Analyses of signal transduction cascades in rat pinealocytes reveal a switch in cholinergic signaling during postnatal development

In the rat pineal gland, norepinephrine activates alpha1- and beta-adrenergic receptors and triggers melatonin production through an increase in the intracellular calcium concentration ([Ca2+]i) and stimulation of the cAMP/cAMP responsive element-binding protein (CREB) cascade. VIP and PACAP also elevate the intracellular cAMP level and promote melatonin formation. Finally, ACh antagonizes the norepinephrine-induced hormone synthesis via nicotinic acetylcholine receptors and subsequent activation of voltage-gated calcium channels. By immuno(cyto)chemical demonstration of phosphorylated CREB and calcium imaging we have investigated the temporal relationship between the maturation of these signaling pathways and the rhythmic onset of melatonin biosynthesis in developing rat pinealocytes. Norepinephrine-regulated calcium signaling and phosphorylation of CREB are already fully developed at birth, i.e., prior to ingrowth of the sympathetic innervation into the pineal parenchyma, and appear to develop in an innervation-independent manner. VIP- and PACAP-induced CREB phosphorylation is restricted to subpopulations of neonatal cells and thus also displays an adult pattern. Cholinergic calcium signaling exhibits a developmental switch within the first three postnatal weeks. In neonatal pinealocytes, acetylcholine elevates [Ca2+]i via muscarinic rather than nicotinic acetylcholine receptors. In the second postnatal week, pinealocytes gain responsiveness to nicotine and gradually lose responsiveness to muscarinic cholinergic stimuli. Voltage-gated calcium channels are absent in neonatal cells and develop during the first postnatal days. ACh-evoked cellular events may be diversified depending on the functional subclass of receptor that is present. The transient existence of muscarinic acetylcholine receptors and the subsequent switch to nicotinic receptors would permit ACh to elicit temporary effects in early pineal development.