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

Calcium responses of isolated rat pineal cells to noradrenergic, cholinergic and vasopressinergic stimulations were recorded by use of the fura-2 technique and an image analysis system. Subsequently the recorded cells were identified as pinealocytes by immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker. S-antigen immunoreactive pinealocytes were shown to respond to norepinephrine stimulation with an elevation of the intracellular free calcium concentration ([Ca2+](i)). This response was dose-dependent and consisted of a rapid increase in [Ca2+](i) (primary phase) followed by a decrease to an elevated plateau well above the basal level (secondary phase). The plateau persisted for at least 1 h when cells were constantly exposed to norepinephrine and dropped to basal level upon removal of the stimulus. Analysis of the calcium responses of cells treated with caffeine or thapsigargin suggested that the primary phase reflects mobilization of calcium from inositol 1,4,5-trisphosphate-sensitive intracellular calcium stores. Depletion of these calcium stores was a decisive and sufficient prerequisite to evoke the secondary phase which was apparently elicited 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 different cholinergic agonists and antagonists provided evidence that the acelylcholine-induced calcium response was mediated via nicotinic acetylcholine receptors. Stimulation of isolated rat pineal cells with arginine-vasopressin caused a rise in [Ca2+](i) in approx. 5% of the cells. However, these cells remained unidentified because they contained neither immunoreactive S-antigen nor immunoreactive glial fibrillary acidic protein, a marker for interstitial (glial) cells of the rat pineal organ. Taken together, the results underline the pivotal role of norepinephrine for the regulation of pineal signal transduction, but they also support the notion that other neurotransmitters and neuropeptides are involved in the modulation of pineal calcium signalling.