perfusion period to the baseline CT levels that were collected during a 15-min period immediately prior to the experimental perfusion. Changes in thyroid effluent CT levels that resulted in P values of less than 0.05 were considered significantly different. Results The influence of the ionophores A23187 is illustrated in Fig. 1. With 1.25 mM calcium in the media the perfusion of 19 PM (lo pg/ml) A23187 produced 150% increase in the rate of CT secretion
73
$ c 2
500
0 a?
. 0
5
10
X537A 16 MM
t
A23tS71SirM
5 15
20
25
30
35
Time in Minutes
Fig. 1. Influence of 19 FM A23187 on CT secretion. Each point represents the mean+ SE of the percent change in effluent CT concentration from 4 individual whole thyroids. Effluent fractions were collected at the time indicated. The open horizontal bar indicates the A23187 perfusion period. The shaded horizontal bar represents the mean+ SE of the effluent CT level in the baseline samples that were collected during a 15-min period immediately prior to the experimental perfusion period. *P < 0.025; **p-z0.005; ***P
within 5 min (P < 0.~5) and a 325% increase in CT secretion after 20 min of perfusion (P < 0.025). Following the discontinuation of the A23187 perfusion the level of CT release fell back to the preperfusion control range within 5 min. Perfusion of A23187 in the absence of calcium did not alter the rate of CT secretion. The X537A perfusion at a concentration of 16 PM (10 pg/ml) produced a much greater increase in the level of CT secretion than did A23187 with 1.25 mM calcium present in the perfusion media (see Fig. 2). The X537A perfusion caused a rise in the level of CT secretion which reached a peak of approximately 2000% of control levels at 15 min (P < 0.001). However, with the X537A perfusion, unlike A23187, the level of CT was significantly above control at 15 min post-X537A perfusion, in the presence of 1.25 mM calcium (P < 0.05). In addition, X537A stimulated CT secretion to a maximal level of 260% of control levels in the absence of media calcium (shown in Fig. 2). As illustrated in Fig. 3 increasing the concentration of X573A in the perfusion media from a concentration of 0.16 FM to 16 pB.4 produced a dose-related enhancement of the rate of CT secretion. At 0.16
IO
15
20
25
30
f 35
Tune in Minutes
Fig. 2. Influence of 16 PM X537A on CT secretion. The illustration descriptions are the same as Fig. 1 except: *P -=z 0.05; **P f 0.01; ***p < 0.001. 16m 14cm
C
T
T
Time in Minutes
Fig. 3. Influence of 0.16 PM to 16 PM X537A on CT secretion. The illustration descriptions are the same as Fig. 1 except: 'P < 0.01: **p < 0.005; ***p
FM, X537A produced a slight but not sig~fic~t rise in CT secretion, at 1.6 PM the level of CT secretion was approximately 600% of control level (P < 0.005) and at 16 PM the level of CT release was approximately 1200% of control (P < 0.001). Following the discontinuation of X537A in this experiment, the level of CT secretion fell off rapidly but was still above control level at 10 min postX537A perfusion (P < 0.001). Discussion Calcium ionophores such as X537A and A23187 form a reversible complex with calcium and due to
74
their lipophilic nature, transport calcium across various biological membranes (Pressman, 1973, 1976). In the present study both ionophores significantly enhanced the rate of CT secretion when calcium was present in the perfusion media. Moreover, X537A was found to be substantially more potent than A23187. The secretagogue activity and potency of the calcium ionophores found in this study is similar to that observed in other endocrine tissues. A23187 and/or X537A have been reported to enhance secretory activity in the pancreas (Conway et al., 1976), anterior pituitary (Conn et al., 1979; Ray and Wallis, 1982; Kamel and Krey, 1983), kidney (Schwertschlag et al., 1978), posterior pituitary (Nakazato and Douglas, 1974; Ishikawa and Schrier, 19831, and other secretory tissue (Cochrane and Douglas, 1974; Cochrane et al., 1975; Harty et al., 1983). In the case of parathyroid tissue, in which secretion is known to be inversely related to calcium levels, A23187 and X537A in~bited the secfetory process (Habener et al., 19’77; Brown et al., 1980). As in the present study, Hellman (1975) reported that X537A was a more potent secretagogue than A23187 for insulin secretion from the isolated pancreatic islet cells. In the present study the CT secretory activity of A23187 appears to be completely calcium dependent, in that the elimination of calcium from the perfusate abolished the secretory response. However, the secretagogue activity of X537A was significantly reduced but not eliminated in the absence of calcium. Similarly, X537A was reported to stimulate insulin secretion from islet cells (Hellman, 1975) and renin secretion from the kidney (Schwertschlag et al., 1978) in the absence of extracellular calcium. Accordingly, X537A is known to be a relatively non-specific ionophore (Pressman, 1976). In addition, X537A may act as a transmembrane carrier of biological amines (Colbum et al., 1975; Kafka and Holz, 1976) and as an ionophore for monovalent cations such as sodium and potassium (Pressman, 1976). Therefore, X537A may enhance CT secretion by a combination of several different mechanisms including calcium translocation. This would explain the much greater secretory potency of X537A when compared to A23187 in the current study and its lack of complete calcium dependency. It was observed previously that both ionophores
reduced 45Ca uptake into slices of thyroid tissue (Pento, 1985b). However, since the CT-secreting thyroid C-cells are only a small fraction of the total tissue mass of the thyroid, the 45Ca uptake results may not be reflective of the actual uptake within the C-cell population. Further, the thyroid slices may respond differently to the ionophores than does intact tissue. Another possibility is that the ionophores cause the release of intracellular calcium stores which would stimulate exocytoses and reduce the uptake of extracellular calcium. Similarly, Richardson (1983) reported an X537Aand A23187-induced efflux of 45Ca in mouse pituitary tumor cells and suggests that this may be associated with the mobilization of intracellular calcium. It has been proposed that ionophores-induced exocytosis may be mediated by calmodulin-dependent reactions associated with elevated cytosolic calcium (Kojima et al., 1983; Zawalich et al., 1983). Accordingly, the results of a study by Cooper and Borosky (1985) indicate that calmodulin is involved in calcium-mediated CT secretion in the rat. However, the calcium ionophores are also associated with alterations in intracellular cyclic AMP (Hellman, 1975: Brown et al., 1980) and CT secretion has been reported to be regulated, in part, by cyclic AMP (Care et al., 1970). Whatever the exact mechanism of ionophore action within the secretory C-cell, it is clear from the results of the present study that both A23187 and X537A are potent CT secretagogues in the perfused pig thyroid. Therefore. these agents may be useful probes for the study and elucidation of the CT secretory process.
The author gratefully acknowledges the generous supply of porcine thyroids by Mr. T.F. Andreskowski of Wilson and Co. and the technical assistance of Helen Wuertele. A23187 and X537A were generously provided by Lilly Research Laboratories and Hoffmann-LaRoche, respectivefy. This study was supported in part by NSF Grants GB-43214 and PCM 76-03624. References Brown. EM., Gardner, D.G. and Aurbach, crinology 106.133-138.
G.D. (1980) Endo-
75
Care, A.D., Bates, R.F.L. and Gitelman, H.J. (1970) J. Endocrinol. 48. 1-15. Cochrane. DE. and Douglas, W.W. (1974) Proc. Nat]. Acad. Sci. 71, 408-412. Cochrane, DE., Douglas, W.W.. Mouri,T. and Nakazato, Y. (1975) J. Physiol. 252, 363-378. Colhurn, R.W.. Thoa, Nguyen B. and Kopin, I.J. (1975) Life Sci. 17, 1395-1400. Corm, P.M., Rogers, D.C. and Sandhu, F.S. (1979) Endocrinology 105, 1122-1127. Conway, H.H., Griffey, M.A.. Marks, S.R. and Whitney, J.E. (1976) Worm. Metab. Res. 8. 351-353. Cooper, C.W. (1975) Proc. Sot. Exp. Biol. Med. 148, 449-454. Cooper, C.W. and Borosky, S.A. (1985) Fed. Proc. 44,883. Habener, J.F., Stevens. T.D., Ravazzofa, M., Or& L. and Potts Jr., J.T. (1977) Endocrinology 101, 1524-1537, Harty, R.F., Franklin, P.A. and McGuigan, J.E (1983) Regul. Pept. 7, 171-181. Hellman, B. (1985) Biochim. Biophys. Acta 399, 157-269. Hirsch, P.F. and Munson, P.L. (1969) Physiol. Rev. 49,548-622. Ishikawa, S.-E. and Schrier, R.W. (1983) Am. J. Physiol. 244, R703-R708. Kafka, M.S. and Holz, R.W. (1976) Biochim. Biophys. Acta 426.31-37. Kamel, F. and Krey, L.C. (1983) Mol. Cell, Endocrinol. 32, 283-300.
Kojima, I., Lippes, H., Kojima, K. and Rasmussen, H. (1983) B&hem. Biophys. Res. Commun. 116, 555-562. Lee, M.R., Deftos, L.J. and Poyys, J.T. (1969) Endocrinology 84, 36-40. Nakazato, Y. and Douglas, W.W. (1974) Nature (London) 249, 479-481. Pento, J.T. (1977) Mol. Cell. Endocrinol. 9, 223-226. Pento, J.T. (1980) Chem. Pathol. Pharmacol. 30, 233-243. Pento, J.T. (1985a) J. Pharmacol. Methods 13, 43-51. Pento, J.T. (19856) Fed. Proc. 44, 1649. Pento, J.T., Ghck, S.M. and Kagan, A. (1973) Metabolism 22. 735-744. Pento, J.T., Ghck, SM., Kagan, A. and Gorfien. P.C. (1974a) Endocrinology 94.1176-l 180. Pento, J.T., Walker, J.W. and Estes, R.L. (1974b) Proc. Oklahoma Acad. Sci. S4,73-79. Pressman, B.C. (1973) Fed. Proc. 32,697O. Pressman. B.C. (1976) Annu. Rev. B&hem. 45, 501-529. Ray, K.P. and Wallis, M. (1982) Mol. Cell. Endocrinol. 28, 691-703. Richardson, U.I. (1983) Endocrinology 113, 62-68. Schwertschlag, U., Hackenthal, E., Hackenthal, R. and Rohs, G.H. (1978) Chn. Sci. Mol. Med. 55, 163s-166s. Zawalich, W., Brown, C. and Rasmussen, H. (1983) Biochem. Biophys. Res. Commun. 117, 448-455.
71
MCE 01450
Influence
of the calcium ionophores A23187 and X537A on calcitonin secretion from the isolated perfused porcine thyroid J. Thomas
Pento
Division of Pharmacodynamics and Toxicology, College of Pharmacy, Uniuersity of Oklahoma Health Sciences Center, Oklahoma City, OK 73190 (U.S.A.) (Received
Key words: calcitonin;
calcitonin
secretion;
17 July 1985; accepted
thyroid;
calcium
ionophores;
12 December
A23187;
1985)
X537A.
Summary The present study examined the influence of the calcium ionophores A23187 and X537A on the calcitonin (CT) secretory process. The isolated perfused porcine thyroid was used to evaluate ionophore effects on CT secretion and thyroid slices were used to measure 45Ca uptake. Both A23187 and X537A enhanced the rate of CT release from the perfused thyroid. A23187 at a concentration of 19 PM (10 pg/ml) produced a maximal increase in CT secretion of 325% above control levels. X537A at a concentration of 16 PM (10 pg/ml) produced a peak rise in CT release of approximately 2000% over control levels. The CT secretory response to A23187 was found to be completely calcium dependent; however, the secretory response to X537A was partially, but not completely dependent upon the presence of perfusate calcium. The results demonstrate that these calcium ionophores are very potent CT secretagogues which vary considerably in their calcium dependency.
Introduction Calcitonin acts on bone to lower the circulating levels of calcium (Hirsch and Munson, 1969). This endocrine system is regulated in a negative feedback manner primarily in response to the level of calcium in the circulation (Lee et al., 1969). It has been demonstrated that the calcium ion is the most potent among the alkaline earth calcium in stimulating CT secretions and appears to be the only cation that is involved in the physiological regulation of CT secretory system (Pento et al., 1974a; Cooper, 1975). Further, it has been demonAll correspondence should be addressed to: Dr. J. Thomas Pento, College of Pharmacy, P.O. Box 26901, Oklahoma City, OK 73190 (U.S.A.). 0303-7207/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
strated that both organic and inorganic calcium antagonists inhibit basal and secretagogue-induced CT secretion (Pento, 1977, 1980). Calcium ionophores are antimicrobial derivatives which form a reversible complex with calcium ions and facilitate calcium translocation across biological membranes. The calcium ionophores such as A23187 and X537A have been shown to enhance calcium transport in a number of biological systems including endocrine tissue (Pressman, 1973). Accordingly, the calcium-related secretagogue activity of these ionophores has been demonstrated in a number of calcium-dependent endocrine systems (Pressman, 1976). Sinde the calcium ion is known to be involved in the physiological regulation of calcitonin secretion, the present study was designed to examine Ltd.
12
the influence of the calcium ionophores A23185 and X53lA on the CT secretory process and to compare the secretagogue influence of these two calcium ionophores with differing degrees of calcium ion specificity (Pressman, 1973). This study was conducted using the in vitro whole-thyroid perfusion system which has been shown to mimic both quantitatively and qualitatively the in vivo CT secretory response to a variety of CT secretagogues in the pig (Pento, 1985a). Materials and methods Tissue preparation
Fresh pig thyroids were collected from mature pigs of mixed breeding at a local abattoir (Wilson and Co., Oklahoma City, OK) immediately after slaughter, and returned to the laboratory in an oxygenated Tris-balanced salt (TBS) solution that was chilled on ice. The TBS solution contained 20 mM Tris-HCl, 146 mM NaCl, 1 mM MgSO,, 4.75 mM KCl, 1.25 mM CaCl,, and 5.6 mM glucose, and was titrated to pH 7.4. Intact whole thyroids were cleaned of connective tissue in an ice bath containing the TBS solution. Perfusion
methodology
The thyroid artery was isolated and cannulated and the whole thyroids were perfused as previously described (Pento, 1985a). The thyroids were then placed in a water-jacketed tissue chamber maintained at 37°C. The thyroid tissue was perfused at a rate of 2.5 ml/min with the TBS solution, which was continuously oxygenated with 95% O/5’% CO, and maintained at 37°C. After a preexperimental equilibration perfusion period of 20-30 min, total effluent fractions were collected at 5-min intervals. The various experimental treatments were added to the perfusion media at the times indicated following the initiation of the sample collection period. The calcium ionophores were dissolved in dimethylsulfoxide (DMSO) and added to the TBS perfusion solution so that the final concentration of DMSO was 0.15%. The same concentration of DMSO was added to the control perfusion media. At the conclusion of each experiment, the thyroids were perfused for 10 min with a 10% solution of carbon ink to estimate of the extent of tissue perfusion. Thyroids that appeared
to be less than 75% perfused were removed from experimental consideration. Following collection, tissue effluent samples were immediately frozen at -20°C for a period of up to one month until subsequent CT assay. Calcitonin radioimmunoassay
The CT concentration of the thyroid effluent samples was determined by means of a specific porcine CT radioimmunoassay (Pento et al., 1973). Pure porcine CT (Lederle Laboratories, Lot 4688C-140A) was used as the assay standard and for labelling with high specific activity 13’1 (Union Carbide Corp.). Equilibrium incubations were conducted at 4°C for 6 days in a 50 mM Verona1 buffer containing 20 mM disodium EDTA and 0.5% bovine serum albumin. Bound and free fractions were separated by talc adsorption. The individual bound and free fractions were counted on an autogamma counter (Beckman, Model G 300). Counting times were automatically adjusted to obtain a counting error of less than 1%. Data analysis
The radioimmunoassay data were processed by computer (IBM Systems/370) using an algorism that has been described (Pento et al., 1974b) and expressed as ng CT/ml effluent. Because of the variation in baseline effluent CT concentration between individual thyroids, the data is presented as percent change from baseline CT concentrations. The baseline CT concentration in these experiments ranged from 3 to 30 ng/ml. In this study a non-directional hypothesis was tested using a t-test for correlated data in comparing treatment-induced changes in CT levels during the treatment perfusion period to the baseline CT levels that were collected during a 15-min period immediately prior to the experimental perfusion. Changes in thyroid effluent CT levels that resulted in P values of less than 0.05 were considered significantly different. Results The influence of the ionophores A23187 is illustrated in Fig. 1. With 1.25 mM calcium in the media the perfusion of 19 PM (lo pg/ml) A23187 produced 150% increase in the rate of CT secretion
73
$ c 2
500
0 a?
. 0
5
10
X537A 16 MM
t
A23tS71SirM
5 15
20
25
30
35
Time in Minutes
Fig. 1. Influence of 19 FM A23187 on CT secretion. Each point represents the mean+ SE of the percent change in effluent CT concentration from 4 individual whole thyroids. Effluent fractions were collected at the time indicated. The open horizontal bar indicates the A23187 perfusion period. The shaded horizontal bar represents the mean+ SE of the effluent CT level in the baseline samples that were collected during a 15-min period immediately prior to the experimental perfusion period. *P < 0.025; **p-z0.005; ***P
within 5 min (P < 0.~5) and a 325% increase in CT secretion after 20 min of perfusion (P < 0.025). Following the discontinuation of the A23187 perfusion the level of CT release fell back to the preperfusion control range within 5 min. Perfusion of A23187 in the absence of calcium did not alter the rate of CT secretion. The X537A perfusion at a concentration of 16 PM (10 pg/ml) produced a much greater increase in the level of CT secretion than did A23187 with 1.25 mM calcium present in the perfusion media (see Fig. 2). The X537A perfusion caused a rise in the level of CT secretion which reached a peak of approximately 2000% of control levels at 15 min (P < 0.001). However, with the X537A perfusion, unlike A23187, the level of CT was significantly above control at 15 min post-X537A perfusion, in the presence of 1.25 mM calcium (P < 0.05). In addition, X537A stimulated CT secretion to a maximal level of 260% of control levels in the absence of media calcium (shown in Fig. 2). As illustrated in Fig. 3 increasing the concentration of X573A in the perfusion media from a concentration of 0.16 FM to 16 pB.4 produced a dose-related enhancement of the rate of CT secretion. At 0.16
IO
15
20
25
30
f 35
Tune in Minutes
Fig. 2. Influence of 16 PM X537A on CT secretion. The illustration descriptions are the same as Fig. 1 except: *P -=z 0.05; **P f 0.01; ***p < 0.001. 16m 14cm
C
T
T
Time in Minutes
Fig. 3. Influence of 0.16 PM to 16 PM X537A on CT secretion. The illustration descriptions are the same as Fig. 1 except: 'P < 0.01: **p < 0.005; ***p
FM, X537A produced a slight but not sig~fic~t rise in CT secretion, at 1.6 PM the level of CT secretion was approximately 600% of control level (P < 0.005) and at 16 PM the level of CT release was approximately 1200% of control (P < 0.001). Following the discontinuation of X537A in this experiment, the level of CT secretion fell off rapidly but was still above control level at 10 min postX537A perfusion (P < 0.001). Discussion Calcium ionophores such as X537A and A23187 form a reversible complex with calcium and due to
74
their lipophilic nature, transport calcium across various biological membranes (Pressman, 1973, 1976). In the present study both ionophores significantly enhanced the rate of CT secretion when calcium was present in the perfusion media. Moreover, X537A was found to be substantially more potent than A23187. The secretagogue activity and potency of the calcium ionophores found in this study is similar to that observed in other endocrine tissues. A23187 and/or X537A have been reported to enhance secretory activity in the pancreas (Conway et al., 1976), anterior pituitary (Conn et al., 1979; Ray and Wallis, 1982; Kamel and Krey, 1983), kidney (Schwertschlag et al., 1978), posterior pituitary (Nakazato and Douglas, 1974; Ishikawa and Schrier, 19831, and other secretory tissue (Cochrane and Douglas, 1974; Cochrane et al., 1975; Harty et al., 1983). In the case of parathyroid tissue, in which secretion is known to be inversely related to calcium levels, A23187 and X537A in~bited the secfetory process (Habener et al., 19’77; Brown et al., 1980). As in the present study, Hellman (1975) reported that X537A was a more potent secretagogue than A23187 for insulin secretion from the isolated pancreatic islet cells. In the present study the CT secretory activity of A23187 appears to be completely calcium dependent, in that the elimination of calcium from the perfusate abolished the secretory response. However, the secretagogue activity of X537A was significantly reduced but not eliminated in the absence of calcium. Similarly, X537A was reported to stimulate insulin secretion from islet cells (Hellman, 1975) and renin secretion from the kidney (Schwertschlag et al., 1978) in the absence of extracellular calcium. Accordingly, X537A is known to be a relatively non-specific ionophore (Pressman, 1976). In addition, X537A may act as a transmembrane carrier of biological amines (Colbum et al., 1975; Kafka and Holz, 1976) and as an ionophore for monovalent cations such as sodium and potassium (Pressman, 1976). Therefore, X537A may enhance CT secretion by a combination of several different mechanisms including calcium translocation. This would explain the much greater secretory potency of X537A when compared to A23187 in the current study and its lack of complete calcium dependency. It was observed previously that both ionophores
reduced 45Ca uptake into slices of thyroid tissue (Pento, 1985b). However, since the CT-secreting thyroid C-cells are only a small fraction of the total tissue mass of the thyroid, the 45Ca uptake results may not be reflective of the actual uptake within the C-cell population. Further, the thyroid slices may respond differently to the ionophores than does intact tissue. Another possibility is that the ionophores cause the release of intracellular calcium stores which would stimulate exocytoses and reduce the uptake of extracellular calcium. Similarly, Richardson (1983) reported an X537Aand A23187-induced efflux of 45Ca in mouse pituitary tumor cells and suggests that this may be associated with the mobilization of intracellular calcium. It has been proposed that ionophores-induced exocytosis may be mediated by calmodulin-dependent reactions associated with elevated cytosolic calcium (Kojima et al., 1983; Zawalich et al., 1983). Accordingly, the results of a study by Cooper and Borosky (1985) indicate that calmodulin is involved in calcium-mediated CT secretion in the rat. However, the calcium ionophores are also associated with alterations in intracellular cyclic AMP (Hellman, 1975: Brown et al., 1980) and CT secretion has been reported to be regulated, in part, by cyclic AMP (Care et al., 1970). Whatever the exact mechanism of ionophore action within the secretory C-cell, it is clear from the results of the present study that both A23187 and X537A are potent CT secretagogues in the perfused pig thyroid. Therefore. these agents may be useful probes for the study and elucidation of the CT secretory process.
The author gratefully acknowledges the generous supply of porcine thyroids by Mr. T.F. Andreskowski of Wilson and Co. and the technical assistance of Helen Wuertele. A23187 and X537A were generously provided by Lilly Research Laboratories and Hoffmann-LaRoche, respectivefy. This study was supported in part by NSF Grants GB-43214 and PCM 76-03624. References Brown. EM., Gardner, D.G. and Aurbach, crinology 106.133-138.
G.D. (1980) Endo-
75
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