Sarcoplasmic reticulum Ca2+ depletion by caffeine and changes of [Ca2+](i) during refilling in bovine airway smooth muscle cells.
BACKGROUND
In airway smooth muscle (ASM), Ca2+ influx in response to the Ca2+ depletion of the sarcoplasmic reticulum (SR) seems to play a role in the regulation of intracellular free Ca2+ concentrations ([Ca2+](i)). This study evaluates some possible Ca2+ entry pathways activated during SR-Ca2+ depletion induced by 10 mM caffeine.


METHODS
Enzymatically dispersed bovine ASM cells were loaded with Fura-2/AM to permit measurement of [Ca2+](i) changes in single cells.


RESULTS
Caffeine (10 mM) induced a transient increase in [[Ca2+](i) that depleted SR-Ca(2)+ content. After caffeine washout, a decrease in basal [Ca2+](i) (undershoot) was invariably observed, followed by a slow recovery. This phenomenon was inhibited by cyclopiazonic acid (5 microM). External Ca(2)+ removal in depolarized and nondepolarized cells induced a decrease in basal [Ca2+](i) that continued until depletion of the SR-Ca2+ content. The decrease in [Ca2+](i) induced by Ca2+-free physiological saline solution (PSS) was accelerated in caffeine-stimulated cells. Recovery from undershoot was not observed in Ca2+-free PSS. Depolarization with KCl and addition of D600 (30 microM) did not modify recovery. Similar results were obtained when the Na(+)/Ca2+ exchanger was blocked by substituting NaCl with KCl in normal PSS (Na(+)-free PSS) or by adding benzamil amiloride (25 microM).


CONCLUSIONS
SR-Ca2+ content plays an important role in the Ca2+ leak induced by Ca2+-free medium, and does not depend on membrane potential. Additionally, recovery from undershoot after caffeine depends on extracellular Ca2+, and neither voltage-dependent Ca2+ channels nor the Na(+)/Ca2+ exchanger are involved.
Departamento de Investigación en Asma, Instituto Nacional de Enfermedades Respiratorios (INER), Tlalpan #4502, 14080 Mexico City, D.F., Mexico. lmmr@servidor.unam.mx
B
B
Bazán-Perkins
E
E
Sánchez-Guerrero
V
V
Carbajal
C
C
Barajas-López
L M
LM
Montaño
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects* / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects*; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D005711:Gallopamil / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects*; D011189:Potassium Chloride / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000187:drug effects / Q000502:physiology; D014132:Trachea / Q000166:cytology*
D015220:Calcium Channels; D007211:Indoles; D007476:Ionophores; D019831:Sodium-Calcium Exchanger; C013407:benzamil; D005711:Gallopamil; D002110:Caffeine; D015759:Ionomycin; D011189:Potassium Chloride; D000584:Amiloride; D002217:Carbachol; D002118:Calcium; C000543:cyclopiazonic acid