Secretory pathway Ca(2+)-ATPase (SPCA1) Ca(2)+ pumps, not SERCAs, regulate complex [Ca(2+)](i) signals in human spermatozoa.
The sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors thapsigargin (0.1-1 microM) and cyclopiazonic acid (10 microM), failed to affect resting [Ca(2+)] in human spermatozoa. Slow progesterone-induced [Ca(2+ i)](i) oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca(2+) store were also insensitive to these inhibitors. Non-selective doses of thapsigargin (5-30 microM, 50-300 times the saturating dose for SERCA inhibition), caused elevation of resting [Ca(2+)](i) and partial, dose-dependent disruption of oscillations. A 10-40 microM concentration of bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane (bis-phenol), which inhibits both thapsigargin-sensitive and -insensitive microsomal Ca(2+) ATPases, caused elevation of resting [Ca(2+)](i) and inhibition of [Ca(2+)](i) oscillations at doses consistent with inhibition of thapsigargin-resistant, microsomal ATPase and liberation of stored Ca(2+). Low doses of bis-phenol had marked effects on [Ca(2+)](i) oscillation kinetics. Application of the drug to cells previously stimulated with progesterone had effects very similar to those observed when it was applied to unstimulated cells, suggesting that the sustained Ca(2+) influx induced by progesterone is not mediated via mobilisation of Ca(2+) stores. Western blotting for human sperm proteins showed expression of secretory pathway Ca(2+) ATPase (SPCA1). Immunolocalisation studies revealed expression of SPCA1 in all cells in an area behind the nucleus, extending into the midpiece. Staining for SERCA, carried out in parallel, detected no expression with either technique. We conclude that: (1) intracellular Ca(2+) store(s) and store-dependent [Ca(2+)](i) oscillations in human spermatozoa rely primarily on a thapsigargin/cyclopiazonic acid-insensitive Ca(2+) pump, which is not a SERCA as characterised in somatic cells; (2) effects of high-dose thapsigargin on spermatozoa primarily reflect non-specific actions on non-SERCAs and; (3) secretory pathway Ca(2+) ATPases contribute at least part of this non-SERCA Ca(2+) pump activity.
Harper
Claire
C
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Wootton
Laura
L
Michelangeli
Francesco
F
Lefièvre
Linda
L
Barratt
Christopher
C
Publicover
Stephen
S
D002084:Butylated Hydroxytoluene / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007424:Intracellular Fluid / Q000378:metabolism; D008297:Male; D008345:Manganese / Q000378:metabolism; D011374:Progesterone / Q000494:pharmacology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D002084:Butylated Hydroxytoluene; D008345:Manganese; D011374:Progesterone; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C403624:ATP2C1 protein, human; D000252:Calcium-Transporting ATPases; C008437:2,2'-methylenebis(4-methyl-6-tert-butylphenol); D002118:Calcium