Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intra-adrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.

Paneth cells located at the bottom of intestinal crypts may play a role in controlling the bacterial milieu of the intestine. Using morphometry to clarify the secretory mechanism of the Paneth cells, we studied the ultrastructural changes in mouse Paneth cells produced following intra-arterial perfusion with Hanks' balanced salt solution containing a cholinergic muscarinic secretagogue (bethanechol), a neuroblocking agent (tetrodotoxin), or a G-protein activator (NAF/AlCl3). Bethanechol (2 x 10(-4) mol/l) induced Paneth-cell secretion. Many Paneth cells massively exocytosed their secretory material into the crypt lumen; the enhanced secretion caused degranulation and vacuole formation. However, tetrodotoxin (2 x 10(-6) mol/l) did not prevent the bethanechol-enhanced secretion by the Paneth cells. NaF (1 x 10(-2) mol/l) and AlCl3 (1 x 10(-5) mol/l) induced massive exocytosis of the Paneth cells; the exocytotic figures were similar to those observed in mice stimulated by bethanechol. G-protein activation was followed by a sequence of intracellular events, resulting in exocytosis.

Prolonged perfusion of isolated rat adrenal glands with high K produced a transient increase in adrenaline release that reached a maximum within 1 min and then declined with a half-time of about 5 min. The higher the [K]o used, the larger was the release of adrenaline elicited. There was a linear relation between the mean rate of adrenaline release in the initial 5 min of continuous stimulation with excess K and logarithmic increase in the [K]o. The higher the [Ca] o used, the steeper was relation obtained: the slope determined in the presence of 5 mM-Ca was nearly twice as much as that obtained in the presence of 0.5 mM-Ca. The release of adrenaline in response to excess K was virtually abolished by the omission of [Ca]o. The chromaffin cells were depolarized in proportion to logarithmic increase in the [K]o. Linear relations were thus obtained between the mean rates of adrenaline release and the membrane potential of chromaffin cells in the presence of different [Ca]o .

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.

To confirm whether the Paneth cells of mice (ICR, male, 10-12 weeks old) have the same secretory response to hormonal and cholinergic stimulation as do pancreatic acinar cells, ultrastructural changes of Paneth cells and pancreatic acinar cells 1 hr after administration of various doses of cholecystokinin (octapeptide, CCK-8) and carbamylcholine were morphometrically assessed. After maximal (1.5 micrograms/kg intraperitoneally [i.p.]) and supramaximal (15 micrograms/kg, i.p.) stimulation by CCK-8, pancreatic acinar cells showed, respectively, degranulation or disturbance of secretion (e.g., an increase in lysosome-like bodies, aggregation of zymogen granules). The Paneth cells, however, were almost unchanged in the parameters examined. After carbamylcholine injection (1,000 micrograms/kg, subcutaneously [s.c.]), both pancreatic acinar cells and Paneth cells showed degranulation. Paneth cells sometimes developed large vacuoles, probably formed after massive exocytosis; such vacuoles were not observed in pancreatic acinar cells. It is suggested that Paneth cells and pancreatic acinar cells have different secretory responses. Paneth cell secretion, which possibly plays a role in controlling the intestinal bacterial milieu, may be stimulated by cholinergic rather than hormonal mechanisms.