Localisation, function and composition of primary Ca2+ spark discharge region in isolated smooth muscle cells from guinea-pig mesenteric arteries

Smooth muscle cells (SMCs) contain numerous calcium release domains, grouped into regions discharging as a single unit. Laser scanning confocal microscopy, voltage clamp and immunocytochemistry of single SMCs from small mesenteric arteries of guinea-pig were used to study the localisation, function and macromolecular composition of such calcium discharge regions (CDRs). Use of the Ca2+-sensitive fluorescent dye fluo-3 or fluo-4 with BODIPY TR-X ryanodine (BTR), a fluorescent derivative of ryanodine, showed spontaneous Ca2+ sparks originating from regions stained by BTR, located immediately under the plasma membrane, in the arch formed by the sarcoplasmic reticulum surrounding the nucleus. Membrane depolarisation or application of noradrenaline or α,β-methylene ATP, a P2X purinoceptor agonist, elicited Ca2+ sparks from the same, spontaneous Ca2+ spark-discharging region. The most active (primary) CDR accounted for nearly 60% of spontaneous transient outward currents at −40 mV and these were of significantly higher amplitude than the ones discharged by secondary CDRs. Immunocytochemical staining for type 1 IP3 receptors, BKCa channels, P2X1 purinoceptors or α1 adrenoceptors revealed their juxtaposition with BTR staining at the location typical of the primary CDR. These data suggest the existence of a primary calcium discharge region in SMCs; its position can be predicted from the cell's structure, it acts as a key region for the regulation of membrane potential via Ca2+ sparks and is a potential link between the external, neurohumoral and the cell's internal, calcium signalling system.