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Fura-2-loaded human platelets were immobilized on a fibrinogen-coated surface and the cytosolic free calcium concentration ([Ca2+]i) was measured in single platelets by low-light-level video-ratio image-processing of the optical probe signal. Some fibrinogen-bound platelets showed repetitive spiking in [Ca2+]i with a mean frequency of about 2/min, which increased to 5/min in the presence of ADP. Other cells showed no activity until the addition of agonist. When immobilized in the presence of prostaglandin I2 and the fibrinogen antagonist Arg-Gly-Asp-Ser, the platelets adhered less firmly to fibrinogen, and in many [Ca2+]i remained low and constant. Subsequent activation of such platelets with ADP evoked oscillations in [Ca2+]i with a peak frequency of about 5/min and which persisted for at least 5 min. These results indicate that human platelets, like many other non-excitable cells, have an elaborate system of calcium signalling involving spiking.

Vasoconstrictor agonists stimulate smooth muscle contraction by inducing a rise in intracellular free Ca2+. Digital-imaging microscopy of fura-2 fluorescence from single vascular smooth muscle cells cultured from the human internal mammary artery has allowed us to record the subcellular alterations in Ca2+ that occur immediately after stimulation by receptor agonists. The thrombin-induced rise in cytoplasmic free Ca2+ begins in a discrete region typically located close to the end of the cell. Subsequently, this region of elevated Ca2+ expands until Ca2+ is elevated throughout the cell cytoplasm. The rate of spreading in the region of elevated Ca2+ in a linear direction averaged 10.1 microns/s, enabling it to traverse the length of most cells within approximately 5 s, and involved rises in Ca2+ of between 200 and 500 nM. In some cells, the Ca2+ rise began at both ends and collided midway. Similar dynamic changes in the spatial distribution of Ca2+ were recorded in cells stimulated by acetylcholine. The novel observation that vasoconstrictor agonists induce an elevation of Ca2+ in a localized region which subsequently expands throughout the cytoplasm of single smooth muscle cells may provide new insight into the nature of Ca2+ signaling in vascular tissue.

SUMMARY1. Changes in intracellular ionized calcium [Ca2+]i induced by human growth hormone releasing factor (hGRF) were analysed by quantitative fluorescent microscopy using a dual-wavelength, ratiometric video imaging system and low light level charge-coupled device (CCD) camera visualizing Fura-2 in dispersed male rat anterior pituitary cells.2. In cells responding to hGRF, spontaneous basal oscillations in [Ca2+]i were frequently observed, and these were usually characterized by a gradient of [Ca21] localized in the subplasmalemmal region of the cell. 9. From these results, taken together with previous findings, we propose the possibility that hGRF activates tetrodotoxin-insensitive Na' (or non-selective cationic) channels via cyclic AMP, which in turn causes depolarization of the somatotroph leading to activation of Ca21 channels, Ca2" influx and exocytotic secretion of growth hormone.