CALCIUM RESPONSES OF ISOLATED, IMMUNOCYTOCHEMICALLY IDENTIFIED RAT PINEALOCYTES TO NORADRENERGIC, CHOLINERGIC AND VASOPRESSINERGIC STIMULATIONS

Calcium responses of isolated rat pineal cells to noradrenergic, choli nergic and vasopressinergic stimulations were recorded by use of the f ura-2 technique and an image analysis system. Subsequently the recorde d cells were identified as pinealocytes by immunocytochemical demonstr ation of S-antigen, a pinealocyte-specific marker. S-antigen immunorea ctive pinealocytes were shown to respond to norepinephrine stimulation with an elevation of the intracellular free calcium concentration ([C a2+](i)). This response was dose-dependent and consisted of a rapid in crease in [Ca2+](i) (primary phase) followed by a decrease to an eleva ted plateau well above the basal level (secondary phase). The plateau persisted for at least 1 h when cells were constantly exposed to norep inephrine and dropped to basal level upon removal of the stimulus. Ana lysis of the calcium responses of cells treated with caffeine or thaps igargin suggested that the primary phase reflects mobilization of calc ium from inositol 1,4,5-trisphosphate-sensitive intracellular calcium stores. Depletion of these calcium stores was a decisive and sufficien t prerequisite to evoke the secondary phase which was apparently elici ted by calcium influx. These data suggest that a capacitative calcium entry is involved in pineal calcium signalling. Acetylcholine induced an increase in [Ca2+](i) in rat pinealocytes. Experiments with differe nt cholinergic agonists and antagonists provided evidence that the ace lylcholine-induced calcium response was mediated via nicotinic acetylc holine receptors. Stimulation of isolated rat pineal cells with argini ne-vasopressin caused a rise in [Ca2+](i) in approx. 5% of the cells. However, these cells remained unidentified because they contained neit her immunoreactive S-antigen nor immunoreactive glial fibrillary acidi c protein, a marker for interstitial (glial) cells of the rat pineal o rgan. Taken together, the results underline the pivotal role of norepi nephrine for the regulation of pineal signal transduction, but they al so support the notion that other neurotransmitters and neuropeptides a re involved in the modulation of pineal calcium signalling.