Verhandelingen - Koninklijke Academie voor Geneeskunde van België. vol:55 issue:5 pages:425-56
The cytoplasmic free calcium concentration ([Ca2+]i) is one of the most important factors in the regulation of the cellular activity. We therefore studied in this work the changes of [Ca2+]i during muscle excitation with optical techniques in intact smooth muscle preparations. Also the relation between [Ca2+]i, the phosphorylation of the myosin light chain and the force development in phasic and tonic smooth muscle during excitation-contraction coupling was investigated. We concluded that the changes of [Ca2+]i and force are not invariably tightly coupled during stimulation in spite of the fact that the increase of [Ca2+]i is the primary regulator of smooth muscle contraction. Agonists may, besides their Ca2+ mobilizing effect, also modulate the sensitivity of the regulatory-contractile apparatus for Ca2+. Ca(2+)-release from the internal stores during activation may modulate its further depletion. We demonstrated that the Ins(1,4,5)P3 receptor is controlled by luminal Ca2+. These observations allowed us to develop a model whereby Ca2+ oscillations during submaximal stimulation could be explained. This model of control by luminal Ca2+ also explained why the release in response to a low Ins(1,4,5)P3 concentration suddenly stops at a time when there is still plenty of Ca2+ in the internal stores. We furthermore showed that these Ca2+ channels in the SR are controlled by the redox-status of the cell. The study of Ca2+ extrusion was mainly focused on the Ca2+ pump of the plasma-membrane and its regulation by negatively charged phospholipids. Our main finding was that the plasma-membrane Ca2+ pump was stimulated by negatively charged phospholipids. Finally, we started the investigation of the existence of Ca2+ gradients between nucleus and cytoplasm in intact cells with confocal laser microscopy. We demonstrated in different cell types the existence of spatio-temporal differences in the Ca2+ distribution between nucleus and cytosol in basal and stimulated conditions. These observations indicate that a Ca2+ barrier must be present at the nuclear envelope, that different Ca2+ mobilizing phenomena are present and are in favour of the existence of a specific regulation of nuclear Ca2+.