Chromaffin Cells Of Adrenal Medulla Research Articles

Rodent noradrenergic chromaffin cells contain calbindin D28K immmunoreactivity.

The calcium binding protein calbindin D28K is heterogeneously distributed in neurons throughout the body. We have investigated the distribution of calbindin in the chromaffin cells of the adult rodent adrenal medulla, which share the same developmental origin as peripheral sensory and autonomic neurons. Calbindin immunoreactivity was present in all noradrenergic chromaffin cells (defined by their lack of the adrenaline synthesizing enzyme, phenylethanolamine N-methyl transferase) in both the rat and mouse. It was also present in a very few adrenergic chromaffin cells in both rat and mouse. Calbindin-immunoreactivity is present in rat noradrenergic chromaffin cells from the day of birth and so is a useful marker for identifying rodent noradrenergic chromaffin cells.

Chromogranins: current concepts. Structural and functional aspects.

The large dense core secretory granules of adrenal medulla chromaffin cells synthesize, store and release catecholamines. Upon cell stimulation intragranular stored material is liberated into circulation to activate target organs. In addition to catecholamines, numerous peptides and proteins are present in chromaffin secretory granules and are exocytotically co-secreted with the catecholamines (Aunis, 1997).

A preferential pole for exocytosis in cultured chromaffin cells revealed by confocal microscopy

Histological studies suggest that adrenal medulla chromaffin cells in situ are polarised, but functional evidence is lacking. We present here the first demonstration for polarisation of exocytosis in isolated, spherical, bovine chromaffin cells. Cells were stimulated with 70 mM K + to cause a marked enhancement of catecholamine release, monitored amperometrically. FM1-43 and dopamine β-hydroxylase antibodies provided fluorescence confocal pictures that were 2–3-fold more intense in the bottom of the cells, as compared to equatorial or apex planes. This suggests that the solid phase to which the cell attaches serves as a ‘trophic’ signal for the polarisation of its secretory apparatus.

Cytoplasmic dynein and dynactin as likely candidates for microtubule-dependent apical targeting of pancreatic zymogen granules

The critical role of microtubules in vectorial delivery of post-Golgi carrier vesicles to the apical cell surface has been established for various polarized epithelial cell types. In the present study we used secretory granules of the rat and chicken pancreas, termed zymogen granules, as model system for apically bound post-Golgi carrier vesicles that underlie the regulated exocytotic pathway. We found that targeting of zymogen granules to the apical cell surface requires an intact microtubule system which contains its colchicine-resistant organizing center and, thus, the microtubular minus ends close to the apical membrane domain. Purified zymogen granules and their membranes were found to be associated with cytoplasmic dynein intermediate and heavy chain and to contain the major components of the dynein activator complex, dynactin, i.e. p150Glued, p62, p50, Arp1, and beta-actin. Kinesin heavy chain and the kinesin receptor, 160 kD kinectin, were not detected as components of zymogen granules. Immunofluorescence staining showed a zymogen granule-like distribution for dynein and dynactin (p150Glued, p62, p50, Arpl) in the apical cytoplasm, whereas kinesin and kinectin were largely concentrated in the basal half of the cells in a pattern similar to the distribution of calreticulin, a component of the endoplasmic reticulum. Secretory granules of non-polarized chromaffin cells of the bovine adrenal medulla, that are assumed to underlie microtubular plus end targeting from the Golgi apparatus to the cell periphery, were not found to be associated with dynein or dynactin. To our knowledge, this is the first demonstration of major components of the dynein-dynactin complex associated with the membrane of a biochemically and functionally well-defined organelle which is considered to underlie a vectorial minus end-driven microtubular transport critically involved in precise delivery of digestive enzymes to the apically located acinar lumen.

Characterization of Antibacterial COOH-terminal Proenkephalin-A-derived Peptides (PEAP) in Infectious Fluids: IMPORTANCE OF ENKELYTIN, THE ANTIBACTERIAL PEAP209–237 SECRETED BY STIMULATED CHROMAFFIN CELLS

Proenkephalin-A (PEA) and its derived peptides (PEAP) have been described in neural, neuroendocrine tissues and immune cells. The processing of PEA has been extensively studied in the adrenal medulla chromaffin cell showing that maturation starts with the removal of the carboxyl-terminal PEAP209-239. In 1995, our laboratory has shown that antibacterial activity is present within the intragranular chromaffin granule matrix and in the extracellular medium following exocytosis. More recently, we have identified an intragranular peptide, named enkelytin, corresponding to the bisphosphorylated PEAP209-237, that inhibits the growth of Micrococcus luteus (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516-525). As a continuation of this previous study, in order to characterize the biological function of antibacterial PEAP, we have here examined whether this COOH-terminal fragment is released from stimulated chromaffin cells and whether it could be detected in wound fluids and in polymorphonuclear secretions following cell stimulation. The antibacterial spectrum shows that enkelytin is active against several Gram-positive bacteria including Staphylococcus aureus, but it is unable to inhibit the Gram-negative bacteria growth. In order to relate the antibacterial activity of enkelytin with structural features, various synthetic enkelytin-derived peptides were tested. We also propose a computer model of synthetic PEAP209-237 deduced from 1H NMR analysis, in order to relate the antibacterial activity of enkelytin with the three-dimensional structure. Finally, we report the high phylogenetic conservation of the COOH-terminal PEAP, which implies some important biological function and we discuss the putative importance of enkelytin in the defensive processes.

A Novel Protein Containing cdc10/SWI6 Motifs Regulates Expression of mRNA Encoding Catecholamine Biosynthesizing Enzymes

Catecholaminergic (dopaminergic, noradrenergic, and adrenergic) transmitter phenotypes require the cooperative actions of four biosynthetic enzymes: tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase. Mechanisms that control expression of these enzymes in a transmitter phenotype-specific manner, however, are poorly understood. Here, we provide evidence that overexpression of a novel cdc10/SWI6 motif-containing protein, V-1, elicits the coordinate up-regulation of tyrosine hydroxylase, aromatic L-amino acid decarboxylase, and dopamine beta-hydroxylase mRNAs in the neuronal cell line PC12D, and as a result, catecholamine levels are increased. Furthermore, V-1 is strongly expressed in the cytoplasm of rat chromaffin cells of adrenal medulla. Thus, V-1 may act as a cytoplasmic protein/protein adapter and be involved in control of the catecholaminergic phenotype expression via an intracellular pathway signaling to the nucleus.

Neuronal differentiation of chromaffin cells in vitro, induced by extremely low frequency magnetic fields or nerve growth factor: A histological and ultrastructural comparative study

The application of nerve growth factor (NGF) to primary adrenal medulla chromaffin cell cultures induces phenotypic changes characterized mainly by the presence of neurites. A similar effect has been seen when these cells are stimulated by extremely low frequency magnetic fields (ELFMF). In this study, newborn rat chromaffin cells were cultured and subjected to NGF or ELFMF in order to compare their histological and ultrastructural characteristics. Cells cultured in the presence of NGF developed cytoplasmic projections and their distal ends showed growth cones as well as filopodia. With scanning and transmission electron microscopy, an increased submembranous electron density was observed in the nuclei of cells as well as irregular, wavy neuritic projections with a moderate number of varicosities, as well as the prevalence of intermediate filaments among the cytoskeleton components. Cells stimulated with ELFMF presented straighter neuritic extensions with a greater number of varicosities. With the transmission electron microscope, numerous neurotubules were observed, both in the cell soma and in their neuritic extensions. In both groups, growth cones were clearly identified by their ultrastructural characteristics. The differences seen in the cytoskeleton of cells stimulated with NGF or ELFMF suggest differential stimulation mechanisms possibly determining the biochemical, electrophysiological, and morphological characteristics in both types of cell cultures. J. Neurosci. Res. 53:569–582, 1998. © 1998 Wiley-Liss, Inc.

Human metalloprotease-disintegrin Kuzbanian regulates sympathoadrenal cell fate in development and neoplasia.

The development of the sympathetic nervous system involves cell-cell interactions that regulate the fate and migration of progenitor neural cells. Recent evidence shows that focal membrane-bound protease activity is critical for such interactions. The Drosophila kuzbanian (kuz) gene is required in neurogenesis and encodes a highly conserved, membrane-bound metalloprotease- disintegrin closley related to theTNF-alphaconvertingenzyme (TACE). We have characterized the human and mouse kuz homologs and mapped human kuz to chromosome 15q22. During mouse embryonic development Kuz is expressed mainly in the sympathoadrenal and olfactory neural precursors. Once sympathoadrenal cells differentiate into chromaffin cells in the adult adrenal medulla, they no longer express Kuz. However, we found that tumors of sympathoadrenal origin, such as pheochromocytomas and neuroblastomas, overexpress Kuz. Further, transfection of a kuz construct lacking the protease domain, but not the full-length construct, induces neurite formation in PC12 chromaffin tumor cells. Taken together our results suggest a critical role for Kuz in regulation of sympathoadrenal cell fate.

Oxygen-sensing mechanisms are present in the chromaffin cells of the sheep adrenal medulla before birth.

1. The ability of the fetal adrenal medulla to respond directly to hypoxaemia and secrete catecholamines before the development of a functional innervation of the gland is essential for intrauterine survival. The cellular mechanisms involved in this response to low PO2 are not known, although the presence of oxygen-sensitive K+ channels in carotid body chemoreceptor cells and other sites suggests that these might underlie the chromaffin cell response. 2. Whole-cell patch-clamp techniques have been used to study K+ currents during normoxia and hypoxia in chromaffin cells isolated from the adrenal glands of fetal sheep. 3. Two types of chromaffin cells were observed, those with a fast inactivating K+ current and a larger capacitance and those with a delayed K+ current and smaller capacitance. No cell showed both types of current. The fast inactivating current showed voltage-dependent inactivation and was blocked by 1 mM 4-aminopyridine, characteristics of an IA-type current. The delayed current had two components, a TEA-sensitive, Ca2+-dependent current and a component with the kinetic behaviour of a delayed rectifier. 4. Both types of current were oxygen sensitive. The IA-type current was reduced by 27.4 +/- 3.2 % when the PO2 was reduced to about 15 mmHg. With the delayed current, hypoxia reduced the amplitude by 26.9 +/- 2.4 %, largely by reduction of the Ca2+-dependent component. 5. In the presence of hypoxia, reduction in the amplitude of these oxygen-sensitive K+ currents would increase the frequency and duration of action potentials, leading to increased activation of the L-type Ca2+ channels, influx of Ca2+ and the subsequent secretion of catecholamines.

Cruciform-extruding regulatory element controls cell-specific activity of the tyrosine hydroxylase gene promoter.

Tyrosine hydroxylase (TH) is expressed specifically in catecholaminergic cells. We have identified a novel regulatory sequence in the upstream region of the bovine TH gene promoter formed by a dyad symmetry element (DSE1;-352/-307 bp). DSE1 supports TH promoter activity in TH-expressing bovine adrenal medulla chromaffin (BAMC) cells and inhibits promoter activity in non-expressing TE671 cells. DNase I footprinting of relaxed TH promoter DNA showed weak binding of nuclear BAMC cell proteins to a short sequence in the right DSE1 arm. In BAMC cells, deletion of the right arm markedly reduced the expression of luciferase from the TH promoter. However, deletion of the left DSE1 arm or its reversed orientation (RevL) also inactivated the TH promoter. In supercoiled TH promoter, DSE1 assumes a cruciform-like conformation i.e., it binds cruciform-specific 2D3 antibody, and S1 nuclease-cleavage and OsO4-modification assays have identified an imperfect cruciform extruded by the DSE1. DNase I footprinting of supercoiled plasmid showed that cruciformed DSE1 is targeted by nuclear proteins more efficiently than the linear duplex isomer and that the protected site encompasses the left arm and center of DSE1. Our results suggest that the disruption of intrastrand base-pairing preventing cruciform formation and protein binding to DSE1 is responsible for its inactivation in DSE1 mutants. DSE1 cruciform may act as a target site for activator (BAMC cells) and repressor (TE671) proteins. Its extrusion emerges as a novel mechanism that controls cell-specific promoter activity.

5] Noncovalent and covalent labeling of vesicular monoamine transporter with tetrabenazine and ketanserin derivatives: Purification of photolabeled protein

The vesicular monoamine transporter (VMAT) accumulates monoamines inside secretory granules or synaptic vesicles by catalyzing an H+/ monoamine antiport. Monoamine uptake is coupled to the generation of a proton electrochemical gradient by an ATP-dependent H + pump of the vacuolar type. The uptake is extremely efficient and in the adrenal medulla chromaffin cells, the monoamine gradient between the cytosol and the secretory granules has been estimated to five orders of magnitude. This chapter discusses noncovalent and covalent labeling of vesicular monoamine transporter with tetrabenazine and ketanserin derivatives protein. The vesicular monoamine transporter from various origins is cloned and expressed in various cell types. Two isoforms, VMAT1 and VMAT2, are identified; they have different localizations and slightly different properties. Two different binding sites are also identified: the first one binds reserpine, whereas the second one binds tetrabenazine (TBZ) and ketanserin (KET). The H+ electrochemical gradient generated in the presence of ATP greatly accelerates reserpine binding, but has no effect on TBZ and KET binding.

NaG: a sodium channel-like mRNA shared by Schwann cells and other neural crest derivatives.

NaG, a member of subfamily 2, was originally characterized as a "glial" sodium channel, and is expressed at high levels in Schwann cells in vivo. However, NaG has also been shown by in situ hybridization to be highly expressed in rat dorsal root ganglion (DRG) neurons, which, like Schwann cells, are neural crest derivatives. In the present study, we used non-isotopic in situ hybridization with a riboprobe for NaG, in conjunction with RT-PCR, to determine whether NaG is expressed in tissues related to the DRG either by neural crest origin or sensory function. We found that the expression of significant levels of NaG mRNA was restricted to derivatives of the neural crest (neurons of DRG, trigeminal ganglion and mesencephalic nucleus of the trigeminal nerve, and Schwann and satellite cells); the absence of NaG from superior cervical ganglion neurons and adrenal medulla chromaffin cells indicates that not all neural crest derived neural elements express NaG. NaG was not observed in sensory neurons (retina, vestibular ganglion, spiral ganglion, olfactory epithelium) that are not of neural crest origin. Our results indicate that NaG mRNA is expressed not only in Schwann cells, but also in a spectrum of neuronal cell types with afferent function of neural crest origin. NaG thus appears to represent a transcript encoding a sodium channel or sodium channel-like protein that is uniquely expressed by both Schwann cells and afferent neurons of neural crest origin.

Short term effect of steroids on catecholamine secretion from bovine adrenal medulla chromaffin cells

Bovine chromaffin cells were used to examine neuronal modulation, as their function is similar to sympathetic post-ganglionic neurons. The effect of steroids on evoked catecholamine secretion from primary culture of bovine adrenal medullary cells was investigated. A wide range of progestins, androgens and estrogens was found to have a significant effect on catecholamine secretion induced by the natural neurotransmitter acetylcholine (ACh). The androgens (especially androstandione and androsterone), as a class were the most effective in inhibition of stimulated secretion, while the estrogens had little, to no, effect. Among all steroids tested, progesterone had the most significant effect, other progestins were less potent. Progesterone inhibited catecholamine secretion evoked by ACh, nicotine and oxotremorine-M in a dose-dependent manner with similar IC 50 values in the μM range. It also blocked the secretion evoked by high potassium concentration (59 nM) or veratradine (100 μM), but no effect was seen on the secretion evoked by the calcium ionophore A-23187 (10 μM). Progesterone inhibition of ACh or oxotremorine-M stimulation was immediate and sustained. These results suggest that progesterone and other steroids might have a membrane effect probably acting through blockade of calcium influx necessary for the secretory response.

Developmental regulation of preproenkephalin (PENK) gene expression in the adrenal gland of the ovine fetus and newborn lamb: effects of hypoxemia and exogenous cortisol infusion.

Development of the fetal adrenal gland is crucial not only for maturation of several fetal organ systems and the initiation of parturition, but also for the development of the fetal response to stress. The enkephalin-related peptides are present in the chromaffin cells of the fetal adrenal medulla and are secreted in response to stress and with sympathetic stimulation. However, changes in expression of preproenkephalin (PENK) with gestation and in response to stress have not been studied in detail. Therefore we examined the developmental pattern of PENK gene expression in the adrenal gland of fetal and newborn lambs, and of adult sheep. We also determined whether levels of PENK mRNA in the fetal adrenal gland changed in response to exogenous glucocorticoids in late gestation, or in response to hypoxemia. Adrenal glands were removed from fetal sheep, lambs and adult sheep at different stages of development for measurement of PENK mRNA. Cortisol was infused (5 micrograms/min) for 12, 24 or 96 h beginning on day 124-129 of gestation. Moderate hypoxemia was induced for 48 h beginning on day 126-130, or at day 134-136 of gestation, by lowering the maternal fractional inspired oxygen. At the end of the treatment periods, the ewes and fetuses were euthanized. Adrenal PENK mRNA were measured by Northern blot analysis. PENK mRNA levels in fetal adrenals were significantly higher (P < 0.05) on days 140-141 of gestation than earlier in pregnancy, and then decreased significantly with the onset of parturition (days 142-146). After cortisol infusion to the fetus for 96 h there was a significant reduction in adrenal PENK mRNA levels. Hypoxemia resulted in a significant increase in PENK mRNA levels in fetuses at day 126-130 of gestation, but not at the later time in pregnancy when endogenous plasma cortisol concentrations were higher. We conclude that there is a decrease in levels of PENK mRNA in the fetal adrenal gland before parturition at the time of the endogenous prepartum rise in plasma cortisol. Hypoxemia led to an elevation of PENK mRNA levels in fetuses at less than 130 days, but after that time, when the basal and stimulated cortisol responses had risen, there was no significant effect of hypoxemia on PENK mRNA. Cortisol infusion to the fetus at this stage of pregnancy resulted in a decrease in adrenal PENK mRNA levels. We suggest that cortisol may play an important role in the regulation of fetal adrenal PENK mRNA levels and enkephalin synthesis by the adrenal gland of the fetal sheep.

Heterogeneous distribution of neurocalcin-immunoreactive nerve terminals in the mouse adrenal medulla.

Neurocalcin is a novel calcium-binding protein found in bovine brain tissue. We investigated immunoreactivity for neurocalcin in the mouse adrenal medulla using light and electron microscopy. The immunoreactivity was present in nerve fibers, nerve terminals, and ganglion cells in the adrenal medulla, but chromaffin cells, sustentacular cells, and Schwann cells were negative in reaction. Nerve bundles containing neurocalcin-immunoreactive fibers passed through the adrenal cortex and extended into the medulla. Immunopositive nerve fibers branched off and projected varicose terminals around the chromaffin cells. These varicose terminals contained small and large-cored vesicles and made synapses with the chromaffin cells. We performed paraformaldehyde-induced fluorescence-histochemical studies for catecholamine combined with immunohistochemical studies for neurocalcin. Neurocalcin-immunoreactive nerve terminals were more abundant at noradrenaline (fluorescent) cell-rich regions than at adrenaline (non-fluorescent) cell-rich regions. These results show that neurocalcin-immunoreactive nerves mainly innervate noradrenaline-containing chromaffin cells in the mouse adrenal medulla and that neurocalcin may regulate synaptic function in the nerve terminals.

A caffeine-sensitive Ca2+ store modulates K+-evoked secretion in chromaffin cells

Catecholamine release from bovine adrenal medulla chromaffin cells superfused with a Krebs-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution was monitored on-line with an electrochemical detector. Caffeine (10 mM) progressively depressed the magnitude of secretory responses to depolarizing pulses of 70 mM K+ and 2 mM Ca2+ (70 K+/2 Ca2+) in cells superfused with a Krebs-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution containing 0 mM Ca2+ + 0.5 mM EGTA; blockade reached 80% at the third 70 K+/2 Ca2+ challenge given in the presence of caffeine. A similar effect was obtained when, instead of continuous superfusion, prepulses of caffeine were applied (10 mM for 60 s). The blocking effects of caffeine on K+-induced secretion depended on the time of exposure to the drug: the longer the exposure time the greater the blockade. The recovery of the K+ secretory responses previously impaired by caffeine was always gradual and followed a staircase mode. This contrasts with the effects of caffeine on various parameters measuring Ca2+ entry through Ca2+ channels, which did not parallel its effects on K+-evoked secretion. The secretion data, however, are compatible with the disappearance and recovery of an intracellular Ca2+ concentration signal triggered by K+ in single chromaffin cells loaded with fura 2 and treated with 10 mM caffeine. Thus, contrary to previous views, the depression of secretion by caffeine does not seem to be associated with inhibition of extracellular Ca2+ entry through Ca2+ channels. These functional data are, rather, compatible with the view that the degree of filling of a caffeine-sensitive intracellular Ca2+ store might regulate the extent of exocytosis. When emptied, such a store might act as a sink for the external Ca2+ entering through Ca2+ channels during cell depolarization, thus decreasing the intracellular Ca2+ concentration available for exocytosis.

Pharmacological characterization and autoradiographic localization of dopamine receptors in the rat adrenal medulla

The pharmacological profile and the anatomical localization of dopamine D 1-like and D 2-like receptors were studied in sections of rat adrenal medulla, with radioligand binding and autoradiographic techniques, respectively. [ 3H]([ R]-(+)-chloro-2,3,4,5-tetrahydro-5-phenyl-1 H-3benzazepin-al hemimaleate) (SCH 23390) was used as a ligand for dopamine D 1-like receptors and [ 3H]spiperone was used as a ligand for dopamine D 2-like receptors. Radioligand binding and light microscope autoradiography did not show specific [ 3H]SCH 23390 binding in sections of rat adrenal medulla. This suggests that rat adrenal medulla does not express dopamine D 1-like receptors. [ 3H]Spiperone was specifically bound to sections of rat adrenal medulla. The binding was time-, temperature- and concentration-dependent, with a dissociation constant ( K d) of 1.05 nM and a maximum density of binding sites ( B max) of 100.2 ± 3.8 fmol/mg tissue. The pharmacological profile of [ 3H]spiperone binding to rat adrenal medulla was similar to that displayed by neostriatum, which is known to express dopamine D 2 receptors. Light microscope autoradiography showed the accumulation of specifically bound [ 3H]spiperone as silver grains within sections of adrenal medulla. Silver grains were found primarily over the cellular membrane of chromaffin cells. The above data indicate that chromaffin cells of the rat adrenal medulla express dopamine receptors belonging to the dopamine D 2 receptor subtype. These receptors are probably involved in the modulation of catecholamine release from chromaffin cells, as documented by functional studies.

A morphometric analysis of exocytosis in KCl-stimulated bovine chromaffin cells.

Transmission electron microscopy has been used to morphometrically evaluate exocytosis in bovine adrenal medulla chromaffin cells as the mechanism of catecholamine release. Purified cell suspensions were stimulated with KCl at varying strengths and durations and then conventionally processed for ultrastructural analysis. Quantitation of exocytotic images of dense cored chromaffin granules was a major objective and such images were found in all preparations, attesting to the efficacy of chemical fixation to preserve this event. However, because hundreds of cell profiles had to be screened to find a single granule in the process of release this low frequency precluded any meaningful correlations with estimates of granular involvement based on catecholamine release. Neither KCl molarity nor duration altered this finding nor did these variables significantly affect other parameters linked to exocytotic activity. For example, cell size and numbers of "empty' granules and vesicles remained constant and attempts to label "any' organelle with 30-nm colloidal gold or lanthanum precipitate proved unsuccessful. In short, if exocytosis is responsible for release, it would appear to function without leaving a morphological trace. An alternative hypothesis, therefore, is outlined which better accommodates existing data.

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.

Thapsigargin-sensitive Ca(2+)-ATPases account for Ca2+ uptake to inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive Ca2+ stores in adrenal chromaffin cells.

In chromaffin cells of adrenal medulla, heterogeneity of Ca2+ stores has been suggested with respect to the mechanisms of Ca2+ uptake and release. We have examined Ca(2+)-ATPases responsible for loading of Ca2+ stores in these cells for their sensitivity to thapsigargin, a highly selective inhibitor of the SERCA [sarco(endo)plasmic reticulum calcium ATPase] family of intracellular Ca2+ pumps. Using immunostaining, we studied the distribution of Ca(2+)-ATPases, and of receptors for inositol 1,4,5-trisphosphate (InsP3) and ryanodine, in the density-gradient fractions of microsomes from bovine adrenal medulla. In parallel, we examined distribution profiles of ATP-dependent Ca2+ uptake in the same fractions, along with subcellular markers for plasma membranes and endoplasmic reticulum (ER). Two Ca(2+)-ATPase-like proteins (116 and 100 kDa) were detected, consistent with the presence of SERCA 2b and SERCA 3 isoenzymes of Ca2+ pumps. The distribution of these putative Ca(2+)-ATPase iso-enzymes paralleled that of InsP3 and ryanodine receptors. This distribution of ER Ca(2+)-ATPases, as determined immunologically, was consistent with that of thapsigargin-sensitive, but not of thapsigargin-insensitive, ATP-dependent Ca2+ uptake. In contrast, the distribution profile of the thapsigargin-insensitive Ca2+ uptake was strongly correlated to that of plasma membranes, and co-distributed with plasma membrane Ca(2+)-ATPase detected immunologically. In isolated, permeabilized chromaffin cells, InsP3 and caffeine induced Ca2+ release following an ATP-dependent Ca2+ accumulation to the stores. This accumulation was abolished by thapsigargin. Together, these data strongly indicate that the thapsigargin-sensitive, presumably SERCA-type Ca(2+)-ATPases account for Ca2+ uptake to InsP3-sensitive, as well as to caffeine-sensitive, Ca2+ stores in bovine adrenal chromaffin cells.