Functional separation of deep cytoplasmic calcium from subplasmalemmal space calcium in cultured human uterine smooth muscle cells.
For smooth muscle, two important functions of free intracellular calcium (Ca(2+)(i)) are modulation of plasma membrane excitability properties and modulation of the contractile apparatus. As proposed by van Breemen, Ca(2+)(i) can be divided into the subplasmalemmal space (Ca(2+)(sps)) and the deep cytosol (Ca(2+)(d)) by the superficial calcium buffer barrier. Using these distinctions, Ca(2+)(sps) activates the large conductance calcium-activated potassium channel (BK), and Ca(2+)(d) binds calcium-dependent fluorescent probes in the cytoplasm. We present here combined fluorescence-patch clamp experiments designed to simultaneously assess Ca(2+)(d) and Ca(2+)(sps) in cultured human uterine smooth muscle cells. Open probabilities (P(o)) of the BK channel were measured using the cell-attached patch clamp technique. P(o) was used to approximate changes of [Ca(2+)(sps)]. Relative concentrations of Ca(2+)(d) were approximated by observing fluorescence of Calcium green-1 (F). Under control conditions, we found similar time courses for rises of P(o) and F following 10nM oxytocin (OT) addition. In parallel experiments, but with lanthanum (La(3+)) added to the bath to block transmembrane calcium flux, P(o) was only slightly affected, but F increases were delayed and blunted. These data paradoxically indicate that following OT stimulation, the primary source of calcium for Ca(2+)(sps) is internal stores, and calcium entry from the extracellular space is required to raise Ca(2+)(d). When cells were exposed to cyclopiazonic acid (CPA) to release SR calcium stores, P(o) increased slowly, then persisted at large values. The persistence of P(o) rises suggests that removal of calcium from the subplasmalemmal space is primarily via reuptake into the SR. In the presence of La(3+), OT-induced rises of F were slightly prolonged, suggesting that transmembrane calcium flux contributes to decreasing Ca(2+)(d), but is not the primary mechanism. In summary, these data demonstrate that Ca(2+)(d) and Ca(2+)(sps) are not always intimately linked, but indicate a functional separation of the deep cytosol and the subplasmalemmal space that is consistent with the existence of a barrier to calcium diffusion between these two regions.
Young
Roger C
RC
Department of Obstetrics and Gynecology, Dartmouth School of Medicine, Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA. roger.c.young@hitchcock.org
Zhang
PeiSheng
P
D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002451:Cell Compartmentation / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism / Q000502:physiology*; D005260:Female; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D009119:Muscle Contraction / Q000502:physiology*; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000502:physiology; D011336:Probability; D014599:Uterus / Q000166:cytology / Q000502:physiology*
D002121:Calcium Channel Blockers; D007211:Indoles; D015221:Potassium Channels; D007811:Lanthanum; D002118:Calcium; C000543:cyclopiazonic acid