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Nearly 90% of premature infants experience the stress of intermittent hypoxia (IH) as a consequence of recurrent apneas (periodic cessation of breathing). In neonates, catecholamine secretion from the adrenal medulla is critical for maintaining homeostasis under hypoxic stress. We recently reported that IH treatment enhanced hypoxia-evoked catecholamine secretion and [Ca2+]iresponses in neonatal rat adrenal chromaffin cells and involves reactive oxygen species (ROS). The purpose of the present study was to identify the source(s) of ROS generation and examine the mechanisms underlying the enhanced catecholamine secretion by IH. Neonatal rats of either sex (postal day 0–5) were exposed to either IH or normoxia. IH treatment increased NADPH oxidase (NOX) activity, upregulated NOX2 and NOX4 transcription in adrenal medullae, and a NOX inhibitor prevented the effects of IH on hypoxia-evoked chromaffin cell secretion. IH upregulated Cav3.1 and Cav3.2 T-type Ca2+channel mRNAs via NOX/ROS signaling and augmented T-type Ca2+current in IH-treated chromaffin cells. Mibefradil, a blocker of T-type Ca2+channels attenuated the effects of hypoxia on [Ca2+]iand catecholamine secretion in IH-treated cells. In Ca2+-free medium, IH-treated cells exhibited higher basal [Ca2+]ilevels and more pronounced [Ca2+]iresponses to hypoxia compared with controls, and blockade of ryanodine receptors (RyRs) prevented these effects. RyR2 and RyR3 mRNAs were upregulated, RyR2 was S-glutathionylated in IH-treated adrenal medullae, and NOX/ROS inhibitors prevented these effects. These results demonstrate that neonatal IH treatment leads to NOX/ROS-dependent recruitment of T-type Ca2+channels and RyRs, resulting in augmented [Ca2+]imobilization and catecholamine secretion.

Calcium-activator protein for secretion (CAPS) is a cytosolic protein that associates with large dense-core vesicles and is involved in their secretion. Mammals express two CAPS isoforms, which share a similar domain structure including a Munc13 homology domain that is believed to be involved in the priming of secretory vesicles. A variety of studies designed to perturb CAPS function indicate that CAPS is involved in the secretion of large dense-core vesicles, but where in the secretory pathway CAPS acts is still under debate. Mice in which one allele of theCAPS-1gene is deleted exhibit a deficit in catecholamine secretion from chromaffin cells. We have examined catecholamine secretion from chromaffin cells in which bothCAPSgenes were deleted and show that the deletion of both CAPS isoforms causes a strong reduction in the pool of rapidly releasable chromaffin granules and of sustained release during ongoing stimulation. We conclude that CAPS is required for the adequate refilling and/or maintenance of a rapidly releasable granule pool.

An emerging area of research has documented a novel role for the plasminogen activation system in the regulation of neurotransmitter release. Prohormones, secreted by cells within the sympathoadrenal system, are processed by plasmin to bioactive peptides that feed back to inhibit secretagogue-stimulated release. Catecholaminergic cells of the sympathoadrenal system are prototypic prohormone-secreting cells. Processing of prohormones by plasmin is enhanced in the presence of catecholaminergic cells, and the enhancement requires binding of plasmin(ogen) to cellular receptors. Consequently, modulation of the local cellular fibrinolytic system of catecholaminergic cells results in substantial changes in catecholamine release. However, mechanisms for enhancing prohormone processing and cell-surface molecules mediating the enhancement on catecholaminergic cells have not been investigated. Here we show that plasminogen activation was enhanced >6.5-fold on catecholaminergic cells. Carboxypeptidase B treatment decreased cell-dependent plasminogen activation by ∼90%, suggesting that the binding of plasminogen to proteins exposing C-terminal lysines on the cell surface is required to promote plasminogen activation. We identified catecholaminergic plasminogen receptors required for enhancing plasminogen activation, using a novel strategy combining targeted specific proteolysis using carboxypeptidase B with a proteomics approach using two-dimensional gel electrophoresis, radioligand blotting, and tandem mass spectrometry. Two major plasminogen-binding proteins that exposed C-terminal lysines on the cell surface contained amino acid sequences corresponding to β/γ-actin. An anti-actin monoclonal antibody inhibited cell-dependent plasminogen activation and also enhanced nicotine-dependent catecholamine release. Our results suggest that cell-surface-expressed forms of actin bind plasminogen, thereby promoting plasminogen activation and increased prohormone processing leading to inhibition of neurotransmitter release.

Identification of the molecules that regulate the priming of synaptic vesicles for fusion and the structural coupling of the calcium sensor with the solubleN-ethyl maleimide sensitive factor adaptor protein receptor (SNARE)-based fusion machinery is critical for understanding the mechanisms underlying calcium-dependent neurosecretion. Snapin binds to synaptosomal-associated protein 25 kDa (SNAP-25) and enhances the association of the SNARE complex with synaptotagmin. In the present study, we abolishedsnapinexpression in mice and functionally evaluated the role of Snapin in neuroexocytosis. We found that the association of synaptotagmin-1 with SNAP-25 in brain homogenates ofsnapinmutant mice is impaired. Consequently, the absence of Snapin in embryonic chromaffin cells leads to a significant reduction of calcium-dependent exocytosis resulting from a decreased number of vesicles in releasable pools. Overexpression of Snapin fully rescued this inhibitory effect in the mutant cells. Furthermore, Snapin is relatively enriched in the purified large dense-core vesicles of chromaffin cells and associated with synaptotagmin-1. Thus, our biochemical and electrophysiological studies usingsnapinknock-out mice demonstrate that Snapin plays a critical role in modulating neurosecretion by stabilizing the release-ready vesicles.

During exocytosis, certain phospholipids may act as regulators of secretion. Here, we used several independent approaches to perturb the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] level in bovine chromaffin cells to investigate how changes of plasmalemmal PI(4,5)P2affect secretion. Membrane levels of PI(4,5)P2were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5)P2. The specific PI(4,5)P2signal was enriched in submicrometer-sized clusters. In parallel patch-clamp experiments on intact cells, we measured the secretion of catecholamines. Overexpression of phosphatidylinositol-4-phosphate-5-kinase Iγ, or infusion of PI(4,5)P2through the patch pipette, increased the PI(4,5)P2level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5)P2from the membrane and abolished secretion. An inhibitor of phosphatidylinositol-3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, led to a transient increase in the PI(4,5)P2level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5)P2decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5)P2levels led to correlated changes in the size of two releasable vesicle pools, whereas their fusion kinetics remained unaffected. We conclude that during both short- and long-term manipulations of PI(4,5)P2level secretion scales with plasma membrane PI(4,5)P2content and that PI(4,5)P2has an early effect on secretion by regulating the number of vesicles ready for release.

We examined the role of SNAPs, soluble proteins that attachN-ethylmaleimide-sensitive factor (NSF), in regulating exocytosis in single rat adrenal chromaffin cells. Whole-cell dialysis of Ca2+-buffered solution or photolysis of caged-Ca2+was used to manipulate cytosolic Ca2+concentration ([Ca2+]i), whereas exocytosis was measured via carbon fiber amperometry or membrane capacitance. Buffering [Ca2+]ito ∼170 nmproduced a mean rate of exocytosis of approximately one amperometric event per minute. Including α-SNAP (60 or 500 nm) in the intracellular solution dramatically increased the mean rate of exocytosis. The stimulatory action of α-SNAP requires ATP hydrolysis mediated via NSF, because this action was blocked by intracellular dialysis of ATP-γ-S (2 mm) and could not be mimicked by a mutant α-SNAP that does not stimulate the ATPase activity of NSF. This action of α-SNAP was significant only at [Ca2+]ibetween 100 and 300 nmand was not shared by β-SNAP (500 nm), suggesting that α-SNAP enhanced a component of exocytosis that is regulated by a high-affinity Ca2+sensor. In cells dialyzed with both α- and β-SNAP, the rate of exocytosis was smaller than that produced by α-SNAP alone, suggesting that α- and β-SNAP interact competitively. Although only α-SNAP stimulated exocytosis at [Ca2+]ibetween 100 and 300 nm, both α- and β-SNAP isoforms equally slowed the time-dependent rundown of the exocytic response. Our results indicate that α- and β-SNAP have different actions in exocytosis. Thus, the ratio of different isoforms of SNAPs can determine release probability at the levels of [Ca2+]ithat are involved in regulation of exocytosis.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease that affects neurotransmitter release at peripheral synapses. LEMS antibodies inhibit Ca2+currents in excitable cells, but it is not known whether there are additional effects on stimulus-secretion coupling. The effect of LEMS antibodies on Ca2+currents and exocytosis was studied in bovine adrenal chromaffin cells using whole-cell voltage clamp in perforated-patch recordings. Purified LEMS IgGs from five patients inhibited N- and P/Q-type Ca2+current components to different extents. The reduction in Ca2+current resulted in smaller exocytotic responses to single depolarizing pulses, but the normal relationship between integrated Ca2+entry and exocytosis (Engisch and Nowycky, 1996) was preserved. The hallmark of LEMS is a large potentiation of neuromuscular transmission after high-frequency stimulation. In chromaffin cells, stimulus trains can induce activity-dependent enhancement of the Ca2+–exocytosis relationship. Enhancement during trains occurs most frequently when pulses are brief and evoke very small amounts of Ca2+entry (Engisch et al., 1997). LEMS antibody treatment increased the percentage of trains eliciting enhancement through two mechanisms: (1) by reducing Ca2+entry and (2) through a Ca2+-independent effect on the process of enhancement. This leads to a paradoxical increase in the amount of exocytosis during stimulus trains, despite inhibition of Ca2+currents.

Adrenomedullary chromaffin cells express at least two subtypes of acetylcholine nicotinic receptors, which differ in their sensitivity to the snake toxin α-bungarotoxin. One subtype is involved in the activation step of the catecholamine secretion process and is not blocked by the toxin. The other is α-bungarotoxin-sensitive, and its functional role has not yet been defined. The α7 subunit is a component of this subtype. Autoradiography of bovine adrenal gland slices with α-bungarotoxin indicates that these receptors are restricted to medullary areas adjacent to the adrenal cortex and colocalize with the enzyme phenylethanolamine N-methyl transferase (PNMT), which confers the adrenergic phenotype to chromaffin cells. Transcripts corresponding to the α7 subunit also are localized exclusively to adrenergic cells. To identify possible transcriptional regulatory elements of the α7 subunit gene involved in the restricted expression of nicotinic receptors, we isolated and characterized its 5′ flanking region, revealing putative binding sites for the immediate early gene transcription factor Egr-1, which is known to activate PNMT expression. In reporter gene transfection experiments, Egr-1 increased α7 promoter activity by up to sevenfold. Activation was abolished when the most promoter-proximal of the Egr-1 sites was mutated, whereas modification of a close upstream site produced a partial decrease of the Egr-1 response. Because Egr-1 was found to be expressed exclusively in adrenergic cells, we suggest that this transcription factor may be part of a common mechanism involved in the induction of the adrenergic phenotype and the differential expression of α-bungarotoxin-sensitive nicotinic receptors in the adrenal gland., This work was supported by Grants from the Ministries of Education (Direccion General de Investigacion Cientifica yTecnica:PB92-0346,PB95-0690, and PB930931) and Health (Fis95/1672) of Spain,the Commission of the European Economic Community (SC1*CT91-0666),Generalitat Valenciana(GV-D-VS-20-15896), and the WellcomeTrust(039284).E.D. del T. was the recipient of a predoctoral fellowship from the Ministry of Education of Spain.F.I.S.was a postdoctoral fellow of theWellcomeTrust.C.C.-S. wasthe recipient of a fellowship from Generalitat Valenciana.

Submembrane [Ca2+]ichanges were examined in rat chromaffin cells by monitoring the activity of an endogenous Ca2+-dependent protein: the large conductance Ca2+- and voltage-activated K+channel (also known as the BK channel). The Ca2+and voltage dependence of BK current inactivation and conductance were calibrated first by using defined [Ca2+]isalines. This information was used to examine submembrane [Ca2+]ielevations arising out of Ca2+influx and muscarine-mediated release of Ca2+from intracellular stores. During Ca2+influx, some BK channels are exposed to [Ca2+]iof at least 60 μm. However, the distribution of this [Ca2+]ielevation is highly nonuniform so that the average [Ca2+]idetected when all BK channels are activated is only ∼10 μm. Intracellular dialysis with 1 mmor higher EGTA spares only the BK channels activated by the highest [Ca2+]iduring influx, whereas dialysis with 1 mmor higher BAPTA blocks activation of all BK channels. Submembrane [Ca2+]ielevations fall rapidly after termination of short (5 msec) Ca2+influx steps but persist above 1 μmfor several hundred milliseconds after termination of long (200 msec) influx steps. In contrast to influx, the submembrane [Ca2+]ielevations produced by release of intracellular Ca2+by muscarinic actetylcholine receptor (mAChR) activation are much more uniform and reach peak levels of 3–5 μm. Our results suggest that during normal action potential activity only 10–20% of BK channels in each chromaffin cell see sufficient [Ca2+]ito be activated.

Previous investigations have demonstrated that adrenal chromaffin cells survive poorly when grafted into the striatum of rodents, nonhuman primates, and patients with Parkinson's disease. This poor survival has been attributed to the low levels of endogenous NGF within the striatum. However, chromaffin cells isolated from the nonchromaffin constituents of the adrenal medulla (fibroblasts and endothelial cells) have recently been demonstrated to survive grafting into a number of CNS sites. The present study determined whether nonchromaffin constituents of the adrenal medulla may be responsible for poor graft survival. We compared the survival of intrastriatally grafted isolated bovine chromaffin cells with that observed following implantation of either perfused adrenal medullary suspensions containing all adrenal medullary cell types or isolated chromaffin cells that were then reseeded with autologous fibroblasts and endothelial cells. Implants of perfused adrenal medullary cells survived poorly and most graft sites were infiltrated with macrophages. The chromaffin cells in this group that did survive appeared to be in the process of degeneration. In contrast, large numbers of isolated chromaffin cells survived for up to 2 months following transplantation. These cells maintained their endocrine phenotype and stained for all enzymatic markers of catecholamine synthesis as well as chromogranin A. Morphologically, these cells resembled chromaffin cells seen in situ and the perigraft region was essentially devoid of macrophages. When isolated chromaffin cells were reseeded with autologous fibroblasts and endothelial cells, the implants degenerated and few, if any, surviving chromaffin cells were observed. Interestingly, these latter grafts induced a host- derived sprouting response of tyrosine hydroxylase-immunoreactive fibers. These data demonstrate that large numbers of adrenal chromaffin cells can survive intrastriatal implantation in the absence of exposure to exogenous NGF. Rather, the nonchromaffin cells of the adrenal medulla (fibroblasts and endothelial cells) appear to compromise the viability of grafted chromaffin cells. Once they are eliminated from the graft, robust survival of chromaffin cells occurs. If clinical trials employing adrenal medullary grafts are still to be considered for the treatment of Parkinson's disease, isolation of the chromaffin cells should be considered to enhance graft viability.

Adrenal medullary chromaffin cells are a potential source of neuroactive substances for transplantation into the CNS to alleviate neurochemical deficits. In particular, work in our laboratory has suggested that adrenal medullary transplants in the spinal subarachnoid space can alleviate pain by providing sustained local delivery of catecholamines and opioid peptides. One of the major limitations for clinical application of neural transplantation is the availability of donor material in sufficient quantities. This limitation may be overcome by the use of xenogeneic donors if long-term graft rejection can be prevented. The purpose of this study was to assess whether xenogeneic chromaffin cells immunologically isolated by semipermeable membranes could survive and continue to reduce pain when transplanted into the CNS. Isolated bovine chromaffin cells were encapsulated by semipermeable polymer membranes and implanted into the rat spinal subarachnoid space. Pain sensitivity was assessed at several intervals up to 3 months following implantation. Results indicated that encapsulated bovine chromaffin cell implants, but not empty control capsules, could repeatedly reduce pain sensitivity with nicotine stimulation for the duration of the study. This response was dose related, indicating that pharmacologic integrity of the transplanted chromaffin cells is retained. The analgesia induced by encapsulated chromaffin cell implants could be attenuated by the opiate antagonist naloxone and the alpha-adrenergic antagonist phentolamine, suggesting the involvement of both opioid peptides and catecholamines in mediating this response. In addition, in vitro neurochemical studies of recultured capsules revealed sustained release of Met-enkephalin and catecholamines from encapsulated cells 3 months following implantation into the spinal subarachnoid space.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult rat chromaffin cells in vitro show a large proliferative response to NGF, followed by neuronal differentiation. Infection of replicating chromaffin cells with a retrovirus carrying the Escherichia coli beta- galactosidase (beta-gal) gene demonstrates beta-gal expression in cells that continue to multiply, that differentiate into neurons, and that become static. The effects of NGF on proliferation and differentiation are abolished by the protein kinase inhibitors K252a and staurosporine, and by cholera toxin, an activator of adenylate cyclase. They are diminished, but not abolished, by high concentrations of dexamethasone. Both cholera toxin alone and phorbol myristate acetate (PMA), an activator of protein kinase C, elicit small and inconsistent mitogenic responses. The responses to PMA cannot be shown to be additive with the effects of NGF. NGF is a known mitogen and neuritogen for chromaffin cells from neonatal rats, but has not previously been believed to affect similarly chromaffin cells from adults. The present findings suggest that portions of NGF signaling pathways might continue to be involved in regulating proliferation of adult rat chromaffin cells in vivo, and might be constitutively activated in PC12 cells and other adrenal medullary tumors. They further suggest that rat chromaffin cells might be propagated in vitro to obtain large numbers of sympathetic neurons expressing normal or exogenous genes.

Most catecholaminergic cells derived from the sympathoadrenal lineage of the neural crest contain one or more neuropeptides. Although a great deal is known about the development and regulation of catecholaminergic properties in these cells, relatively little is known about the developmental control of their neuropeptidergic properties. We have investigated the possible role of glucocorticoids and preganglionic innervation in the regulation of leucine-enkephalin (L-Enk) expression in cultures of embryonic and neonatal adrenal chromaffin cells and in mature chromaffin cells in vivo. Exposure of embryonic and neonatal chromaffin cells to the synthetic glucocorticoid dexamethasone increases L-Enk content. Neonatal chromaffin cells grown in medium containing elevated levels of potassium to mimic depolarization also exhibited increased L-Enk levels. The depolarization-induced increase in L-Enk was selectively inhibited by treatment with the enkephalin analog [D-Ala, d-Leu]-enkephalin to mimic the enkephalinergic component of the preganglionic innervation. Denervation of the adrenal gland in vivo resulted in a dramatic increase in L-enk expression that could be partially mimicked by selectively blocking enkephalinergic transmission with administration of the opiate receptor antagonist naloxone. Taken together with the developmental time course and pattern of L-Enk expression in vivo, our results suggest that glucocorticoids and the preganglionic innervation regulate the developmental expression of this peptide in adrenal chromaffin cells and therefore participate in the generation of the mature neurochemical phenotypes present in the adrenal medulla. Further, in adult chromaffin cells similar factors appear to regulate the expression of L-Enk, which could in turn participate in physiological responses to stress.

The rat gene encoding phenylethanolamine N-methyltransferase (PNMT) was cloned and a consensus sequence for a glucocorticoid response element (GRE) was found at -513 bp, 5′ to the transcriptional start site. In order to define the function of this element, fusion genes containing the PNMT promoter and a chloramphenicol acetyltransferase (CAT) reporter gene were constructed. These constructs did not express after transfection into any of 7 continuous cell lines, none of which endogenously produce PNMT. A system for transfecting chromaffin cells in primary culture was therefore devised using constructs containing 200 bp of the proenkephalin (ENK) promoter, whose expression characteristics are well known. pENK beta GAL-1, containing the ENK promoter with a lac Z reporter, was introduced into these cells and beta-galactosidase activity was visualized in situ. Approximately 90% of cells transfected were chromaffin; transfection efficiency was 5%. High levels of CAT activity were measured in chromaffin cells transfected with pENKAT12, possessing a CAT reporter. In contrast to tumor cell lines, pENKAT12 induction in these cells by forskolin and phorbol esters did not require a phosphodiesterase inhibitor. In this chromaffin system, both basal and regulated expression of the PNMT fusion genes were detected. Dexamethasone (dex) induced expression of pPNMT3000 and pPNMT900, containing the putative GRE and 3000 bp or 863 bp of PNMT promoter sequence, 4- to 10-fold. Expression of pPNMT300 and pPNMT100, which lack the GRE and contain 273 bp or 99 bp of PNMT promoter sequence, was unaffected by dex. Addition of the PNMT region spanning -490 to -863 bp conferred full dex responsiveness to a thymidine kinase promoter. Deletion of the putative GRE sequence by site-directed mutagenesis abolished the dex response. These data identify the sequence at -513 bp in the rat PNMT gene as a functional, positively acting GRE. Primary cultures of bovine chromaffin cells provide a biologically relevant expression system for transcriptional studies of catecholamine genes and their related neuropeptides.

Neurotransmitters are released from nerve terminals and neuroendocrine cells by calcium-dependent exocytosis of vesicles. Before fusion, vesicles are docked to the plasma membrane and rendered release competent through a process called priming. Electrophysiological methods such as membrane capacitance measurements and carbon fiber amperometry accurately measure the fusion step of exocytosis with high time resolution but provide only indirect information about priming and docking. Total internal reflection fluorescence microscopy (TIRFM) enables the real-time visualization of vesicles, near the plasma membrane, as they undergo changes from one molecular state to the other. We devised a new method to analyze the mobility of vesicles, which not only allowed us to classify the movement of vesicles in three different categories but also to monitor dynamic changes in the mobility of vesicles over time. We selectively enhanced priming by treating bovine chromaffin cells with phorbol myristate acetate (PMA) or by overexpressing Munc13-1 (mammalian Unc) and analyzed the mobility of large dense-core vesicles. We demonstrate that nearly immobile vesicles represent primed vesicles because the pool of vesicles displaying this type of mobility was significantly increased after PMA treatment and Munc13-1 overexpression and decreased during tetanus toxin expression. Moreover, we showed that the movement of docked but unprimed vesicles is restricted to a confined region of ∼220 nm diameter. Finally, a small third population of undocked vesicles showed a directed and probably active type of mobility. For the first time, we can thus distinguish the molecular state of vesicles in TIRFM by their mobility.

Transient fusion (“kiss-and-run”) is accepted as a mode of transmitter release both in central neurons and neuroendocrine cells, but the prevalence of this mechanism compared with full fusion is still in doubt. Using a novel double patch-clamp method (whole cell/cell attached), permitting the recording of unitary capacitance events while stimulating under a variety of conditions including action potentials, we show that transient fusion is the predominant (>90%) mode of secretion in calf adrenal chromaffin cells. Raising intracellular Ca2+concentration ([Ca]i) from 10 to 200 μmincreases the incidence of full fusion events at the expense of transient fusion. Blocking rapid endocytosis that normally terminates transient fusion events also promotes full fusion events. Thus, [Ca]icontrols the transition between transient and full fusion, each of which is coupled to different modes of endocytosis.

α-Latrotoxin (α-Ltx), a component of black widow spider venom, stimulates secretion from nerve terminals and from PC12 cells. In this study we examine the effects of expression of a newly cloned Ca2+-independent receptor for α-Ltx (CIRL) on secretion from bovine chromaffin cells. We first characterized the effect of α-Ltx on secretion from untransfected cells. α-Ltx, by binding in a Ca2+-independentmanner to an endogenous receptor, causes subsequent Ca2+-dependent secretion from intact cells. The stimulation of secretion is correlated with Ca2+influx caused by the toxin. In permeabilized cells in which the Ca2+concentration is regulated by buffer, α-Ltx also enhances Ca2+-dependent secretion, indicating a direct role of the endogenous receptor in the secretory pathway. Expression of CIRL increased the sensitivity of intact and permeabilized cells to the effects of α-Ltx, demonstrating that this protein is functional in coupling to secretion. Importantly, in the absence of α-Ltx, the expression of CIRL specifically inhibited the ATP-dependent component of secretion in permeabilized cells without affecting the ATP-independent secretion. This suggests that this receptor modulates the normal function of the regulated secretory pathway and that α-Ltx may act by reversing the inhibitory effects of the receptor.

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.

Primary cultures of bovine adrenal medullary chromaffin cells were used to study the regulation of opioid peptide (OP) synthesis. Chromaffin cells continuously exposed to tetrabenazine, a drug that depletes cellular catecholamine stores, increase their OP contents between 32 hr and 6 days of treatment. At no time following tetrabenazine addition were increases in opiate receptor-inactive enkephalin-containing peptides (IECPs) observed. Because IECPs may serve as precursors to OPs, these results suggest increased processing of OP precursors following treatment with catecholamine-depleting drugs in addition to an increased rate of OP precursor synthesis. The increases in cellular OP levels induced by tetrabenazine were approximately proportional to the depletion in cellular catecholamines produced by this drug. Also, the effects of tetrabenazine on chromaffin cell OP and IECP contents were mimicked by inhibitors of catecholamine biosynthesis and other agents that decreased catecholamine stores, but not by supplementing the culture medium with catecholamines or catecholamine receptor agonists. Addition of 8-bromo-cAMP or forskolin, an activator of adenylate cyclase, to chromaffin cell cultures increased both OP and IECP stores. Inhibitors of cyclic nucleotide phosphodiesterase also increase chromaffin cell OP and IECP contents, although it is unclear whether these increases result from increased cyclic nucleotide levels. Hence, both alterations in some intracellular catecholamine pool and elevations of cAMP levels may trigger increases in the synthesis and processing of OPs and IECPs in the adrenal medullary chromaffin cell.

Primary cultures of bovine adrenal chromaffin cells provide large quantities of a homogeneous population of target cells for nerve growth factor (NGF) and, thus, are a suitable system for studying the molecular mechanism of action of NGF. In this study, we have shown that NGF mediates the specific induction of the key enzymes in catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). Acetylcholinesterase (AChE), an enzyme which catalyzes the breakdown of acetylcholine, is also induced by NGF. We have compared NGF-mediated TH and AChE induction and have provided pharmacological evidence that TH induction involves a post-transcriptional, polyadenylation-dependent event (blockable by 9-beta-arabinofuranosyladenine but not by alpha- amanitin), whereas AChE induction requires transcription (blockable by alpha-amanitin). DBH and PNMT appear to be regulated via the same mechanism as TH. The time course of TH induction is such that NGF must be continuously present for at least the first 36 hr (during which time TH levels remain unchanged), and then the entire increase takes place during the subsequent 12 hr. In contrast, AChE induction proceeds linearly with time of NGF exposure. These data suggest that there may be multiple mechanisms by which NGF regulates enzyme induction. We have also compared the effects of cAMP with those of NGF. As compared to NGF, cAMP produces a different pattern of enzyme induction (in addition to TH, DBH, PNMT, and AChE, dopa decarboxylase (DDC) is also induced), it acts rapidly (a 12-hr exposure produces the full effect), and it acts only at the transcriptional level (its effects are blocked by alpha-amanitin). These data provide evidence that cAMP does not act as a second messenger for NGF with regard to enzyme induction.

Glucocorticoids are known to regulate the enzyme phenylethanolamine-N-methyltransferase (PNMT) in the adrenal medulla of the rat and are thereby thought to control the synthesis of epinephrine. We have examined the details of this relationship in a simplified system, chromaffin cell primary cultures derived from bovine adrenal medulla. Cultured chromaffin cells were found to have a cytosolic, high affinity, saturable glucocorticoid-binding protein with the steroid specificity of a classical glucocorticoid receptor and a Kd of approximately 1 nM. Treatment of cultured cells with dexamethasone or hydrocortisone at any time up to 21 days in culture increased PNMT activity in the soluble fraction of the cell. The concentration of hormone required to produce a half-maximal response was 10 nM dexamethasone when cells were cultured in the presence of 5% fetal calf serum, or 1 nM in a defined serum-free medium. These dose-response relationships are consistent with mediation of this effect by the glucocorticoid receptor. Unexpectedly, however, the glucocorticoid-induced increment in PNMT activity was not inhibited by cycloheximide at concentrations up to 50 microM, and an acceleration of protein synthesis by insulin treatment did not augment the glucocorticoid effect on PNMT. Treatment of the cells with dexamethasone (100 microM) prevented the decline in the epinephrine-to-norepinephrine ratio seen over time in culture, an effect consistent with increased PNMT activity. However, there was no effect of dexamethasone on the ability of the cells to secrete catecholamines in response to stimulation with high KCl or 30 microM nicotine.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Bovine adrenal medullary chromaffin cells cultured in serum-free medium were examined for changes in their catecholamine and opioid peptide stores following exposure to dexamethasone, ascorbic acid, or insulin for 2 to 12 days. Dexamethasone failed to alter cellular catecholamine levels, measured by high performance liquid chromatography with electrochemical detection, or cellular opioid peptide content, measured by an enkephalin radioreceptor assay. Chromaffin cells cultured in medium supplemented with ascorbic acid retained high ascorbate contents for 2 to 3 days, despite the rapid loss of this vitamin from the culture medium (approximately 50% lost in 2 hr). The epinephrine and norepinephrine contents of chromaffin cell cultures supplemented with ascorbate for 7 days were increased approximately 10% compared to cultures without added ascorbic acid; ascorbate had no effect on chromaffin cell opioid peptide levels. Addition of insulin to chromaffin cell cultures produced a doubling of cellular protein and opioid peptide levels by 6 days and produced a concentration-dependent increase in the dopamine and norepinephrine contents of the cells with only a slight elevation in cell epinephrine. Chromaffin cells were also tested for the ability to resynthesize their catecholamine and opioid peptide stores following depletion as the result of secretion evoked by acetylcholine or nicotine. The cellular stores of norepinephrine and dopamine were resynthesized within 3 days following secretagogue-evoked depletion. Epinephrine stores were incompletely replenished with only 20% of the epinephrine lost via secretion recovered in 7 days. Opioid peptide levels were maximally recovered (50% of the amount secreted) within 1 day. Addition of ascorbic acid, dexamethasone, or insulin to the medium failed to enhance the recovery of catecholamine stores.

A nicotinic acetylcholine receptor (AChR) on bovine adrenal chromaffin cells in culture has previously been identified using the alpha- neurotoxin n-Bgt and the monoclonal antibody mAb 35. Here, we report that the cells have 2 classes of high-affinity binding sites for 3H- nicotine, one being associated with the AChR and the other being associated with the alpha-bungarotoxin binding component that is distinct from the AChR. Scatchard analysis of 3H-nicotine binding to the AChR site yields a KD of 20 +/- 3 nM and a Bmax of 104 +/- 12 fmol/mg protein. Nicotinic antagonists block 3H-nicotine binding to the AChR site with the same rank order of potency and affinity with which they block nicotine-induced catecholamine release from the cells. About 80% of the AChRs are on the cell surface, as judged by the distribution of both 3H-nicotine binding and 125I-mAb 35 binding to the receptor, and the ratio of nicotine/mAb 35 binding to the AChR on the cell surface is approximately 1:1. Chronic treatment of the cells with mAb 35 results in receptor modulation such that all of AChR-related nicotine binding is lost from the cell surface, and all of the functional response to nicotine is lost as well. The results confirm that 3H-nicotine binding is associated with AChRs on the cells. The 3H- nicotine binding observed to the AChR represents binding to a desensitized form of the receptor having increased affinity for agonists and unchanged affinity for antagonists. This conclusion derives from the following observations. The KiS for agonist competition of 3H-nicotine binding indicate agonist affinities several orders of magnitude greater than do the KDS measured for receptor activation. Exposing cultures to low concentrations of nicotine and substance P causes receptor densensitization, and the concentration dependence of the nicotine-induced desensitization displays an EC50 of 20 nM, in good agreement with the KD obtained from equilibrium binding studies with 3H-nicotine. In addition, the rate of 3H-nicotine binding is increased both by substance P, which enhances the rate of agonist- induced desensitization on adrenal chromaffin cells, and by preincubation with nicotine. The increased rate of association, together with the dissociation rate, yields a kinetically derived KD of 19 nM, again in good agreement with the KD obtained from equilibrium binding studies. These results demonstrate that the bovine adrenal chromaffin AChR is similar to AChRs from muscle and electric organ in undergoing an agonist-induced conversion to a desensitized state having increased affinity for agonists.

Two probes previously shown to distinguish the nicotinic ACh receptor of chick ciliary ganglion neurons also recognize a component on the surface of bovine chromaffin cells in culture that displays the properties expected for the chromaffin nicotinic ACh receptor. The first probe is a monoclonal antibody, mAb 35, raised against ACh receptor from Electrophorus electric organ, and the second is an alpha- neurotoxin, Bgt 3.1, purified from B. multicinctus venom. mAb 35 binds specifically to a single class of high-affinity sites on the chromaffin cells in culture. Scatchard analysis indicates a KD of 2.1 +/- 0.2 nM for the binding and a Bmax of 1.6 +/- 0.1 X 10(4) mAb 35 sites per cell. The number of sites on the cells can be reduced through modulation by exposure of the cells to Bgt 3.1. The modulation can be blocked by the cholinergic ligands d-tubocurarine and carbamylcholine. Long-term exposure to the agonist carbamylcholine alone also reduces the number of mAb 35 binding sites. Bgt 3.1 inhibits nicotine-induced catecholamine release from the cells to the same extent and with the same concentration dependence that it modulates the number of mAb 35 sites on the cells. In addition, mAb 35 treatment of the cells causes a specific and almost complete blockade of nicotine-induced catecholamine release, apparently through a modulation of the receptor. These results indicate that the bovine chromaffin component recognized by mAb 35 and Bgt 3.1 is likely to be the nicotinic ACh receptor on the cells and that it has many similarities to ACh receptors on chick autonomic neurons.