Isolated Chromaffin Cells Research Articles

European Neuroscience Association Pre‐ and post‐synaptic muscarinic receptors in thin slices of rat adrenal gland

AbstractThe effects of activation of muscarinic receptors on chromaffin cells and splanchnic nerve terminals were studied in a rat adrenal slice preparation. In chromaffin cells, muscarine induced a transient hyperpolarization followed by a depolarization associated with cell spiking. The hyperpolarization was blocked by charybdotoxin (1 μm) and tetraethylammonium chloride (TEA, 1 mm), but was not affected by 200 μm Cd2+ or removal of external Ca2+, consistent with activation of BK channels. This would follow internal Ca2+ mobilization, as shown by Ca2+ imaging with fura‐2 on isolated chromaffin cells in culture. Under voltage‐clamp, outward BK currents were insensitive to MT3 toxin, a specific muscarinic m4 receptor antagonist. In contrast, muscarine‐induced depolarization was due to a m4 receptor‐mediated inward current blocked by MT3 toxin. This current was permeable to cations and was associated with Ca2+ entry and subsequently, Ca2+‐induced Ca2+ release. Finally, both muscarine (25 μm) and oxotremorine (10 μm) decreased the amplitude and frequency of KCl‐evoked excitatory postsynaptic currents, without affecting quantal size, consistent with a presynaptic inhibitory effect. Taken together, our data suggest that activation of m4 and probably m3 muscarinic receptors results in a strong, long‐lasting excitation of chromaffin cells, as well as an uncoupling of synaptic inputs onto these cells.

Uptake of [ 3H]dopamine in isolated chromaffin cells of the mouse: modulation by intra- and extra-adrenal peptides and other secretagogues

The effects of intra- and extra-adrenal peptides on [ 3H]dopamine uptake in adrenal chromaffin cells of the mouse were examined in vitro. Dopamine uptake was inhibited by acetylcholine, high potassium, reserpine, imipramine and desmethylimipramine as was in noradrenaline uptake. Among the intra-adrenal peptides, vasoactive intestinal peptide (VIP, 100 pmol/l) and neurotensin inhibited [ 3H]dopamine uptake by approximately 25%. Somatostatin, enkephalin, and neuropeptide Y did not cause any significant inhibition. An extra-adrenal peptide, bradykinin, inhibited the uptake while angiotensin II showed no significant effect. Intra-adrenal peptides which cause catecholamine secretion inhibit catecholamine uptake probably to extend its effect. Extra-adrenal peptide which causes catecholamine secretion also inhibits catecholamine uptake.

Relief of intractable cancer pain by human chromaffin cell transplants: Experience at two medical centers

In addition to its possible role as a replacement source in CNS degenerative diseases, neural tral)splantation may be used to augment the normal production of neuroactive substances. Our laboratory at the University of Illinois at Chicago has shown, in both acute and chronic pain models, that transplantation of adrenal medullary tissue or isolated chromaffin cells into CNS pain modulatory regions can reduce pain sensitivity in rodents. Chromaffin cells were chosen as the donor source since they produce high levels ofboth opioid peptides and catecholamines, substances which reduce pain sensitivity when injected locally intq the spinal subarachnoid space. The analgesia produced by these transplants probably results from the release of both opioid peptides and catecholamines since it can be blocked or attenuated by both opiate and adrenergic antagonists. Studies indicate that even over long periods there is no apparent development of tolerance. Promising results have been obtained in preliminary clinical studies using allografts of adrenal medulla to relieve cancer pain. This clinical review encompasses results at two Medical Centers-University of Illinois at Chicago and University Paul Sabatier, Toulouse, France- in assessing efficacy of subarachnoid adrenal medullary transplantation for alleviating cancer pain. Our clinical and autopsy data strongly support our previous laboratory studies, i.e., that chromaffin cell transplants into the subarachnoid space represent a promising new approach to the alleviation of chronic pain. It is suggested that further clinical studies are now warranted. [Neural Res 1997; 19: 71-77]

Calcium signalling in isolated single chromaffin cells of the rainbow trout (Oncorhynchus mykiss)

Chromaffin cells were isolated from the posterior cardinal vein of rainbow trout (Oncorhynchus mykiss) to assess their suitability as a model system for studying mechanisms of catecholamine secretion in fish and to evaluate intracellular calcium changes associated with cholinoreceptor stimulation. Immunocytochemistry in concert with fluorescence microscopy was employed to identify characteristic chromaffin cell proteins and thus to confirm the presence of these specific cells in suspensions and cultures. Dopamine-β-hydroxylase, an enzyme of the catecholamine-synthesising Blaschko pathway, was identified in cytoplasmic vesicles of the isolated chromaffin cells. The actin filament-severing protein, scinderin, was co-localized with actin in the sub-plasmalemmal membrane of these chromaffin cells. Intracellular calcium [Ca2+]i was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca2+]i were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K+ or by the stimulation of the cell by the cholinergic receptor agonists, nicotine, acetylcholine or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexamethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible cholinergic agonist, carbachol. These results demonstrate the presence of functional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these cells as a model system for studying aspects of catecholamine secretion in fish.

Bovine chromaffin cells for CNS transplantation do not elicit xenogeneic T cell proliferative responses in vitro

Adrenal chromaffin cells have been utilized for several neural grafting applications, but limitations in allogeneic donor availability and dangers inherent in autografting limit the widespread use of this approach clinically. While xenogeneic donors offer promise as a source for cell transplantation in the central nervous system (CNS), immunologic responses to cellular components of the adrenal medulla have not been well characterized. To further study the host T cell xenogeneic response to chromaffin and passenger cells of the adrenal medulla, an in vitro lymphocyte proliferation assay was used. Lymphocyte proliferation was determined by mixing rat lymphocytes with potential stimulator cell subpopulations of the bovine adrenal medulla: isolated chromaffin cells, isolated endothelial cells, or passenger nonchromaffin cells, which include a mixture of fibroblasts, smooth muscle cells, and endothelial cells. As a positive control, bovine aortic endothelial cells were also used. 3[H]-thymidine incorporation, corresponding to lymphocyte proliferation, was measured. Results indicated that isolated bovine chromaffin cells produce only a mild, statistically insignificant stimulation of rat lymphocytes. In contrast, there was a significant response to passenger nonchromaffin cells of the adrenal medulla, especially endothelial cells. The inclusion of low levels of cyclosporin A in the cultures completely eliminated the mild proliferative response to isolated bovine chromaffin cells, while near toxic levels were necessary to abrogate the response to endothelial cells. Immunocytochemical analysis revealed that routine chromaffin cell isolation procedures result in the inclusion of a small percentage of endothelial cells, which may be responsible for the slight lymphocyte stimulation. The results of this study indicate that isolated chromaffin cells possess low immunogenicity, and suggest that passenger cells in the adrenal medulla, particularly endothelial cells, may be primarily responsible for progressive rejection in CNS grafts. Thus, removal of passenger nonchromaffin cells from xenogeneic donor tissues prior to transplantation may produce a more tolerated graft in rodent models of neural transplantation.

Bovine chromaffin cells for CNS transplantation do not elicit xenogeneic T cell proliferative responses in vitro.

Adrenal chromaffin cells have been utilized for several neural grafting applications, but limitations in allogeneic donor availability and dangers inherent in autografting limit the widespread use of this approach clinically. While xenogeneic donors offer promise as a source for cell transplantation in the central nervous system (CNS), immunologic responses to cellular components of the adrenal medulla have not been well characterized. To further study the host T cell xenogeneic response to chromaffin and passenger cells of the adrenal medulla, an in vitro lymphocyte proliferation assay was used. Lymphocyte proliferation was determined by mixing rat lymphocytes with potential stimulator cell subpopulations of the bovine adrenal medulla: isolated chromaffin cells, isolated endothelial cells, or passenger nonchromaffin cells, which include a mixture of fibroblasts, smooth muscle cells, and endothelial cells. As a positive control, bovine aortic endothelial cells were also used. 3[H]-thymidine incorporation, corresponding to lymphocyte proliferation, was measured. Results indicated that isolated bovine chromaffin cells produce only a mild, statistically insignificant stimulation of rat lymphocytes. In contrast, there was a significant response to passenger nonchromaffin cells of the adrenal medulla, especially endothelial cells. The inclusion of low levels of cyclosporin A in the cultures completely eliminated the mild proliferative response to isolated bovine chromaffin cells, while near toxic levels were necessary to abrogate the response to endothelial cells. Immunocytochemical analysis revealed that routine chromaffin cell isolation procedures result in the inclusion of a small percentage of endothelial cells, which may be responsible for the slight lymphocyte stimulation. The results of this study indicate that isolated chromaffin cells possess low immunogenicity, and suggest that passenger cells in the adrenal medulla, particularly endothelial cells, may be primarily responsible for progressive rejection in CNS grafts. Thus, removal of passenger nonchromaffin cells from xenogeneic donor tissues prior to transplantation may produce a more tolerated graft in rodent models of neural transplantation.

ADP exerts a protective effect against rundown of the Ca2+ current in bovine chromaffin cells.

In isolated chromaffin cells, the high-voltage-activated Ca2+ current, recorded using 5 mM Ca2+ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5'-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5'-triphosphate (ITP) could not replace ATP, whereas guanosine 5'-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl2 and the liberation of a phosphate group since the ATP analogue 5'-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5'-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg2+ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5'-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca2+ concentration (pCa 6 instead of pCa 7.7, where pCa = -log10[Ca2+]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca2+ channels when going to the inside-out patch recording configuration.(ABSTRACT TRUNCATED AT 250 WORDS)

Natriuretic peptides inhibit nicotine-induced whole-cell currents and catecholamine secretion in bovine chromaffin cells: evidence for the involvement of the atrial natriuretic factor R2 receptors.

There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C-type natriuretic peptide (CNP) and found that these cells specifically express the ANF-R1C but not the ANF-R1A receptor subtype. Evidence for the existence of ANF-R2 receptors was obtained from patch-clamp experiments where C-ANF, an ANF-R2-specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF-R2 receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide-bridged structure of C-ANF. This linearized form of C-ANF also displayed a lower binding affinity for ANF-R2 receptors. Like the patch-clamp studies, secretion experiments demonstrated that both CNP and C-ANF are equally effective in reducing nicotine-evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that the effect of CNP is predominantly mediated by the ANF-R2 and not the ANF-R1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine- nor KCl-induced secretions were affected by natriuretic peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term viability of isolated bovine adrenal medullary chromaffin cells following intrastriatal transplantation

Adrenal medullary grafts generally exhibit poor viability when grafted into the striatum. Previous work in our laboratory demonstrated that chromaffin cells can survive well for up to 2 mo following grafting into the intact rat striatum after cells are isolated from the nonchromaffin supporting cells (fibroblasts and endothelial cells) of the adrenal medulla. The aim of the present study was to assess the long-term viability of isolated bovine chromaffin cells following grafting into the intact rat striatum. The viability of grafted bovine adrenal medullary chromaffin cells was compared in rats receiving either (a) perfused adrenal medulla; (b) isolated chromaffin cells; or (c) isolated chromaffin cells that were subsequently recombined with their nonchromaffin supporting cells. One year postimplantation, all graft types which included fibroblasts and endothelial cells were infiltrated with macrophages and demonstrated an abundance of cellular debris. No viable chromaffin cells were observed. In contrast, healthy tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DβH) immunoreactive chromaffin cells survived for 1 yr posttransplantation when grafted in isolation from the nonchromaffin constituents of the adrenal medulla. Good xenograft survival was achieved in this group despite the fact that these rats were only immunosuppressed for 1 mo postimplantation. Grafted cells demonstrated morphological characteristics of chromaffin cells in situ and these implants were not accompanied by macrophage infiltration. These data demonstrate that long-term survival of chromaffin cells can be achieved following intra-striatal implantation and the viability of grafted chromaffin cells is dependent upon the removal of the nonchromaffin supporting cells.

Long-term viability of isolated bovine adrenal medullary chromaffin cells following intrastriatal transplantation.

Adrenal medullary grafts generally exhibit poor viability when grafted into the striatum. Previous work in our laboratory demonstrated that chromaffin cells can survive well for up to 2 mo following grafting into the intact rat striatum after cells are isolated from the nonchromaffin supporting cells (fibroblasts and endothelial cells) of the adrenal medulla. The aim of the present study was to assess the long-term viability of isolated bovine chromaffin cells following grafting into the intact rat striatum. The viability of grafted bovine adrenal medullary chromaffin cells was compared in rats receiving either (a) perfused adrenal medulla; (b) isolated chromaffin cells; or (c) isolated chromaffin cells that were subsequently recombined with their nonchromaffin supporting cells. One year postimplantation, all graft types which included fibroblasts and endothelial cells were infiltrated with macrophages and demonstrated an abundance of cellular debris. No viable chromaffin cells were observed. In contrast, healthy tyrosine hydroxylase (TH) and dopamine beta hydroxylase (D beta H) immunoreactive chromaffin cells survived for 1 yr posttransplantation when grafted in isolation from the nonchromaffin constituents of the adrenal medulla. Good xenograft survival was achieved in this group despite the fact that these rats were only immunosuppressed for 1 mo postimplantation. Grafted cells demonstrated morphological characteristics of chromaffin cells in situ and these implants were not accompanied by macrophage infiltration. These data demonstrate that long-term survival of chromaffin cells can be achieved following intrastriatal implantation and the viability of grafted chromaffin cells is dependent upon the removal of the nonchromaffin supporting cells.

Processing of tetanus and botulinum A neurotoxins in isolated chromaffin cells.

Tetanus and botulinum A neurotoxins were introduced into the cytosol of chromaffin cells by means of an electric field in which the plasma membrane is forced to form pores of approximately 1 micron at the sites facing the electrodes. As demonstrated by electron microscopy, both [125I] and gold-labelled tetanus toxin (TeTx) diffuse through these transient openings. Dichain-TeTx, with its light chain linked to the heavy chain by means of a disulfide bond, causes the block of exocytosis to develop more slowly than does the purified light chain. The disulfide bonds, which in both toxins hold the subunits together, were cleaved by the intrinsic thioredoxin-reductase system. Single chain TeTx, in which the heavy and light chains are interconnected by an additional peptide bond, was far less effective than dichain-TeTx at blocking exocytosis, which indicates that proteolysis is the rate-limiting step. The toxins were degraded further to low-molecular weight fragments which, together with intact toxins and subunits, were released by the cells. The intracellular half-life of [125I] dichain-TeTx was approximately three days. The number of light-chain molecules required to maintain exocytosis block in a single cell, as calculated by two different methods, was less than 10. The long duration of tetanus poisoning may result from the persistence of intracellular toxin due to scarcity of free cytosolic proteases. This may also hold for the slow recovery from botulism.

Synaptic transmission from splanchnic nerves to the adrenal medulla of guinea‐pigs.

1. Membrane potentials were recorded with conventional intracellular microelectrodes from chromaffin cells in isolated, bisected adrenal glands from guinea-pigs. 2. All cells were electrically excitable and responded to depolarizing current with all-or-nothing action potentials that were blocked by tetrodotoxin. 3. Input resistance was 180 +/- 14 M omega and this was lower than that reported for isolated chromaffin cells using patch electrodes. 4. All cells responded to transmural stimulation with action potentials that arose from excitatory synaptic potentials in response to the excitation of one or more preganglionic fibres, many having strong synaptic action. Other fibres had weaker synaptic action but in all cases, maximal transmural stimulation caused depolarization well above threshold for action potential initiation. 5. Spontaneous excitatory synaptic potentials were observed whose frequency was greatly increased by repetitive stimulation at 10 or 30 Hz. 6. No evidence was found for the desensitization of nicotinic receptors in response to acetylcholine released from presynaptic nerve terminals. 7. These experiments show that there are many similarities between the responses to splanchnic nerve stimulation of guinea-pig chromaffin cells in situ and sympathetic ganglion cells from the same species.

Robust survival of isolated bovine adrenal chromaffin cells following intrastriatal transplantation: a novel hypothesis of adrenal graft viability

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.

Ionic components of the electrical response of chromaffin cells from the toad ( Caudiverbera caudiverbera) adrenal gland

1. Ultra fine tip muelectrodes (300 MOhm) were used to study the electrical properties of the chromaffin cell membrane in situ in the intact toad adrenal gland. 2. In the presence of physiologic [K +] o(2 mM) the resting membrane potential ( V m) was − 53 ± 3.2 mV. V m, depended on [K +] o as predicted by the constant field equation with P Na P K of 0.16 3. A small fraction (20%) of the impaled cells exhibited spontaneous electrical activity, though in all the cells examined, the injection of depolarizing current pulses elicited repetitive spikes. 4. Our measurements of the chromaffin cell input resistance (326 ± 35 MOhm) is substantially smaller than the values reported for bovine isolated chromaffin cells, suggesting that the toad adrenal chromaffin cells might be electrically coupled. 5. Tetraethylammonium (TEA) increased the amplitude and duration of spikes, probably inhibiting outward K + current. In the presence of tetrodotoxin (TTX) action potentials were abolished, although they reappeared if TEA was added, suggesting the participation of both Na + and Ca 2+ currents in the genesis of spikes. 6. As expected, acetylcholine (ACh) and nicotine depolarized the cells, though they did not always elicit electrical activity. 7. Muscarine (10–100 μM) had no effect on both V m and on the depolarization induced by ACh or nicotine. Since muscarine inhibits catecholamine (CA) secretion induced by ACh and nicotine, we concluded that the inhibition of CA release by muscarine in the toad probably occurs at a level other than the membrane.

Isolation of chromaffin cell thapsigargin-sensitive Ca 2+ store in light microsomes from bovine adrenal medulla

1. 1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca 2+ stores in chromaffin cells. 2. 2. The thapsigargin-sensitive compartment of Ca 2+ stores was found to be highly enriched in a light microsomal fraction (LMF) on a 15–30% linear sucrose gradient, and was found to be essentially devoid of contamination by plasma, mitochondrial or secretory granule membranes. 3. 3. A Ca 2+-pumping ATPase was identified in this LMF as a 97 kDa protein forming an acid-stable, Ca 2+-dependent, thapsigargin-sensitive phosphorylated intermediate upon incubation with [γ- 32P]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca 2+ ATPases. 4. 4. A major 162 kDa protein, previously demonstrated in the isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca 2+ uptake. 5. 5. LMF appears to represent a part of the thapsigargin-sensitive Ca 2+ store of chromaffin cells, and should be useful for further studies of the store properties at the subcellular and molecular level.

Alleviation of pain and depression by specific neural transplants: Fine structural correlates

We have been interested in the use of neural transplants mainly as a local source of neuroactive substances, rather than as a replacement for damaged neural circuities. In particular, we have been exploring the possibilities of reducing pain by transplants of opioid peptide producing cells, and reducing depression by transplants of monoamine-producing cells. For the past several years, work in our laboratory has demonstrated in both acute and chronic pain models that transplantation of adrenal medullary tissue or isolated chromaffin cells into CNS pain modulatory regions can reduce pain sensitivity in rodents. Chromaffin cells were chosen as donor source since they produce high levels of both opioid peptides and catecholamines, substances which independently, and probably synergistically, reduce pain sensitivity when injected locally into the spinal cord. The analgesia produced by these transplants most likely results from the release of both opioid peptides and catecholamines, since it can be blocked or attenuated by opiate or adrenergic antagonists, respectively. Furthermore, CSF levels of met-enkephalin and catecholamines are increased by the transplants.

Reserpine‐Induced Processing of Chromogranin A in Cultured Bovine Adrenal Chromaffin Cells

The effect of reserpine on the processing of the secretory granule protein chromogranin A (CgA) in isolated bovine adrenal chromaffin cells was investigated using two radioimmunoassays employing site-specific antisera. The two antisera were directed against closely associated regions of the CgA molecule which would be exposed by specific processing: antiserum L331 was raised against the C-terminus of the regulatory peptide pancreastatin, and the second antiserum, L300, was raised against the synthetic peptide [Tyr0]CgA306-313 (YLSKEWEDA), a sequence that lies immediately C-terminal to pancreastatin and adjacent to a dibasic amino acid cleavage site. Chronic reserpine treatment of chromaffin cells produced a time- and dose-dependent increase in processing, as demonstrated by an increase in pancreastatin- and YLSKEWEDA-immunoreactivity (ir). The reserpine-induced rise in pancreastatin-ir was due predominantly to an increase in pancreastatin 1-47, whereas the rise in YLSKEWEDA-ir was due to increases in three polypeptides: a 51-kDa YLSKEWEDA-ir polypeptide, CgA297-313, and CgA248-313. The latter predominated. The action of reserpine on both pancreastatin- and YLSKEWEDA-ir was found to be largely inhibited by the protein synthesis inhibitor cycloheximide. The results show that treatment of isolated chromaffin cells with reserpine induces both the selective proteolytic processing and peptidyl-glycine amidation of CgA and its derived fragments. As reserpine has a similar effect on proenkephalin in chromaffin cells, the results suggest that reserpine induces a general increase in the activity of the processing enzymes, partially by an increase in protein synthesis.

Presence of [formula omitted] exchange activity and its role in regulation of intracellular calcium concentration in bovine adrenal chromaffin cells

The presence of a Na + Ca 2+ exchanger in bovine adrenal chromaffin cells was demonstrated by measuring the efflux of 45Ca 2+ which had been preloaded into cells by a brief depolarization. The efflux of 45Ca 2+ was dependent on extracellular Na + (Na + o); 45Ca 2+ efflux was significantly decreased by replacing Na + o with N-methylglucamine (NMG), or Li +. Replacement of Na + o by NMG increased the resting intracellular Ca 2+ concentration ([Ca 2+] i) of freshly isolated chromaffin cells. This could be reversed by adding Na +, suggesting that Na + Ca 2+ exchanger activity was involved in maintaining [Ca 2+] i at its resting level. The initial rate of Na +-dependent [Ca 2+] i recovery after Ca 2+ loading by depolarization was dependent on the level of [Ca 2+] i. There was an apparent linear relationship between the activity of the Na + Ca 2+ exchanger and [Ca 2+] i both in the presence and absence of Na + o. When cells were treated with other stimuli, including 10 μM DMPP or 40 mM caffeine, the ability of the stimulated cells to decrease [Ca 2+] i was significantly reduced upon replacing Na + o with NMG. Our data show that the Na + Ca 2+ exchanger is one of the major pathways for regulating [Ca 2+] i in chromaffin cells in both resting and stimulated states.

Ionic mechanisms involved in muscarinic regulation of neuronal and paraneuronal activity.

The characteristics of neuronal and paraneuronal muscarinic inhibition and excitation were analyzed using rat caudate nucleus (CN) slices and isolated chromaffin cells obtained from the rat adrenal medulla. In CN neurons, either acetylcholine (ACh), carbachol, or muscarine inhibited orthodromically activated firing, while nicotine had no effect on neuronal activity. Muscarine decreased the amplitude of EPSPs without altering the resting membrane potential (RMP), input impedance and EPSP time courses. These results indicate that muscarinic receptors produce the presynaptic inhibition of synaptic transmission in the CN. In adrenal chromaffin cells, it was found that ACh, muscarine, and nicotine all increased extracellularly recorded firing. During voltage clamp recording at the RMP, ACh induced a transient inward current (fast response) followed by a long-lasting current (slow response). Muscarine induced the slow response, whereas nicotine induced the fast response. Muscarine reduced the inward K+ current produced by the application of a high K+ medium to cells. During patch clamp recording, muscarine decreased the opening rate of the single K+ channels. These results indicate that the muscarinic excitation of adrenal chromaffin cells was triggered by a reduction in the number of active K+ channels at the RMP.

Ionic Mechanisms Involved in Muscarinic Regulation of Neuronal and Paraneuronal Activity†

The characteristics of neuronal and paraneuronal muscarinic inhibition and excitation were analyzed using rat caudate nucleus (CN) slices and isolated chromaffin cells obtained from the rat adrenal medulla. In CN neurons, either acctylcholine (ACh), carbachol, or muscarine inhibited orthodromically activated firing, while nicotine had no effect on neuronal activity. Muscarine decreased the amplitude of EPSPs without altering the resting membrane potential (RMP), input impedance and EPSP time courses. These results indicate that muscarinic receptors produce the presynaptic in hibition of synaptic transmission in the CN. In adrenal chromaffi n cells, it was found that ACh, muscarine, and nicotine all increased extracellularly recorded firing. During voltage clamp recording at the RMP, ACh induced a transient inward current (fast response) followed by a long-lasting cu rrent (slow response). Muscarine induced the slow response, whereas nicotine induced the fast response. Muscarine reduced the inward K+ current produced by the application of a high K+ medium to cells. During patch clamp recording, muscarine decreased the opening rate of the single K+ channels. These resu lts indicate that the muscarinic excitation of adrenal chromaffin cells was triggered by a reduction in the number of active K+ channels at the RMP.