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 acetylcholine-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.