Presence of Kisspeptin-like Immunoreactivity in Human Adrenal Glands and Adrenal Tumors

Kisspeptins are neuropeptides which activate the hypothalamo-pituitary gonadal axis and are considered to play important physiological roles in the reproduction. Kisspeptins have also been reported to stimulate the aldosterone secretion from the adrenal cortex. However, the expression of kisspeptins in human adrenal glands and adrenal tumors has not been clarified yet. We, therefore, studied the presence of kisspeptin-like immunoreactivity (LI) in human adrenal glands and adrenal tumors (adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas) by radioimmunoassay and immunocytochemistry. Kisspeptin-LI was detected in all the tissues examined; normal portions of adrenal glands (3.0?±?2.3 pmol/g wet weight, n?=?21, mean ± SD), aldosterone-producing adenomas (4.6?±?3.3 pmol/g wet weight, n?=?10), cortisol-producing adenomas (2.7?±?1.4 pmol/g wet weight, n?=?14), adrenocortical carcinomas (1.7?±?0.2 pmol/g wet weight, n?=?4), and pheochromocytomas (1.8?±?0.8 pmol/g wet weight, n?=?6). There was no significant difference in kisspeptin-LI levels among them. Immunocytochemistry showed positive kisspeptin-immunostaining in normal adrenal glands, with stronger immunostaining found in the medulla. Furthermore, positive kisspeptin-immunostaining was found in all types of adrenal tumors examined; adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas. The intensity of kisspeptin-immunostaining in these adrenal tumors was, however, not so strong as that in normal adrenal medulla. The present study has shown for the first time the presence of kisspeptin-LI in adrenal glands and adrenal tumors.     

Substance P in the adrenal gland: Origin and species distribution

Substance P-like immunoreactivity (I-SP) was found in adrenal glands of cat, rabbit, guinea-pig, rat, cattle and man, as well as in a pheochromocytoma. I-SP was reduced in chronically denervated adrenal glands of rats and in adrenal glands of guinea-pigs pretreated with capsaicin, suggesting innervation of the adrenal glands by I-SP containing nerves, possibly of sensory origin. I-SP could partially be recovered from the chromaffin granule fraction of bovine adrenal medulla and a pheochromocytoma tumor. HPLC analysis of bovine chromaffin granules and a pheochromocytoma extract showed that the measured immunoreactivity corresponded to authentic substance P. Therefore, a dual localization of I-SP in nerve fibers and chromaffin cells can be assumed. The presence of somatostatin-like immunoreactivity in human adrenal medullae and in a pheochromocytoma is also reported. No neurotensin could be detected in the pheochromocytoma.     

Reduction of endogenous TGF-beta increases proliferation of developing adrenal chromaffin cells in vivo

Chromaffin cells and sympathetic neurons are derivatives of the sympathoadrenal cell lineage of the neural crest. Although they are similar with respect to many cell biological aspects, chromaffin cells, in contrast to sympathetic neurons, continue to synthesize DNA and proliferate through their whole life span. Large numbers of neural and hormonal signals have been implicated in the regulation of chromaffin cell proliferation based on in vitro studies. We have previously shown that chromaffin cells synthesize and release transforming growth factor-beta (TGF-beta) and that exogenously applied TGF-beta suppresses chromaffin cell proliferation in vitro. We show now that TGF-beta is also a physiologically relevant factor in the control of cell division in developing chromaffin cells. We have neutralized endogenous TGF-beta in quail embryos using a monoclonal antibody recognizing all three TGF-beta isoforms, TGF-beta1, -beta2, and -beta3. Embryos deprived of TGF-beta show a prominent increase in numbers of tyrosine hydroxylase (TH)-immunoreactive adrenal chromaffin cells and TH-positive cells incorporating 5'-bromo-2'deoxyuridine. This is the first documentation of the physiological significance of a factor that has been suggested to play a role in the regulation of chromaffin cell mitosis based on in vitro experiments.     

Transient axonal glycoprotein-1 (TAG-1) and laminin-α1 regulate dynamic growth cone behaviors and initial axon direction in vivo

Background

Adrenal chromaffin cells and sympathetic neurons both originate from the neural crest, yet signals that trigger chromaffin development remain elusive. Bone morphogenetic proteins (BMPs) emanating from the dorsal aorta are important signals for the induction of a sympathoadrenal catecholaminergic cell fate.

Results

We report here that BMP-4 is also expressed by adrenal cortical cells throughout chick embryonic development, suggesting a putative role in chromaffin cell development. Moreover, bone morphogenetic protein receptor IA is expressed by both cortical and chromaffin cells. Inhibiting BMP-4 with noggin prevents the increase in the number of tyrosine hydroxylase positive cells in adrenal explants without affecting cell proliferation. Hence, adrenal BMP-4 is likely to induce tyrosine hydroxylase in sympathoadrenal progenitors. To investigate whether persistent BMP-4 exposure is able to induce chromaffin traits in sympathetic ganglia, we locally grafted BMP-4 overexpressing cells next to sympathetic ganglia. Embryonic day 8 chick sympathetic ganglia, in addition to principal neurons, contain about 25% chromaffin-like cells. Ectopic BMP-4 did not increase this proportion, yet numbers and sizes of 'chromaffin' granules were significantly increased.

Conclusion

BMP-4 may serve to promote specific chromaffin traits, but is not sufficient to convert sympathetic neurons into a chromaffin phenotype.     

Actions of somatostatin on perfused bovine adrenal glands and cultured bovine adrenal medullary cells

The effects of somatostatin on catecholamine secretion and inositol phosphate accumulation have been studied using isolated perfused bovine adrenal glands and cultured bovine adrenal medullary cells. Somatostatin had no effect on basal adrenaline or noradrenaline secretion from either preparation. At concentrations above 1 microM, somatostatin inhibited the secretion of both catecholamines induced by 5 microM nicotine from cultured chromaffin cells. In contrast, over the concentration range 0.1 nM-10 microM, somatostatin had no effect on the secretory responses produced by 10 nM angiotensin II or 1 microM histamine. Inositol phosphate accumulation in cultured bovine adrenal medullary cells was unaffected by 0.1 nM-0.1 microM somatostatin, however at 1 and 10 microM somatostatin it was significantly increased, by 23% and 103% respectively. The effects of somatostatin (0.1 nM-10 microM) and of 50 microM muscarine on inositol phosphate accumulation were simply additive. Similarly, somatostatin at 0.1 nM and 10 nM together with 10 nM angiotensin II or 1 microM histamine produced additive inositol phosphate responses. In contrast, 1 microM somatostatin gave significantly more-than-additive (synergistic) inositol phosphate responses with angiotensin II and histamine. The results suggest that some adrenal medullary cells possess several types of receptors, and that these receptors may interact to produce non-additive responses.     

The nigrostriatal dopaminergic system in MPTP-treated mice shows more prominent recovery by syngeneic adrenal medullary graft than by allogeneic or xenogeneic graft

Following systemic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), young (2-month-old) C57BL/6 mice show decreased dopaminergic (DA) nigrostriatal fibers and DA concentration in the striatum. We transplanted syngeneic, allogeneic and xenogeneic adrenal medullary grafts into the striatum of the MPTP-treated young mice and compared the survivability of grafted chromaffin cells and the recovery of intrinsic host DA fibers using computerized image analysis of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers and high performance liquid chromatography with electrochemical detection (LCEC). The grafted syngeneic adrenal chromaffin cells survived better than allogeneic or xenogeneic chromaffin cells, and host DA nigrostriatal fiber recovery was more prominent in mice with a syngeneic graft than in mice with an allogeneic or xenogeneic graft. However, the degree of host fiber recovery in mice with allogeneic or xenogeneic mice was greater than in mice with a sham operation alone, even though the allografts and xenografts had no surviving chromaffin cells. Allografts and xenografts showed prominent rejection responses, with T lymphocyte infiltration in addition to macrophages. We conclude that a syngeneic adrenal graft survives better than an adrenal allograft or xenograft and promotes recovery of the intrinsic host nigrostriatal DA fibers. We also conclude that grafted chromaffin cell survivability influences the degree of host DA fiber recovery following MPTP depletion. Adrenal medullary grafts to Parkinsonian patients are currently under way in a large number of hospitals; we suggest that greater attention be paid to methods which lead to enhanced survival of the grafted chromaffin cells, since survivability might be closely related to the functional recovery of these patients.     

Age-related changes in the localization of P2X (nucleotide) receptors in the rat adrenal gland

Observation of the changes in the occurrence and distribution of nucleotide (P2X) receptors in the adrenal gland during development and ageing, and correlation with the changes in adrenal status at similar stages may give morphological insights into the functions of purine nucleotides in the gland. Age-related changes in the localization of all seven subtypes of the P2X receptor in the adrenal gland of rat were therefore investigated immunohistochemically. In the adrenal glands of prenatal rats, immunoreactivity to P2X receptor subtypes was not observed. In glands of the postnatal rat at the developmental stages studied, only immunoreactivity for the P2X(5) receptor subtype was observed. A small number of faintly P2X(5)-immunoreactive chromaffin cells were found in the adrenal glands of 1-day-old rats; the frequency of localization and intensity of staining of immunoreactive cells had increased by day 4 and was further increased at day 7. P2X(5) immunoreactivity was not observed in the adrenal glands from 14- and 21-day-old rats. At 8 weeks of age, immunoreactivity with a specific distribution for each of the seven receptor subtypes was observed. Except for the P2X(4) receptor, adrenal glands at 24 months showed a similar pattern of immunoreactivity for the receptor subtypes as that observed at 8 weeks. Immunoreactivity for P2X(4) was first observed in the adrenal cortical cells of the zona reticularis at 8 weeks, but was absent in 24-month-old rats. However, several P2X(4)-immunoreactive chromaffin cells appeared at 24 months. Such immunoreactive cells were not seen in rats of any of the other ages studied. It was concluded that the greater expression of P2X(5) receptor at an early developmental stage and of P2X(4) in ageing might reflect functional roles for purines in cellular proliferation and/or differentiation, and in cellular degeneration, respectively, in adrenal glands of rat.     

Adrenal medullary graft induces recovery of the host nigrostriatal dopaminergic system in MPTP-induced mouse model of Parkinson's disease

C57BL/6 mice show decreased dopaminergic fibers and dopamine concentration in the striatum following systemic injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). We have investigated the effect of adrenal medullary grafts into the striatum of young mice treated with MPTP. Enhanced recovery of the host nigrostriatal dopaminergic system was observed in those adrenal medullary grafted mice. However, this recovery was influenced by the survivability of grafted chromaffin cells and adrenal chromaffin cells from younger donors survived better than those from older donors. Since adrenal chromaffin cells contain several kinds of neurotrophic factors such as basic fibroblast growth factor and gangliosides, survivability of those grafted chromaffin cells may play an important role concerning recovery of the host intrinsic dopaminergic fibers. Adrenal medullary grafts to the patients with Parkinson's disease are currently under way in a large number of hospitals and we suggest more consideration be given to methods which lead to enhance the grafted chromaffin cell survival, since those survivability might be closely related to the functional recovery of these patients.     

Chromaffin cell proliferation in the adult rat adrenal medulla

Epinephrine and norepinephrine-containing chromaffin cells proliferate in the adrenal glands of normal adult rats throughout life. Moreover, their rate of proliferation is markedly increased by short-term administration of reserpine, one of many agents which in long-term experiments are associated with the development of adrenal medullary tumors. Current data suggest that chromaffin cell proliferation in the adult rat adrenal is mediated by the interaction of neurogenic and hormonal signals. Reserpine is known to directly deplete catecholamine stores, and to reflexively increase the activity of the splanchnic nerve endings innervating the adrenal medulla to stimulate both secretion and synthesis of catecholamines and other secretory granule constituents. Its effect on chromaffin cell proliferation suggests that the same signals may regulate chromaffin cell number to meet physiological needs. The reserpine model might shed light on signal transduction mechanisms which normally promote or prevent proliferation of chromaffin cells and of other neuroendocrine cells during development or in adult life, and on ways in which such mechanisms are altered in the course of the development and progression of tumors. It also suggests the possibility that chromaffin cells might be propagated in vitro for use in basic biological studies or in transplants for the treatment of Parkinson's disease.     

Herpesviruses in brains in Alzheimer's and Parkinson's diseases

We evaluated the association of HSV-1, HHV-6, and VZV with Alzheimer's disease (AD) and Parkinson's disease (PD). Brain specimens for viral DNA polymerase chain reaction represented 34 patients with AD, 40 with PD, and 40 controls. One AD patient (2.9%) was positive for HSV-1 DNA, 88.2% for HHV-6 DNA, and 26.5% for VZV DNA; 17.5% of PD patients were HSV-1 DNA-positive and 75% HHV-6-positive, whereas 40% had VZV DNA. Twenty-five percent of the controls were positive for HSV-1 DNA, 87.5% for HHV-6, and 27.5% for VZV. HSV-1, VZV, or HHV-6 DNA in brains was no additional risk factor for AD.     

Autoradiographic Localization of Insulin-Like Growth Factor I Binding Sites in the Bovine Adrenal Gland and on Cultured Bovine Adrenal Chromaffin Cells

Previous studies have reported the presence of binding sites for insulin-like growth factor I (IGF-I) in membranes prepared from isolated bovine adrenal medullary cells, and IGF-I was found to regulate the secretory function of bovine chromaffin cells. In the present study, binding sites for IGF-I have been localized in sections of bovine adrenal gland and on cultured bovine adrenal medullary cells, using [¹²⁵ l][Thr⁵⁹]-IGF-l as the ligand in conjunction with qualitative autoradiographic techniques. Binding sites were present throughout the adrenal gland and were distributed evenly over all cortical cell layers and over both adrenaline and noradrenaline cell types in the medulla. They were also present at lower density over blood vessels and nerve bundles and over the capsule. The binding of radioligand was to a single class of sites with K_d 0.61 nM, and was completely displaced by excess unlabelled [Thr⁵⁹]-IGF-l and by insulin (Actrapid, K_d 1.04μM). Binding sites were also identified on single cells in primary monolayer cultures of bovine adrenal medullary cells. More than 96% of the cells possessed binding sites, although only 85% of such cells were chromaffin cells, as previously determined from dopamine β-hydroxylase immunohistochemical staining. The results suggest that IGF-I may affect the maturation, growth or function not only of adrenal chromaffin cells but also of many others cell types in this tissue.     

Search for varicella zoster virus and herpes simplex virus-1 in normal human cerebral arteries

Virological confirmation of varicella zoster virus (VZV) vasculopathy is provided by presence of virus in the cerebral arteries, frequently associated with inflammation. Yet, cerebral arteries from normal subjects have never been studied for VZV DNA or antigen. We analyzed 63 human cerebral arteries from 45 subjects for VZV DNA and antigen, control herpes simplex virus (HSV)-1 DNA and antigen, and leukocyte-specific CD45 antigen. No cerebral arteries contained VZV or HSV-1 DNA or antigen; eight arteries from seven subjects contained leukocytes expressing CD45. Thus, the presence of VZV antigen in cerebral arteries of patients with stroke is likely to be clinically significant.     

Dopamine release from bovine adrenal medullary cells in culture.

The present study tested whether release of dopamine from isolated bovine adrenal medullary cells in culture could be stimulated or inhibited by secretagogues and modulators known to affect noradrenaline and adrenaline release from adrenal medullary chromaffin cells. K+ depolarization or activation of voltage-sensitive Na+ channels by veratridine both stimulated dopamine release. Ca2+-dependent dopamine release was also stimulated by the mixed nicotinic-muscarinic agonist, carbachol. Carbachol-induced dopamine release was inhibited by a nicotinic but not by a muscarinic antagonist and dopamine release was also stimulated by a selective nicotinic agonist, 1,1-dimethyl-4-phenyl-piperazinium. Carbachol-induced dopamine release was inhibited by substance P and by neuropeptide Y. Histamine also stimulated dopamine release, while angiotensin II and glutamate produced no significant stimulation of dopamine release. Noradrenaline and adrenaline were released in response to the above agents with a profile almost identical to that of dopamine. The results indicate that dopamine can be directly released from adrenal medullary cells in response to stimulation of those cells and suggest that the dopamine release originates from chromaffin cells similar or identical to those storing noradrenaline and adrenaline. A possible role for dopamine, released from adrenal chromaffin cells, in modulating catecholamine release from the chromaffin cells and/or contributing to circulating plasma dopamine is discussed.     

Cografts of adrenal medulla with peripheral nerve enhance the survivability of transplanted adrenal chromaffin cells and recovery of the host nigrostriatal dopaminergic system in MPTP-treated young adult mice

Schwann cells from transected peripheral nerve segments are known to produce nerve growth factor (NGF). We performed adrenal medullary grafts or cografts of adrenal medulla and sciatic nerve into the striatum of MPTP-treated young adult mice, and compared the survivability of grafted chromaffin cells and the recovery of intrinsic host DA fibers using computerized image analysis of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers and neurochemical analysis with high performance liquid chromatography (HPLC). Adrenal medullary chromaffin cells cografted with sciatic nerve survived better than those in adrenal grafts alone; host DA fiber recovery was more prominent in mice with cografts than in mice with adrenal grafts alone. A large number of TH-IR surviving cells in cografted mice showed long neuronal processes which were rarely seen in the mice receiving adrenal graft alone. We conclude that cograft of adrenal medulla and sciatic nerve promotes intrinsic host DA fiber recovery better than adrenal medulla grafts alone, and that survivability of grafted chromaffin cell may promote host DA fiber recovery. Adrenal medullary autografts have been used in patients with Parkinson's disease; we suggest that if this approach is to be used in the future, methods to increase the survivability of grafted chromaffin cells, such as co-grafting with pieces of peripheral nerve, be considered to enhance the survivability of the chromaffin cells, which might be closely related to the functional recovery of the patients by this grafting procedure. Of course, such strategies as the present cografting approach must be demonstrated to work in older animals using older donor tissue before proceeding to this next step in humans.     

Pharmacologic consequences of the vascular permeability of chromaffin cell transplants in CNS pain modulatory regions

The transplantation of peripheral neural tissue into the CNS has been shown to alter blood-brain barrier (BBB) permeability to intravascularly injected proteins such as horseradish peroxidase. The pharmacological consequences of such BBB alterations following the transplantation of adrenal medullary tissue, isolated bovine chromaffin cell suspensions, or PC12 cell suspensions into the pain modulatory regions of the periaqueductal gray (PAG) or subarachnoid space of the lumbar spinal cord were studied using agents that normally do or do not readily pass the BBB. The injection of nicotine in animals with adrenal medullary or chromaffin cell transplants produces potent analgesia, most likely due to the stimulated release of opioid peptides and catecholamines from the transplanted cells. This analgesia could be blocked by nicotinic antagonist mecamylamine, which normally passes the BBB, but not by nicotinic antagonist hexamethonium, which normally does not readily pass the BBB. Furthermore, quaternary nicotinic agonists tetramethylammonium and 1,1-dimethyl-phenyl-piperazinium had no effect on pain sensitivity in animals with adrenal medullary implants. The Met-enkephalin peptide analog, D-Ala-Met-enkephalinamide, which normally does not alter pain sensitivity when injected systemically due to limited penetration to the CNS, produced analgesia in animals with adrenal medullary, bovine chromaffin cell, and PC12 cell implants in the PAG, but not in control gelfoam-implanted animals. This analgesia, as well as analgesia induced by nicotine, was completely blocked by naloxone pretreatment, but not by naloxone methobromide, a quaternary derivative of naloxone that does not normally pass the BBB.(ABSTRACT TRUNCATED AT 250 WORDS)     

The generation of monoclonal antibodies that bind preferentially to adrenal chromaffin cells and the cells of embryonic sympathetic ganglia.

Adrenal chromaffin cells, sympathetic neurons, and small intensely fluorescent (SIF) cells are each derived from the neural crest, produce catecholamines, and share certain morphological features. These cell types are also partially interconvertible in cell culture (Doupe et al., 1985a,b; Anderson and Axel, 1986). Thus, these cells are said to be members of the sympathoadrenal (SA) lineage and could share a common progenitor. To investigate the origins of this lineage further, we used the cyclophosphamide immuno-suppression method (Matthew and Patterson, 1983) to generate five monoclonal antibodies (SA1-5) that bind strongly to chromaffin cells, with little or no labeling of sympathetic neurons or SIF cells in frozen sections from adult rats. Competition experiments indicate that these antibodies bind to at least three distinct epitopes in tissue sections. The SA antibodies also label most of the cells of embryonic sympathetic ganglia and adrenal primordia. Labeling of sympathetic ganglia appears as the cells initially coalesce and express high levels of tyrosine hydroxylase (TH). Not all TH+ cells in the embryo are SA 1-5+, however; carotid body SIF cells, nodose ganglion TH+ cells, and the transiently TH+ cells in the dorsal root ganglia do not display detectable SA 1-5 labeling. Thus, the expression of these markers for the SA 1-5 lineage is selective. SA antigen expression is hormonally controlled; removal of glucocorticoid and addition of NGF to cultured adrenal chromaffin cells result in the loss of SA 1-5 labeling. These results suggest that the presumed precursors for sympathetic neurons and SIF cells initially express chromaffin cell markers.     

Alphaherpesvirus DNA replication in dissociated human trigeminal ganglia

Analysis of the frequency and PCR-quantifiable abundance of herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) DNA in multiple biological replicates of cells from dissociated randomly distributed human trigeminal ganglia (TG) of four subjects revealed an increase in both parameters and in both viruses during 5 days of culture, with no further change by 10 days. Dissociated TG provides a platform to analyze initiation of latent virus DNA replication within 5 days of culture.     

Organization, fine structure, and viability of the human adrenal medulla: considerations for neural transplantation

Recent reports of adrenal medullary autografts in patients with Parkinson's disease raise several important questions with respect to the cell types actually being transplanted as well as the potential for chromaffin cell banking prior to neural transplantation. In this study, we determined the general morphological characteristics of the human adrenal medulla and assessed factors important for the maintenance of cultured chromaffin cells for later use as transplants. The human adrenal medulla contained islands of cortical cells scattered throughout the gland as well as Schwann cells, nerve endings, endothelial cells, pericytes, isolated ganglionic neurons, and connective tissue elements such as fibroblasts and smooth muscle cells. Because many of these cell types are mitotically active, transplantation of medullary fragments that contain these cells could have far-reaching consequences. One approach that could circumvent the problems arising from multiple cell types in the medulla is differential plating of chromaffin cells prior to transplantation. Differential plating yielded relatively pure populations of chromaffin cells that demonstrated excellent viability if processed within 2 hours after cessation of the gland's circulation. Chromaffin cells cultured in the presence of nerve growth factor exhibited a neuronal phenotype, possessed catecholamine histofluorescence, and displayed tyrosine hydroxylase- and dopamine beta-hydroxylase-like immunoreactivity. The sex and age of the donor did not affect cell viability or morphological characteristics.     

Environmental influences in the development of neural crest derivatives: glucocorticoids, growth factors, and chromaffin cell plasticity

The neural crest gives rise to three major adrenergic cell types: sympathetic principal neurons, adrenal chromaffin cells, and small intensely fluorescent (SIF) cells. All of these derivatives synthesize and store catecholamines, but they differ in numerous other characteristics. SIF cells appear intermediate in phenotype between the other two. We have examined the role of several environmental factors in the differentiation of sympathetic principal neurons and adrenal chromaffin cells. In previous studies of young rat adrenal chromaffin cells in dissociated cell culture, differentiated characteristics such as the presence of the enzyme phenylethanolamine N-methyltransferase (PNMT), epinephrine (E) synthesis, and large catecholamine storage vesicles were not well maintained. Here we describe long-term culture of chromaffin cells which, in the presence of micromolar glucocorticoid, maintained all of these characteristics. In addition, chromaffin cells of a variety of ages were found to be dependent on glucocorticoid for long-term survival in culture. In the absence of glucocorticoid, many adrenal chromaffin cells from neonatal rats could be rescued by nerve growth factor (NGF) administration. They extended neurites, as previously described by Unsicker and colleagues (Unsicker, K., B. Krisch, U. Otten, and H. Thoenen (1978) Proc. Natl. Acad. Sci. U.S.A. 75: 3498-3502). In contrast to previous studies, however, with long-term exposure to NGF these cells became indistinguishable from mature sympathetic neurons, as judged by the following morphological and biochemical criteria: increased cell size and loss of intense CA fluorescence in their cell bodies; acquisition of characteristic neuronal ultrastructure, including morphologically specialized synapses; loss of chromaffin granules, PNMT, and E synthesis; and acquisition of neuron markers, including tetanus toxin labeling and immunoreactivity to neurofilament protein. This conversion to neurons was markedly enhanced by addition of a non-neuronal cell conditioned medium (CM) containing a neurite-promoting factor, which acted by increasing the NGF responsiveness of the chromaffin cells. Even chromaffin cells from adult rats, which are known to grow few processes in response to NGF alone, became neuronal in the presence of this CM plus NGF. While converting to neurons, adrenal chromaffin cells transiently assumed an intermediate phenotype resembling type I SIF cells, which suggests particular developmental relationships between the different cell types of the sympathoadrenal lineage.(ABSTRACT TRUNCATED AT 400 WORDS)