Abstract
There is ample evidence of calcium being an intracellular second messenger during volume regulatory processes in various cells including inner medullary collecting duct (IMCD) cells. Therefore, we measured intracellular calcium concentrations (Cai under anisotonic conditions in primary cultures of IMCD cells using the Fura-2 technique. Basal steady-state calcium at 600 mosmol/l was found to be 110±4 nmol/l; n=119. Exposure to hypotonic medium (300 mosmol/l, reduction of sucrose) resulted, within 1 min, in a strong increase in calcium to 563±87 nmol/l (n=7; P<0.01), followed by a decrease over 4–6 min to twice the initial values. The calcium increase was smaller (260±14 nmol/l; n=5; P<0.05) when the osmotic pressure was decreased by reducing NaCl instead of sucrose. Stepwise reduction of osmolarity to either 500 or 400 mosmol/l increased calcium by a significantly smaller extent, suggesting a threshold for calcium influx between 400 and 300 mosmol/l. In hypotonic calcium-free solutions no significant increase in calcium was observed. Verapamil (40 μmol/l), D-600 (40 μmol/l), diltiazem (40 μmol/l), and nifedipine (40 μmol/l) inhibited the hypotonically induced calcium influx in decreasing order of potency. Lanthanum (La3+) and gadolinium (Gd3+) had no effect. Membrane depolarization by incubation in potassium-rich solution diminished calcium influx. Preincubation with cytochalasin B (50 μmol/l for 30 min) resulted in a lower basal calcium level and attenuated the calcium increase during hypotonic shock. These results demonstrate an increased calcium influx during hypotonic shock in IMCD cells in culture mediated by channels whose nature (stretch activated and/ or voltage dependent) remains to be determined. The transient increase in Cai in turn may trigger inorganic and organic osmolyte fluxes observed previously.
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References
Almers W, Neher E (1985) The Ca signal from Fura-2 loaded mast cells depends strongly on the method of dye-loading. FEBS Lett 192:13–17
Bagnasco SM, Murphy HR, Bedford JJ, Burg MB (1988) Osmoregulation by slow changes in aldose reductase and rapid changes in sorbitol flux. Am J Physiol 254:C788-C792
Bagnasco SM, Montrose MH, Handler JS (1993) Role of calcium in organic osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium. Am J Physiol 264:C1165-C1170
Bean BP (1989) Classes of calcium channels in vertebrate cells. Annu Rev Physiol 51:367–384
Bear CE (1990) A nonselective cation channel in rat liver cells is activated by membrane stretch. Am J Physiol 256:C421-C428
Beck FX, Dörge A, Thurau K, Guder WG (1990) Cell osmoregulation in the countercurrent system of the renal medulla: The role of organic osmolytes. Comp Physiol 4:132–158
Beck JS (1990) Cell swelling, cotransport activation and potassium conductance in isolated perfused rabbit kidney proximal tubules. J Physiol (Lond) 425:369–378
Bevan C, Kinne RKH (1990) Choline transport in collecting duct cells isolated from the rat renal inner medulla. Pflügers Arch 417:324–328
Bevan C, Theiß C, Kinne RKH (1990) Role of Ca2+ in sorbitol release from rat inner medullary collecting duct (IMCD) cells under hypoosmotic stress. Biochem Biophys Res Commun 170:563–568
Biagi BA, Enyeart JJ (1990) Gadolinium blocks low- and high threshold calcium currents in pituitary cells. Am J Physiol 259:C515-C520
Blatter LA, Wier WG (1990) Intracellular diffusion, binding and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2. Biophys J 58:1491–1499
Bourdeau JE, Lau K (1990) Basolateral cell membrane Ca-Na exchange in single rabbit connecting tubules. Am J Physiol 258:F1497-F1503
Chamberlin ME, Strange K (1989) Anisomotic cell volume regulation: a comparative view. Am J Physiol 257:C159-C173
Christensen C (1987) Mediation of cell volume regulation by Ca influx through stretch-activated channels. Nature 330:66–68
Czekay RP, Kinne-Saffran E, Kinne RKH (1994) Membrane traffic and sorbitol release during osmo- and volume regulation in isolated rat renal inner medullary collecting duct cells. Eur J Cell Biol 63:20–31
Filipovic D, Sackin H (1991) A calcium permeable stretch-activated cation channel in renal proximal tubule. Am J Physiol 260:F119-F129
Foskett JK, Spring KR (1985) Involvement of calcium and cytoskeleton in gall-bladder epithelial cell volume regulation. Am J Physiol 248:C27-C36
Garcia-Perez A, Burg MB (1991) Renal medullary organic osmolytes. Physiol Rev 71:1081–1115
Grunewald RW, Kinne RKH (1989) Intracellular sorbitol content in isolated rat inner medullary collecting duct cells. Regulation by extracellular osmolarity. Pflügers Arch 414:178–184
Grunewald JM, Grunewald RW, Kinne RKH (1993) Regulation of ion content and cell volume in isolated rat renal IMCD cells under hypotonic conditions. Kidney Int 44:509–517
Grupp C, Pavenstädt-Grupp I, Kinne RKH (1989) Natrium-, Kalium- und Chlorid-Transport im papillären Sammelrohr. Nieren- und Hochdruckkrankheiten 18:8–12
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
Guharay F, Sachs F (1984) Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. J Physiol (Lond) 352:685–701
Guharay F, Sachs F (1985) Mechanotransducer ion channels in chick skeletal muscle: the effect of extracellular pH. J Physiol (Lond) 363:119–134
Hazama A, Okada Y (1990) Biphasic rises in cytosolic free Ca in association with activation of K and Cl conductance during the regulatory volume decrease in cultured human epithelial cells. Pflügers Arch 416:710–714
Hoffmann EK, Simonsen LO, Lambert IH (1984) Volume-induced increase of K and Cl permeabilities in Ehrlich ascites tumor cells. Role of internal calcium. J Membr Biol 78:211–222
Hurst AM, Hunter M (1990) Stretch-activated channels in single early distal tubule cells of the frog. J Physiol (Lond) 430:13–24
Ishikawa S, Okada K, Saito T (1988) Arginine vasopressin increases cellular free calcium concentration and adenosine 3′,5′-monophosphate production in rat renal papillary collecting tubule cells in culture. Endocrinology 123:1376–1382
Kinne RKH, Czekay RP, Grunewald JM, Mooren FC, Kinne-Saffran E (1993) Hypotonicity-evoked release of organic osmolytes from distal renal cells: systems, signals, and sidedness. Renal Physiol Biochem 16:66–78
Kleinzeller A, Ziyadeh FN (1990) Cell volume regulation in epithelia — with emphasis on the role of osmolytes and the cytoskeleton. Comp Physiol 4:59–86
Lingshaw MA (1991) Role of cytoskeleton in isotonic cell volume control of rabbit proximal tubules. Am J Physiol 261:F60-F69
Malgaroli A, Milani D, Meldolesi J, Pozzan T (1987) Fura-2 measurement of cytosolic free Ca in monolayers and suspensions of various types of animal cells. J Cell Biol 105:2145–2155
McCarty NA, O'Neil RG (1990) Dihydropyridine-sensitive cell volume regulation in proximal tubule: the calcium window. Am J Physiol 259:F950-F960
McCarty NA, O'Neil RG (1991a) Calcium-dependent control of volume regulation in renal proximal tubule cells: I. Swelling-activated Ca entry and release. J Membr Biol 123:149–160
McCarty NA, O'Neil RG (1991b) Calcium-dependent control of volume regulation in renal proximal tubule cells: II. Roles of dihydropyridine-sensitive and -insensitive Ca entry pathways. J Membr Biol 123:161–170
McNeil PL (1989) Incorporation of macromolecules into living cells. Methods Cell Biol 29:153–173
Montrose-Rafizadeh C, Guggino WB (1991) Role of intracellular calcium in volume regulation by rabbit medullary thick ascending limb cells. Am J Physiol 260:F402-F409
Oakes SG, Martin I, Lisek CA, Powis G (1988) Incomplete hydrolysis of the calcium indicator precursor Fura-2 pentaacetoxymethylester by cells. Anal Biochem 169:159–166
Okada Y, Hazama A (1989) Volume-regulatory ion channels in epithelial cells. News Physiol Sci 4:238–242
Okada Y, Hazama A, Yuan W (1990) Stretch-induced activation of Ca-permeable ion channels is involved in the volume regulation of hypotonically swollen epithelial cells. Neurosci Res 12:S5-S13
Opie LH (1987) Calcium channel antagonists. I. Fundamental properties: mechanisms, classification, sites of action. Cardiovasc Drugs Ther 1:411–430
Poenie M, Alderton J, Tsien RG, Steinhardt RA (1985) Changes of free calcium levels with stages of the cell division cycle. Nature 315:147–149
Poenie M, Alderton J, Steinhardt RA, Tsien RG (1986) Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science 233:886–889
Rothstein A, Mack E (1990) Volume-activated K and Cl pathways of dissociated epithelial cells (MDCK):role of Ca. Am J Physiol 258:C827-C834
Sachs F (1986) Biophysics of mechanoreception. Membr Biochem 6:173–195
Sagara Y, Inesi G (1991) Inhibition of the sarcoplasmic reticulum Ca transport ATPase by thapsigargin at subnanomolar concentrations. J Biol Chem 266:13 503–13 506
Schwartz IL, Shlatz LJ, Kinne-Saffran E, Kinne R (1974) Target cell polarity and membrane phosphorylation in relation to the mechanism of action of antidiuretic hormone. Proc Natl Acad Sci USA 71:2595–2599
Slotki IN, Schwartz JH, Alexander EA (1989) Effect of increases in cytosolic Ca2+ on inner medullary collecting duct cell pH. Am J Physiol 257:F210-F217
Smallridge R, Gist ID, Ambroz C (1991) 1-Diethylaminooctyl-3,4,5-trimethoxybenzoate, a calcium store blocker, increases calcium influx, inhibits α-1 adrenergic receptor calcium mobilization, and alters iodide transport in FRTL-5 rat thyroid cells. Endocrinology 129:542–549
Stokes JB, Grupp C, Kinne RKH (1987) Purification of rat papillary collecting duct cells: functional and metabolic assessment. Am J Physiol 253:F251-F262
Taniguchi J, Guggino WB (1989) Membrane-stretch: a physiological stimulator of Ca2+-activated K+ channels in thick ascending limb. Am J Physiol 257:F347-F352
Taniguchi S, Marchetti J, Morel F (1989) Na/Ca-exchangers in collecting cells of rat kidney. Pflügers Arch 415:191–197
Tinel H, Wehner F, Sauer H (1994) Intracellular Ca2+ release and Ca2+ influx during regulatory volume decrease in inner medullary collecting duct cells. Am J Physiol (in press)
Williams DA, Fay FS (1990) Intracellular calibration of the fluorescent calcium indicator Fura-2. Cell Calcium 11:75–83
Wong SME, Chase HS (1986) Role of intracellular calcium in cellular volume regulation. Am J Physiol 250:C841-C852
Yamaguchi DT, Green J, Kleeman CR, Muallem S (1989) Characterization of volume-sensitive, calcium-permeating pathways in the osteosarcoma cell line UMR-106-01. J Biol Chem 264:4383–4390
Yang X-C, Sachs F (1989) Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243:1068–1070
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Mooren, F.C., Kinne, R.K.H. Intracellular calcium in primary cultures of rat renal inner medullary collecting duct cells during variations of extracellular osmolality. Pflügers Arch. 427, 463–472 (1994). https://doi.org/10.1007/BF00374262
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DOI: https://doi.org/10.1007/BF00374262