Neurofilament immunoreactivity and acetylcholinesterase activity in the developing sympathetic tissues of the rat.

In this study, the ontogenetic appearance of three neuronal markers, tyrosine hydroxylase (TH), neurofilament (NF) proteins and acetylcholinesterase (AChE), have been compared in the neural tube and derivatives of the neural crest with special consideration on developing rat sympathetic tissues. The tree markers appeared for the first time on embryonic day E 12.5. At this age, NF immunoreactivity was located in the cells on the ventro- and dorsolateral edges of the neural tube, i.e., in the regions where the cells had reached the postmitotic stage. In addition, on day E 12.5, NF-immunoreactive fibers were located in the dorsal and ventral roots and the spinal and sympathetic ganglia. This suggests rapid extension of neurites. In contrast to NF, AChE first appeared on day E 12.5 in cell somata of spinal and sympathetic ganglia and only after that in axons. Thus, it can be considered as a marker of differentiating neuronal cell bodies. In the developing sympathoadrenal cells, TH is expressed before NF and AChE. However, the migrating TH immunoreactive sympathetic cells are constantly followed by NF immunoreactive fibers, suggesting that sympathetic tissues may receive innervation from preganglionic axons at the very beginning of their ontogeny. During the later development, all sympathetic tissues contain two major cell groups: 1) one with a moderate TH immunoreactivity, NF immunoreactivity and AChE activity and 2) the other with an intense TH immunoreactivity but lacking NF immunoreactivity or AChE activity. The former includes principal neurons, neuron-like cells of the paraganglia and noradrenaline cells of the adrenal medullae, and the latter includes ganglionic small intensely fluorescent (SIF) cells, paraganglionic cells and medullary adrenaline cells.     

Neurofilament immunoreactivity and acetylcholinesterase activity in the developing sympathetic tissues of the rat

Summary In this study, the ontogenetic appearance of three neuronal markers, tyrosine hydroxylase (TH), neurofilament (NF) proteins and acetylcholinesterase (AChE), have been compared in the neural tube and derivatives of the neural crest with special consideration on developing rat sympathetic tissues. The tree markers appeared for the first time on embryonic day E 12.5. At this age, NF immunoreactivity was located in the cells on the ventro- and dorsolateral edges of the neural tube, i.e., in the regions where the cells had reached the postmitotic stage. In addition, on day E 12.5, NF-immunoreactive fibers were located in the dorsal and ventral roots and the spinal and sympathetic ganglia. This suggests rapid extension of neurites. In contrast to NF, AChE first appeared on day E 12.5 in cell somata of spinal and sympathetic ganglia ond only after that in axons. Thus, it can be considered as a marker of differentiating neuronal cell bodies. In the developing sympathoadrenal cells, TH is expressed before NF and AChE. However, the migrating TH immunoreactive sympathetic cells are constantly followed by NF immunoreactive fibers, suggesting that sympathetic tissues may receive innervation from preganglionic axons at the very beginning of their ontogeny. During the later development, all sympathetic tissues contain two major cell groups: 1) one with a moderate TH immunoreactivity, NF immunoreactivity and AChE activity and 2) the other with an intense TH immunoreactivity but lacking NF immunoreactivity or AChE activity. The former includes principal neurons, neuron-like cells of the paraganglia and noradrenaline cells of the adrenal medullae, and the latter includes ganglionic small intensely fluorescent (SIF) cells, paraganglionic cells and medullary adrenaline cells.     

Immunohistochemical localization of the catecholamine-synthesizing enzymes,substance P and enkephalin in the human fetal sympathetic ganglion

Summary The human fetal sympathetic ganglia were studied using the indirect peroxidase-antiperoxidase PAP method for immunocytochemical demonstration of three catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) as well as the neuropeptides leucine (Leu⁵)-enkephalin and substance P. The neuroblasts of the ganglia showed intense peroxidase immunoreactivity for TH, moderate reaction to DBH, and no reaction to PNMT. The small intensely fluorescent (SIF) cells situated along the blood vessels also showed positive labelling for only two enzymes, TH and DBH. The immunocytochemical localization of these enzymes suggests that both neuroblasts and SIF cells synthesize noradrenalin. Neither the neuroblasts nor SIF cells showed a reaction to substance P, and only the SIF cells contained enkephalin-like immunoreactivity. The role of enkephalin in the noradrenalin-containing SIF cells is unknown, but may be related to neuromodulation of ganglionic transmission.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--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 intraadrenal 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.     

Neuroanatomy of morphine-modulating peptides

Antisera against two mammalian peptides related to the molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-NH2 were used to locate immunoreactive neurons in the rat brain, nerve fibres and terminals in the spinal cord, sympathetic ganglion cells and adrenal chromaffin cells. Immunoreactivity for the newly characterised octa- and octadecapeptide was detected in nerve cell bodies in the hypothalamic area, including parts of the dorsomedial, periventricular and paraventricular nuclei, and in the nucleus tractus solitarii. Nerve terminals in the superficial laminae of the spinal cord were also immunoreactive for these peptides, while the sensory ganglia were nonreactive. Some principal ganglion cells in the superior cervical ganglia exhibited bright immunofluorescence for the peptides, and a few adrenal medullary cells were immunoreactive. The presence of these peptides in the substantia gelatinosa of the spinal cord suggests that they may be involved in sensory neurotransmission, especially in the mechanisms mediating pain. In the hypothalamo-hypophysial system these peptides may be involved in the regulation of hormonal systems. They may also act as co-transmitters in the sympathetic nervous system.     

Developmental expression of galanin-like immunoreactivity by members of the avian sympathoadrenal cell lineage

The developmental coexpression of galanin-like immunoreactivity with the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) was studied in the avian embryo sympathoadrenal system using double-labeling immunocytochemistry. Galanin-like immunoreactivity is expressed by various catecholaminergic cell populations, namely sympathoblasts, chromaffin and small intensely fluorescent (SIF) cells, but not by principal neurons of the paravertebral sympathetic ganglia. Both galanin and somatostatin immunoreactivities are coexpressed in the adrenal and sympathetic ganglion primordia by the neural precursors, but the subsequent expression pattern of both peptides differs. Our results support the hypothesis that early sympathoblasts express a large repertoire of neuroactive substances and that the expression of these becomes restricted during further development as the sympathoblasts become principal neurons.     

Localization of L-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

The localization of L-glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, was studied in the rat major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex by indirect immunofluorescence technique with a specific antiserum raised in rabbits. GAD immunoreactivity was demonstrated in small cells of these ganglia. The GAD-immunoreactive small cells were 10-20 microns in diameter and formed clusters or occurred as solitary cells. The principal neurons were non-reactive but they were surrounded by immunoreactive processes. Studies on colocalization of GAD with tyrosine hydroxylase (TH), the rate-limiting enzyme of the catecholamine synthesis, in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex indicated that all GAD-immunoreactive small cells were also labelled with TH. In the major pelvic ganglion all TH-immunoreactive SIF cells were also immunoreactive for GAD. However, in the coeliac-superior mesenteric ganglion complex there occurred TH-immunoreactive small cells which showed no immunoreactivity to GAD. It is suggested that the small GAD-immunoreactive cells represent small intensely fluorescent (SIF) cells.     

Substance P-like immunoreactivity in sympathetic ganglion from toad

Substance P-like immunoreactivity cellular in toad sympathetic ganglia was studied in normal and capsaicin-treated ganglia. In the eighth sympathetic ganglion substance P-like immunoreactive are found in mast cells and SIF cells. The effect of substance P (0.001-0.003 mM) caused increase of compound action potential during tetanical stimulation (50 Hz by 40 sec.) and post-tetanic potentiation (0.1 Hz). Our results show that substance P facilitates synaptic transmission in the sympathetic ganglia from Caudiverbera caudiverbera.     

Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibers in the adrenal gland of rats

Summary The present immunohistochemical study reveals that a small number of chromaffin cells in the rat adrenal medulla exhibit CGRP-like immunoreactivity. All CGRP-immunoreactive cells were found to be chromaffin cells without noradrenaline fluorescence; from combined immunohistochemistry and fluorescence histochemistry we suggest that these are adrenaline cells. In addition, all CGRP-immunoreactive cells simultaneously exhibited NPY-like immunoreactivity. CGRP-chromaffin cells were characterized by abundant chromaffin granules with round cores in which the immunoreactive material was densely localized. These findings suggest the co-existence of CGRP, NPY and adrenaline within the chromaffin granules in a substantial number of chromaffin cells.Thicker and thinner nerve bundles, which included CGRP-immunoreactive nerve fibers, with or without varicosities, penetrated the adrenal capsule. Most of them passed through the cortex and entered the medulla directly, whereas others were distributed in subcapsular regions and among the cortical cells of the zona glomerulosa. Here the CGRP-fibers were in close contact with cortical cells. A few of the fibers supplying the cortex extended further into the medulla. The CGRP-immunoreactive fibers in the medulla were traced among and within small clusters of chromaffin cells and around ganglion cells. The CGRP-fibers were directly apposed to both CGRP-positive and negative chromaffin cells, as well as to ganglion cells. Immunoreactive fibers, which could not be found close to blood vessels, were characterized by the presence of numerous small clear vesicles mixed with a few large granular vesicles. The immunoreactive material was localized in the large granular vesicles and also in the axoplasm. Since no ganglion cells with CGRP-like immunoreactivity were found in the adrenal gland, the CGRP-fibers are regarded as extrinsic in origin. In double-immunofluorescence staining for CGRP and SP, all the SP-immunoreactive fibers corresponded to CGRP-immunoreactive ones in the adrenal gland. This suggests that CGRP-positive fibers in the adrenal gland may be derived from the spinal ganglia, as has been demonstrated with regard to the SP-nerve fibers.     

Sympathetic and sensory innervation of small intensely fluorescent (SIF) cells in rat superior cervical ganglion

The sympathetic ganglion contains small intensely fluorescent (SIF) cells derived from the neural crest. We morphologically characterize SIF cells and focus on their relationship with ganglionic cells, preganglionic nerve fibers and sensory nerve endings. SIF cells stained intensely for tyrosine hydroxylase (TH), with a few cells also being immunoreactive for dopamine β-hydroxylase (DBH). Vesicular acetylcholine transporter (VAChT)-immunoreactive puncta were distributed around some clusters of SIF cells, whereas some SIF cells closely abutted DBH-immunoreactive ganglionic cells. SIF cells contained bassoon-immunoreactive products beneath the cell membrane at the attachments and on opposite sites to the ganglionic cells. Ganglion neurons and SIF cells were immunoreactive to dopamine D2 receptors. Immunohistochemistry for P2X3 revealed ramified nerve endings with P2X3 immunoreactivity around SIF cells. Triple-labeling for P2X3, TH and VAChT allowed the classification of SIF cells into three types based on their innervation: (1) with only VAChT-immunoreactive puncta, (2) with only P2X3-immunoreactive nerve endings, (3) with both P2X3-immunoreactive nerve endings and VAChT-immunoreactive puncta. The results of retrograde tracing with fast blue dye indicated that most of these nerve endings originated from the petrosal ganglion. Thus, SIF cells in the superior cervical ganglion are innervated by preganglionic fibers and glossopharyngeal sensory nerve endings and can be classified into three types. SIF cells might modulate sympathetic activity in the superior cervical ganglion.     

Localization of l-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

Summary The localization of l-glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, was studied in the rat major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex by indirect immunofluorescence technique with a specific antiserum raised in rabbits. GAD immunoreactivity was demonstrated in small cells of these ganglia. The GAD-immunoreactive small cells were 10–20 m in diameter and formed clusters or occured as solitary cells. The principal neurons were non-reactive but they were surrounded by immunoreactive processes. Studies on colocalization of GAD with tyrosine hydroxylase (TH), the rate-limiting enzyme of the catecholamine synthesis, in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex indicated that all GAD-immunoreactive small cells were also labelled with TH. In the major pelvic ganglion all TH-immunoreactive SIF cells were also immunoreactive for GAD. However, in the coeliac-superior mesenteric ganglion complex there occured TH-immunoreactive small cells which showed no immunoreactivity to GAD. It is suggested that the small GAD-immunoreactive cells represent small intensely fluorescent (SIF) cells.     

Immunohistochemical and biochemical study on the development of the noradrenaline- and adrenaline-storing cells of the adrenal medulla of the rat

Summary The pre- and postnatal development of the adrenal medulla was examined in the rat by immunohistochemistry and by assay of catecholamines. Immunohistochemistry involved the use of antibodies to noradrenaline (NA), adrenaline (A) and the biosynthesizing enzymes dopamine -hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Adrenal glands were obtained from animals from the 16th day of gestation to the 7th postnatal day at daily intervals, and at the 14th postnatal day, and from adult rats. Tissues were fixed in ice-cold, 4% paraformaldehyde, buffered at pH 7.3. Cryostat sections (7 m) were stained with the indirect immunofluorescence technique. Adrenals from the same developmental stages were assayed for the presence of DA (dopamine), NA and A by ion-pair reversed-phase liquid chromatography with electrochemical detection.In adult adrenals the majority of the medullary cells (approximately 80%) were highly immunoreactive to A and moderately immunoreactive to NA. They also showed immunoreactivity to both DBH and PNMT, i.e., they are synthesizing and storing A. The remaining cell clusters were only stained by antibodies to DBH and NA (NA-synthesizing and -storing cells). These findings correlate well with the relative concentrations of A and NA as determined by assay.Three developmental phases could be distinguished. In the first phase, the 16th and 17th prenatal day, medullary cells were only immunoreactive to DBH and NA, and only very small amounts of A as compared to NA were found. During the second period, from the 18th prenatal day to 2 or 3 days after birth, all medullary cells were immunoreactive to DBH, NA, PNMT and A, and during this phase the adrenaline concentration increased daily and became the predominant amine on the 20th day of gestation. Adrenaline represented 75% of total catecholamine on the 1st to 3rd day after birth. The third phase started at the 2nd or 3rd postnatal day and was characterized by the presence of an increasing number of medullary cells solely immunoreactive to DBH and NA, hence synthesizing and storing NA. The remaining cells were immunoreactive to DBH, NA, PNMT and A. Postnatally, the relative concentration of A continued to rise reaching 79% by the 4th postnatal day. These results indicate that initially the adrenal medullary cells are synthesizing and storing almost exclusively NA. Probably, adrenaline synthesis begins at the 16th–17th day of gestation and the cells are then capable of synthesizing and storing both NA and A (mixed cell type) with A synthesis and storage rapidly becoming predominant. Finally, after birth, separate NA-synthesizing and -storing cell types are formed and the so-called A cells stored predominantly (probably >90%) adrenaline with a small proportion of noradrenaline.In the medullary blastema and in the sympathetic ganglia of prenatal animals two cell types, only immunoreactive to DBH and NA, were observed. Presumably, these cells represent developing sympathetic neurons and extra-adrenal chromaffin cells; the latter cell type occasionally invades the adrenal gland. Thus, prospective medullary cells are able to synthesize and store NA before they have made contact with the cortical blastema but A-synthesizing cells are found only within the adrenal gland.Low but significant amounts of DA were found in the adrenal before birth and during the first two postnatal weeks but in the adult animal this accounted for less than 0.1% of total catecholamine.Preliminary reports of this study were made to the American Association of Anatomists (Anat. Rec. 196; 196A, 1980), the Dutch Anatomical Society (Acta Morphol. Neerl. Scand. 19; 330, 1981, and the XIIIth Acta Endocrinologica Congress (Acta Endocrinol. 97: Suppl. 243, 285, 1981)     

Effect of hydrocortisone on catecholamines and the enzymes synthesizing them in the developing sympathetic ganglion

Summary Newborn rats were daily injected with 0.2 mg hydrocortisone acetate for seven days. They were killed 1, 7 or 21 days after the last injection, together with untreated controls. Hydrocortisone caused a great increase in the number of the small, intensely fluorescent (SIF) cells and the appearance of similar small cells with intense immunohistochemical reactions for tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine (noradrenaline)N-methyltransferase (PNMT) in the superior cervical ganglion. At the same time, the adrenaline content and the PNMT activity of the ganglion greatly increased, while no significant changes were observed in the dopamine or noradrenaline content or TH or DBH activity. All these changes essentially disappeared after a recovery period of seven or 21 days.It is concluded that hydrocortisone causes a temporary increase in the number of SIF cells by causing a synthesis of TH, DBH and PNMT in previously existing small, non-fluorescent cells, which start to synthesize and store adrenaline, thus becoming intensely fluorescent SIF cells. These SIF cells are different from the normal SIF cells of the same ganglion, most of which appear at a later stage of postnatal development when response to hydrocortisone is lost, which contain TH but neither DBH nor PNMT, and which permanently remain in the ganglion.     

Immunohistochemical localization of WE-14 in the developing porcine sympathoadrenal cell lineage

Immunohistochemical investigation of the post-translational processing of chromogranin A (CgA) to generate WE-14 in the sympathoadrenal cell lineage of the developing porcine fetus (F) detected intense CgA and weak WE-14 immunoreactivity in migrating neuroblast cells of the diffuse sympathetic ganglia adjacent to the dorsal aorta and projecting toward the cortical mass at F24-27. F37-42; WE-14 immunoreactivity was detected in chromaffinoblasts at the periphery of the developing cortex and at F54-56 days gestation WE-14 immunoreactivity was detected in a large population of central medullary cells. From F74 to F76 days and thereafter the number of cells exhibiting intense WE-14 immunostaining decreased, and the majority of chromaffin cells exhibited uniform weak WE-14 immunostaining. At postnatal day 1 (P1) intense WE-14 immunoreactivity was primarily confined to clusters of chromaffin cells with weak immunostaining in the general population. The transitory neuroblasts, chromaffinoblasts, and maturing chromaffin cell population exhibited uniform intense CgA immunostaining through gestation and after birth. Additional observations detected intense CgA and WE-14 immunostaining in extrachromaffin tissue at P1 and in neuronal-like cells in vessels of the aortic arch at F37. This study has demonstrated that CgA is post-translationally processed to generate WE-14 during early fetal development in the migrating progenitor cells of the porcine sympathoadrenal lineage.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

The coexistence of met5- and leu5-enkephalin-like immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Expression of phenylethanolamine N-methyltransferase in rat sympathetic ganglia and extra-adrenal chromaffin tissue

To determine whether similar mechanisms regulate adrenergic phenotypic expression in different cellular populations, the superior cervical sympathetic ganglion (SCG) and extra-adrenal chromaffin tissue were studied in the fetal and neonatal rat; results were compared to those previously obtained with the adrenal medulla. Phenylethanolamine N-methyltransferase (PNMT), the enzyme which converts norepinephrine to epinephrine, was used as an index of adrenergic expression. PNMT catalytic activity was initially detectable in the SCG of normal, untreated fetuses at 17.0 days of gestation (E17.0), and increased three- to fourfold until postnatal day 2. Thereafter activity decreased precipitously, and was undetectable 2 weeks after birth. Immunohistochemical studies, using specific antisera to PNMT, were employed to localize the enzyme. Immunoreactivity (PNMT-IR) was undetectable in sympathetic ganglia of control animals, suggesting that this method is less sensitive than the catalytic assay. Following glucocorticoid treatment, cells heavily stained for PNMT-IR were observed in paravertebral sympathetic ganglia, including the SCG, and in the organ of Zuckerkandl. In the SCG, PNMT-IR was present in small cells presumed to be small, intensely fluorescent (SIF) cells and was never observed in principal ganglion neurons. The increase in PNMT-IR after steroid treatment was strikingly age dependent: initiation of treatment at progressively older ages during the first week of life resulted in fewer and fewer PNMT-IR cells. No response was apparent after 1 week. Moreover, treatment of pregnant rats was associated with appearance of PNMT-IR at E18.5, but not at E16.5. After treatment from days 0 to 6 of life, PNMT-IR gradually disappeared. However, retreatment on days 24–30 caused the reappearance of PNMT-IR, suggesting that exposure to steroids at birth causes (a) an immediate increase in PNMT-IR and (b) responsiveness to steroids during adulthood. Consequently, the disappearance of PNMT-IR after exposure to steroids at birth, is not simply due to death of SIF cells. We conclude that proximity to the adrenal cortex is not necessary for initial expression of PNMT. More generally, the expression of PNMT by ganglion SIF cells parallels that in adrenal chromaffin cells since initial expression was not dependent on high local concentrations of glucocorticoids, whereas subsequent development did require high levels of the hormones. Our observations suggest that similar mechanisms regulate expression and development of the adrenergic phenotype in adrenal and sympathetic ganglia.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

Summary The coexistence of met5- and leu5-enkephalinlike immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Atrial natriuretic polypeptide-like immunoreactivity in the rat pituitary: light and electron microscopic studies

Immunoreactive atrial natriuretic polypeptide (ANP) was investigated in the pituitary of rats by light and electron microscopy using the indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunoreactivity was present in 30-35% of anterior pituitary cells. These cells have two types of secretory granules being characteristic of rat gonadotrophin-storing granules, and were usually adjacent to the capillary endothelium. The results of this study suggest the co-occurrence of ANP and gonadotrophins in the anterior pituitary cells.     

The distribution of cGMP in the adrenal gland in the rat, guinea pig and mouse after stimulation with sodium nitroprusside

Nitric oxide (NO) acts as an intercellular messenger molecule in the nervous system. In the adrenal gland sympathetic preganglionic fibers innervating the medulla, as well as intrinsic neural ganglion cells, contain nitric oxide synthase (NOS). Nitric oxide stimulates the soluble enzyme guanylate cyclase forming cyclic GMP (cGMP). Using sodium nitroprusside (SNP) as nitric oxide donor we have studied the putative target cells for nitric oxide in the rat adrenal gland, both in vivo and in vitro. The guinea pig and a few mouse adrenal glands were studied after SNP perfusion for comparison. Our results show that after vascular perfusion with a high concentration (3 mM) of SNP both noradrenaline and adrenaline chromaffin cells express cGMP-like immunoreactivity in all three species. After incubation of rat adrenal slices with SNP primarily the noradrenaline chromaffin cells are cGMP-positive. In contrast, detectable levels of cGMP-like immunoreactivity were not found in neuronal ganglion cells. In the adrenal cortex cGMP-like immunoreactivity was seen in blood vessel walls, in small cells with processes forming a reticular network, at least partly presumably representing endothelial cells, as well as in some presumable nerve terminals. These findings support the view that chromaffin cells, especially the noradrenergic ones and blood vessels, are targets for nitric oxide in the adrenal gland.