Solitary calcium spike dependent on calmodulin and plasma membrane Ca2+ pump.
Resealed human red cell ghosts were loaded with Fura-2, ATP, Mg2+, and either calmodulin (CaM) or, to prevent CaM activation of the Ca2+ pump, a synthetic peptide that antagonized endogenous CaM (an analogue of the CaM binding domain of protein kinase II, referred to as 'antiCaM'). The ghosts reduced the cytosolic concentration of ionized calcium ([Ca2+]i) to 193 +/- 60 nM (SD, n = 15) in a medium containing 1 mM Ca2+ and to 30 +/- 27 nM (SD, n = 62) in a medium without Ca2+ addition. Without ATP, i.e. no fuelling of the Ca2+ pump, the [Ca2+]i remained high (approx. 5 microM or higher). The simultaneous addition of the ionophore A23187 and Ca2+ rapidly increased the Ca2+ influx, which in the CaM loaded ghosts caused a solitary spike of [Ca2+]i, reaching maximum around 2 microM within 24 +/- 6 s (SD, n = 40). On the contrary, in the ghosts loaded with antiCaM, the addition of A23187 with Ca2+ raised [Ca2+]i during the first 2 min to a high level (2-4 microM) with no preceding spike. Pre-incubation of CaM-ghosts with Ca2+ diminished the height of the Ca2+ spike, and treatment with trypsin even removed the Ca2+ spike. The trypsin treatment activated the Ca2+ pump prior to the rise of [Ca2+]i, making the time-consuming CaM activation unnecessary. In conclusion, the Ca2+ spiking is dependent on a delayed CaM activation of the plasma membrane Ca2+ pump in response to a rapid increase of Ca2+ influx.
Foder
B
B
Department of Clinical Physiology and Nuclear Medicine, Rigshospitalet, University Hospital, Copenhagen, Denmark.
Scharff
O
O
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology*; D003600:Cytosol / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D013268:Stimulation, Chemical
D002147:Calmodulin; D010446:Peptide Fragments; D000001:Calcimycin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium