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. 1978 Feb 1;76(2):386–399. doi: 10.1083/jcb.76.2.386

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. II. Subcellular localization of the fluorescent probe chlorotetracycline

D E Chandler 1, J A Williams 1
PMCID: PMC2109989  PMID: 10605445

Abstract

Subcellular distribution of the divalent cation-sensitive probe chlorotetracycline (CTC) was observed by fluorescence microscopy in isolated pancreatic acinar cells, dissociated hepatocytes, rod photoreceptors, and erythrocytes. In each cell type, areas containing membranes fluoresced intensely while areas containing no membranes (nuclei and zymogen granules) were not fluorescent. Cell compartments packed with rough endoplasmic reticulum or Golgi vesicles (acinar cells) or plasma membrane-derived membranes (rod outer segments) exhibited a uniform fluorescence. In contrast, cell compartments having large numbers of mitochondria (hepatocytes and the rod inner segment) exhibited a punctate fluorescence. Punctate fluorescence was prominent in the perinuclear and peri-granular areas of isolated acinar cells during CTC efflux, suggesting that under these conditions mitochondrial fluorescence may account for a large portion of acinar cell fluorescence. Fluorometry of dissociated pancreatic acini, preloaded with CTC, showed that application of the mitochondrial inhibitors antimycin A, NaCN, rotenone, or C1CCP, or of the divalent cation ionophore A23187 (all agents known to release mitochondrial calcium) rapidly decreased the fluorescence of acini. In the case of mitochondrial inhibitors, this response could be elicited before but not following the loss of CTC fluorescence induced by bethanechol stimulation. Removal of extracellular Ca2+ and Mg2+ or addition of EDTA also decreased fluorescence but did not prevent secretagogues or mitochondrial inhibitors from eliciting a further response. These data suggest that bethanechol acts to decrease CTC fluorescence at the same intracellular site as do mitochondrial inhibitors. This could be due to release of calcium from either mitochondria or another organelle that requires ATP to sequester calcium.

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Selected References

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  1. ALLISON A. C., YOUNG M. R. UPTAKE OF DYES AND DRUGS BY LIVING CELLS IN CULTURE. Life Sci. 1964 Dec;3:1407–1414. doi: 10.1016/0024-3205(64)90082-7. [DOI] [PubMed] [Google Scholar]
  2. Amsterdam A., Jamieson J. D. Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells. J Cell Biol. 1974 Dec;63(3):1037–1056. doi: 10.1083/jcb.63.3.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Argent B. E., Case R. M., Scratcherd T. Amylase secretion by the perfused cat pancreas in relation to the secretion of calcium and other electrolytes and as influenced by the external ionic environment. J Physiol. 1973 May;230(3):575–593. doi: 10.1113/jphysiol.1973.sp010205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Babcock D. F., First N. L., Lardy H. A. Action of ionophore A23187 at the cellular level. Separation of effects at the plasma and mitochondrial membranes. J Biol Chem. 1976 Jul 10;251(13):3881–3886. [PubMed] [Google Scholar]
  5. Borle A. B. Kinetic analyses of calcium movements in HeLa cell cultures. II. Calcium efflux. J Gen Physiol. 1969 Jan;53(1):57–69. doi: 10.1085/jgp.53.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borle A. B. Kinetic analysis of calcium movements in cell culture. V. Intracellular calcium distribution in kidney cells. J Membr Biol. 1972;10(1):45–66. doi: 10.1007/BF01867847. [DOI] [PubMed] [Google Scholar]
  7. Case R. M., Clausen T. The relationship between calcium exchange and enzyme secretion in the isolated rat pancreas. J Physiol. 1973 Nov;235(1):75–102. doi: 10.1113/jphysiol.1973.sp010379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Caswell A. H., Hutchison J. D. Visualization of membrane bound cations by a fluorescent technique. Biochem Biophys Res Commun. 1971 Jan 8;42(1):43–49. doi: 10.1016/0006-291x(71)90359-7. [DOI] [PubMed] [Google Scholar]
  9. Caswell A. H., Pressman B. C. Kinetics of transport of divalent cations across sarcoplasmic reticulum vesicles induced by ionophores. Biochem Biophys Res Commun. 1972 Oct 6;49(1):292–298. doi: 10.1016/0006-291x(72)90043-5. [DOI] [PubMed] [Google Scholar]
  10. Caswell A. H., Warren S. Observation of calcium uptake by isolated sarcoplasmic reticulum employing a fluorescent chelate probe. Biochem Biophys Res Commun. 1972 Mar 10;46(5):1757–1763. doi: 10.1016/0006-291x(72)90047-2. [DOI] [PubMed] [Google Scholar]
  11. Ceccarelli B., Clemente F., Meldolesi J. Secretion of calcium in pancreatic juice. J Physiol. 1975 Mar;245(3):617–638. doi: 10.1113/jphysiol.1975.sp010865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chandler D. E., Williams J. A. Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. I. Use of chlorotetracycline as fluorescent probe. J Cell Biol. 1978 Feb;76(2):371–385. doi: 10.1083/jcb.76.2.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chandler D. E., Williams J. A. Intracellular uptake and alpha-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells. J Membr Biol. 1977 Apr 22;32(3-4):201–230. doi: 10.1007/BF01905220. [DOI] [PubMed] [Google Scholar]
  14. Chandler D. E., Williams J. A. Pancreatic acinar cells: effects of lanthanum ions on amylase release and calcium ion fluxes. J Physiol. 1974 Dec;243(3):831–846. doi: 10.1113/jphysiol.1974.sp010779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Christophe J. P., Frandsen E. K., Conlon T. P., Krishna G., Gardner J. D. Action of cholecystokinin, cholinergic agents, and A-23187 on accumulation of guanosine 3':5'-monophosphate in dispersed guinea pig pancreatic acinar cells. J Biol Chem. 1976 Aug 10;251(15):4640–4645. [PubMed] [Google Scholar]
  16. Clemente F., Meldolesi J. Calcium and pancreatic secretion-dynamics of subcellur calcium pools in resting and stimulated acinar cells. Br J Pharmacol. 1975 Nov;55(3):369–379. doi: 10.1111/j.1476-5381.1975.tb06940.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Clemente F., Meldolesi J. Calcium and pancreatic secretion. I. Subcellular distribution of calcium and magnesium in the exocrine pancreas of the guinea pig. J Cell Biol. 1975 Apr;65(1):88–102. doi: 10.1083/jcb.65.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. DU BUY H. G., SHOWACRE J. L. Selective localization of tetracycline in mitochondria of living cells. Science. 1961 Jan 20;133(3447):196–197. doi: 10.1126/science.133.3447.196. [DOI] [PubMed] [Google Scholar]
  19. Heisler S. Calcium efflux and secretion of alpha-amylase from rat pancreas. Br J Pharmacol. 1974 Nov;52(3):387–392. doi: 10.1111/j.1476-5381.1974.tb08607.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heisler S., Fast D., Tenenhouse A. Role of Ca 2+ and cyclic AMP in protein secretion from rat exocrine pancreas. Biochim Biophys Acta. 1972 Oct 25;279(3):561–572. doi: 10.1016/0304-4165(72)90178-x. [DOI] [PubMed] [Google Scholar]
  21. Kanno T. Calcium-dependent amylase release and electrophysiological measurements in cells of the pancreas. J Physiol. 1972 Oct;226(2):353–371. doi: 10.1113/jphysiol.1972.sp009988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kanno T., Nishimura O. Stimulus-secretion coupling in pancreatic acinar cells: inhibitory effects of calcium removal and manganese addition on pancreozymin-induced amylase release. J Physiol. 1976 May;257(2):309–324. doi: 10.1113/jphysiol.1976.sp011370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kondo S., Schulz I. Ca++ fluxes in isolated cells of rat pancreas. effect of secretagogues and different Ca++ concentrations. J Membr Biol. 1976 Oct 20;29(1-2):185–203. doi: 10.1007/BF01868959. [DOI] [PubMed] [Google Scholar]
  24. Kondo S., Schulz I. Calcium ion uptake in isolated pancreas cells induced by secretagogues. Biochim Biophys Acta. 1976 Jan 8;419(1):76–92. doi: 10.1016/0005-2736(76)90373-4. [DOI] [PubMed] [Google Scholar]
  25. Luthra R., Olson M. S. Studies of mitochondrial calcium movements using chlorotetracycline. Biochim Biophys Acta. 1976 Sep 13;440(3):744–758. doi: 10.1016/0005-2728(76)90056-6. [DOI] [PubMed] [Google Scholar]
  26. Matthews E. K., Petersen O. H., Williams J. A. Pancreatic acinar cells: acetylcholine-induced membrane depolarization, calcium efflux and amylase release. J Physiol. 1973 Nov;234(3):689–701. doi: 10.1113/jphysiol.1973.sp010367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. NILSSON S. E. AN ELECTRON MICROSCOPIC CLASSIFICATION OF THE RETINAL RECEPTORS OF THE LEOPARD FROG (RANA PIPIENS). J Ultrastruct Res. 1964 Jun;10:390–416. doi: 10.1016/s0022-5320(64)80018-6. [DOI] [PubMed] [Google Scholar]
  28. Petersen O. H., Ueda N. Pancreatic acinar cells: the role of calcium in stimulus-secretion coupling. J Physiol. 1976 Jan;254(3):583–606. doi: 10.1113/jphysiol.1976.sp011248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Robberecht P., Christophe J. Secretion of hydrolases by perfused fragments of rat pancreas: effect of calcium. Am J Physiol. 1971 Apr;220(4):911–917. doi: 10.1152/ajplegacy.1971.220.4.911. [DOI] [PubMed] [Google Scholar]
  30. Schreurs V. V., Swarts H. G., De Pont J. J., Bonting S. L. Role of calcium in exocrine pancreatic secretion. II. Comparison of the effects of carbachol and the inophore A-23187 on enzyme secretion and calcium movements in rabbit pancreas. Biochim Biophys Acta. 1976 Jan 21;419(2):320–330. doi: 10.1016/0005-2736(76)90358-8. [DOI] [PubMed] [Google Scholar]
  31. Seglen P. O. Preparation of rat liver cells. I. Effect of Ca 2+ on enzymatic dispersion of isolated, perfused liver. Exp Cell Res. 1972 Oct;74(2):450–454. doi: 10.1016/0014-4827(72)90400-4. [DOI] [PubMed] [Google Scholar]
  32. Shelby H. T., Gross L. P., Lichty P., Gardner J. D. Action of cholecystokinin and cholinergic agents on membrane-bound calcium in dispersed pancreatic acinar cells. J Clin Invest. 1976 Dec;58(6):1482–1493. doi: 10.1172/JCI108605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Williams J. A., Cary P., Moffat B. Effects of ions on amylase release by dissociated pancreatic acinar cells. Am J Physiol. 1976 Nov;231(5 Pt 1):1562–1567. doi: 10.1152/ajplegacy.1976.231.5.1562. [DOI] [PubMed] [Google Scholar]
  34. Williams J. A., Chandler D. Ca++ and pancreatic amylase release. Am J Physiol. 1975 Jun;228(6):1729–1732. doi: 10.1152/ajplegacy.1975.228.6.1729. [DOI] [PubMed] [Google Scholar]
  35. Williams J. A., Lee M. Pancreatic acinar cells: use of Ca++ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling. Biochem Biophys Res Commun. 1974 Sep 23;60(2):542–548. doi: 10.1016/0006-291x(74)90274-5. [DOI] [PubMed] [Google Scholar]

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