Bovine Adrenal Medullary Research Articles

The Inhibition Effects of Shenmai Injection on Acetylcholine-Induced Catecholamine Synthesis and Secretion by Modulating Nicotinic Acetylcholine Receptor Ion Channels in Cultured Bovine Adrenal Medullary Cells.

Shenmai injection (SMI) has been widely used for the treatment of cardiovascular diseases in China. Cardiovascular disorders are often related to excessive catecholamine (CA) secretion. Here, we report the effects of SMI on CA secretion and synthesis in cultured bovine adrenal medullary cells. We found that SMI significantly reduced CA secretion induced by 300 μM acetylcholine (ACh). Cotreatment with SMI (10 μL/mL) and either of the ACh receptor α-subunit inhibitors, HEX (α3) or DhβE (α4β2), did not produce any further inhibition, indicating that SMI may play a role through α3 and α4β2 channels. Furthermore, SMI reduced tyrosine hydroxylase (TH) activity induced by ACh by inhibiting the phosphorylation of TH at Ser19 and Ser40. TH is phosphorylated at Ser19 by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and at Ser40 by protein kinase A (PKA). KN-93 and H89, the antagonists of CaM kinase II and PKA, respectively, inhibited the ACh-induced phosphorylation at Ser19 and Ser40, and the addition of SMI did not augment the inhibitory effect. Taken together, our results show that SMI likely inhibits CA secretion by blocking TH activity at its Ser19 and Ser40 sites.

Effects of Selective Estrogen Receptor Modulators on Plasma Membrane Estrogen Receptors and Catecholamine Synthesis and Secretion in Cultured Bovine Adrenal Medullary Cells

We previously reported the occurrence and function of plasma membrane estrogen receptors in cultured bovine adrenal medullary cells. Here we report the effects of raloxifene and tamoxifen, selective estrogen receptor modulators, on plasma membrane estrogen receptors and catecholamine synthesis and secretion in these cells. Raloxifene caused dual effects on the specific binding of [(3)H]17β-estradiol to the plasma membranes isolated from bovine adrenal medulla; that is, it had a stimulatory effect at 1.0 - 10 nM but an inhibitory effect at 1.0 - 10 μM, whereas tamoxifen (1.0 nM - 10 μM) increased binding at all concentrations (except for 100 nM). Tamoxifen at 100 nM caused a significant increase in basal (14)C-catecholamine synthesis from [(14)C]tyrosine, whereas tamoxifen and raloxifene at higher concentrations attenuated basal and acetylcholine-induced (14)C-catecholamine synthesis. Raloxifene (0.3, 1.0, and 3 - 100 μM) and tamoxifen (10 - 100 μM) also suppressed catecholamine secretion and (45)Ca(2+) and (22)Na(+) influx, respectively, induced by acetylcholine. Raloxifene (1.0 μM) inhibited Na(+) current evoked by acetylcholine in Xenopus oocytes expressing α4β2 neuronal nicotinic acetylcholine receptors. The present findings suggest that raloxifene and tamoxifen at low concentrations allosterically modulate plasma membrane estrogen receptors and at high concentrations inhibit acetylcholine-induced catecholamine synthesis and secretion by inhibiting Na(+) and Ca(2+) influx in bovine adrenal medulla.

Pentazocine Inhibits Norepinephrine Transporter Function by Reducing its Surface Expression in Bovine Adrenal Medullary Cells

(±)-Pentazocine (PTZ), a non-narcotic analgesic, is used for the clinical management of moderate to severe pain. To study the effect of PTZ on the descending noradrenergic inhibitory system, in the present study we examined the effect of [3H]norepinephrine (NE) uptake by cultured bovine adrenal medullary cells and human neuroblastoma SK-N-SH cells. (−)-PTZ and (+)-PTZ inhibited [3H]NE uptake by adrenal medullary cells in a concentration-dependent (3 – 100 μM) manner. Eadie-Hofstee analysis of [3H]NE uptake showed that both PTZs caused a significant decrease in the Vmax with little change in the apparent Km, suggesting non-competitive inhibition. Nor-Binaltorphimine and BD-1047, κ-opioid and σ-receptor antagonists, respectively, did not affect the inhibition of [3H]NE uptake induced by (−)-PTZ and (+)-PTZ, respectively. PTZs suppressed specific [3H]nisoxetine binding to intact SK-N-SH cells, but not directly to the plasma membranes isolated from the bovine adrenal medulla. Scatchard analysis of [3H]nisoxetine binding to SK-N-SH cells revealed that PTZs reduced the Bmax without changing the apparent Kd. Western blot analysis showed a decrease in biotinylated cell-surface NE transporter (NET) expression after the treatment with (−)-PTZ. These findings suggest that PTZ inhibits the NET function by reducing the amount of NET in the cell surface membranes through an opioid and σ-receptor–independent pathway.

Storage of GABA in chromaffin granules and not in synaptic‐like microvesicles in rat adrenal medullary cells

Neurons and certain kinds of endocrine cells, such as adrenal medullary (AM) cells, have large dense-core vesicles (LDCVs) and synaptic vesicles or synaptic-like microvesicles (SLMVs). These secretory vesicles differ in Ca(2+) sensitivity and contain different signaling substances. We have recently reported that GABA functions as a paracrine factor in rat AM cells and modulates catecholamine secretion. The present experiment was undertaken to examine the subcellular localization of the GABA system including GABA itself in AM cells. Fractionation analysis with sucrose density gradient and immunocytochemistry indicated that vesicular GABA transporter (VGAT) was localized in LDCVs and not SLMVs in rat and bovine AM cells. In addition, significant amounts of GABA were detected in high density fractions, which contained LDCVs. When green fluorescence protein-VGAT and green fluorescence protein-vesicular ACh transporter were exogenously expressed in PC12 cells, the former and the latter were selectively targeted to LDCVs and SLMVs, respectively. We conclude that GABA is stored in chromaffin granules in rat and bovine AM cells through VGAT.

Simvastatin Inhibits Catecholamine Secretion and Synthesis Induced by Acetylcholine via Blocking Na+and Ca2+Influx in Bovine Adrenal Medullary Cells

Simvastatin, an inhibitor of HMG-CoA reductase, is a potent inhibitor of cholesterol biosynthesis and has beneficial effects in the primary and secondary prevention of cardiovascular diseases. In this study, we report the effects of simvastatin on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells used as a model of sympathetic neurons. Simvastatin inhibited catecholamine secretion induced by acetylcholine, an agonist of the nicotinic acetylcholine receptor; by veratridine, an activator of voltage-dependent Na(+) channels; and by high K(+), an activator of voltage-dependent Ca(2+) channels (IC(50) = 3.8, 7.8, and 6.1 microM, respectively). Simvastatin also suppressed acetylcholine-induced (22)Na(+) influx (IC(50) = 4.3 microM) and (45)Ca(2+) influx (IC(50) = 6.1 microM), veratridine-induced (22)Na(+) influx (IC(50) = 6.6 microM) and (45)Ca(2+) influx (IC(50) = 12 microM), and high K(+)-induced (45)Ca(2+) influx (IC(50) = 11 microM). The reduction of catecholamine secretion caused by simvastatin was not overcome by increasing the concentration of acetylcholine or by treatment with mevalonate, the first metabolite of HMG-CoA. The inhibitory effect of simvastatin on histamine-induced secretion of catecholamines was observed in the presence of extracellular Ca(2+), but not in a Ca(2+)-free medium, suggesting that simvastatin does not interfere with histamine receptors nonselectively. Simvastatin also suppressed acetylcholine-induced [(14)C]catecholamine synthesis from [(14)C]tyrosine as well as tyrosine hydroxylase activity. These findings suggest that simvastatin inhibits catecholamine secretion and synthesis induced by acetylcholine through suppression of Na(+) and Ca(2+) influx in the adrenal medulla and probably in the sympathetic neurons.

Dual Effects of Daidzein, a Soy Isoflavone, on Catecholamine Synthesis and Secretion in Cultured Bovine Adrenal Medullary Cells

We recently demonstrated the occurrence and functional roles of plasma membrane estrogen receptors in cultured bovine adrenal medullary cells. Here we report the effects of daidzein, a phytoestrogen of soybeans, on catecholamine synthesis and secretion in the cells. Incubation of cells with daidzein for 20 min increased the synthesis of (14)C-catecholamines from [(14)C]tyrosine but not [(14)C]dihydroxyphenylalanine, in a concentration-dependent manner (10-1000 nm). The stimulatory effect of daidzein on (14)C-catecholamine synthesis was not inhibited by ICI182,780, a classical estrogen receptor inhibitor. Acetylcholine, a physiological secretagogue, stimulated the synthesis of (14)C-catecholamines, which was suppressed by daidzein at 1 mum. Daidzein at high concentrations (1-100 microm) suppressed catecholamine secretion induced by acetylcholine. Furthermore, daidzein (10-1000 nm) inhibited the specific binding of [(3)H]17beta-estradiol to plasma membranes isolated from bovine adrenal medulla. The present findings suggest that daidzein at low concentrations stimulates catecholamine synthesis through plasma membrane estrogen receptors but at high concentrations inhibits catecholamine synthesis and secretion induced by acetylcholine in bovine adrenal medulla. The latter effect of daidzein may be a beneficial action on the cardiovascular system.

Characterization of bioactive peptides in bovine adrenal medulla by a combination of fast HPLC and ESI–MS

A method based upon a combination of fast high-performance liquid chromatography (HPLC) and electrospray ionization (ESI)–mass spectrometry (MS) is developed for the analysis of bioactive peptides in bovine adrenal medulla. The fast HPLC uses a short column ( 33 mm×4.6 mm ) packed with nonporous silica-based C-18 stationary phase. Prior to HPLC separation, the medulla was homogenized and the peptide-rich fraction was isolated from it by solid-phase extraction. In-source collision-induced dissociation and tandem MS were used to obtain the sequence of the suspected peptides. Several peptides, including Met–Enk, Leu–Enk, Leu–Enk–Lys, bovine adrenal medullary (BAM)-12 (Met–Enk–RRVGRPE), Leu–Enk–Arg, and YGGT, were unambiguously identified. The first four peptides are the products of proenkephalin A precursor protein and Leu–Enk–Arg belongs to the dynorphin family and is derived from proenkephalin B (prodynorphin) precursor. The database search revealed that YGGT is a part of the sequence of five different precursor proteins.

Pituitary adenylate cyclase-activating polypeptide induces a sustained increase in intracellular free Ca(2+) concentration and catechol amine release by activating Ca(2+) influx via receptor-stimulated Ca(2+) entry, independent of store-operated Ca(2+) channels, and voltage-dependent Ca(2+) channels in bovine adrenal medullary chromaffin cells.

Characteristics of pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increase of Ca(2+) entry and catecholamine (CA) release were studied in bovine adrenal medullary chromaffin cells. PACAP induced intracellular free Ca(2+) concentration ([Ca(2+)](i)), showing an initial transient [Ca(2+)](i) rise followed by a sustained rise and CA release, which were not blocked by the blocking agents for nicotinic acetylcholine receptor (nAChR) channel, the voltage-dependent Ca(2+) channel (VOC), or the Na(+) channel. The sarcoendoplasmic Ca(2+)-ATPase inhibitors thapsigargin and cyclopiazonic acid did not affect the PACAP-induced sustained rise of [Ca(2+)](i), but did inhibit the initial [Ca(2+)](i) rise. In cells pretreated with cyclopiazonic acid or membrane-permeable, low-affinity Ca(2+) chelator N',N',N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, PACAP further stimulated the entry of Ca(2+) or Mn(2+), whereas these treatments masked [Ca(2+)](i) dynamics induced by bradykinin. PACAP-induced sustained [Ca(2+)](i) rise and Mn(2+) entry were enhanced by acidic extracellular solution and reduced by alkalinization, whereas thapsigargin-induced Mn(2+) entry was regulated by the opposite. PACAP-induced [Ca(2+)](i) rise and Mn(2+) entry were not affected by blockers of cAMP-dependent protein kinase, phospholipase C, or protein kinase C. All store-operated Ca(2+) channel (SOC) blocking agents tested inhibited thapsigargin-induced Mn(2+) entry. 1(beta-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenylethyl)-1H-imidazole hydrochloride (SK&F 96365), (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide, and econazole inhibited PACAP-induced Ca(2+) or Mn(2+) entry, whereas GdCl(3), 7,8-benzoflavone, nor-dihydroguaiaretic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, fulfenamic acid, and niflumic acid did not. SK&F 96365 and econazole but not GdCl(3) inhibited PACAP-induced CA release. These results suggest that PACAP activates a novel Ca(2+) entry pathway associated with sustained CA release independent of the nAChR channel, VOC and SOC, activated by acid pH, with different sensitivity to blockers of SOC. This pathway may provide a useful model for the study of receptor-operated Ca(2+) entry.

Differential effects of short and prolonged exposure to carvedilol on voltage-dependent Na(+) channels in cultured bovine adrenal medullary cells.

We examined the effects of short and prolonged exposure to carvedilol, an antihypertensive and beta-adrenoceptor blocking drug, on voltage-dependent Na(+) channels in cultured bovine adrenal medullary cells. Carvedilol (1-100 microM) reduced (22)Na(+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels. Carvedilol also suppressed veratridine-induced (45)Ca(2+) influx and catecholamine secretion in a concentration-dependent manner similar to that of (22)Na(+) influx. Prolonged exposure of the cells to 10 microM carvedilol increased [(3)H]saxitoxin ([(3)H]STX) binding, which reached a plateau at 12 h and was still observed at 48 to 72 h. Scatchard analysis of [(3)H]STX binding revealed that carvedilol increased the B(max) value (control, 14.9 +/- 0.9 fmol/10(6) cells; carvedilol, 23.8 +/- 1.2 fmol/10(6) cells) (n = 3, P < 0.05) without altering the K(d) value, suggesting a rise in the number of cell surface Na(+) channels. The increase in [(3)H]STX binding by carvedilol was prevented by cycloheximide, an inhibitor of protein synthesis, whereas carvedilol changed neither alpha- nor beta(1)-subunit mRNA levels of Na(+) channels. The carvedilol-induced increase of [(3)H]STX binding was abolished by brefeldin A and H-89, inhibitors of intracellular vesicular trafficking of proteins from the trans-Golgi network and of cyclic AMP-dependent protein kinase (protein kinase A), respectively. The present findings suggest that short-term treatment with carvedilol reduces the activity of Na(+) channels, whereas prolonged exposure to carvedilol up-regulates cell surface Na(+) channels. This may add new pharmacological effects of carvedilol to our understanding in the treatment of heart failure and hypertension.

Inhibition by Neuromuscular Blocking Drugs of Norepinephrine Transporter in Cultured Bovine Adrenal Medullary Cells

Inhibition by Neuromuscular Blocking Drugs of Norepinephrine Transporter in Cultured Bovine Adrenal Medullary Cells

Determination of enzyme activity in single bovine adrenal medullary cells by separation of isotopically labeled catecholamines.

A microcolumn liquid chromatography method for determining norepinephrine (NE), epinephrine (E), and phenylethanolamine N-methyltransferase (PNMT) enzyme activity in single bovine adrenal medullary cells is presented. Single cells were isolated and treated with excess deuterated substrate, D3-NE (0.05 mM) for enzyme reaction. After 6 h, the reaction was quenched and the product, D3-E, was quantified along with endogenous NE and E. Separation and detection of deuterated and protiated NE and E were achieved with microcolumns (110-125 cm long, 25 microns inner diameter) packed with 3 microns octadecylsilane-modified particles and operated with amperometric detection. Of the 33 cells reported, most cells containing predominantly E have enzyme activity while cells containing predominantly NE and cells containing a mixture of both NE and E show no enzyme activity. After incubation with 10 microM hydrocortisone, of the 17 cells reported, most cells containing predominantly E and cells containing a mixture of both NE and E have enzyme activity while cells containing predominantly NE have no enzyme activity. Detection limits for NE and E were 42 and 48 amol, respectively.

Effects of External Osmotic Pressure on Vesicular Secretion from Bovine Adrenal Medullary Cells

Secretion of catecholamines from individual vesicles of bovine adrenal medullary cells was studied with amperometry in media of various osmolarities and compared with results obtained in isotonic physiological buffer (315 mosM). Hypotonic solutions caused an increase in the number of amperometric spikes evoked by brief exposure to 5 mM Ba2+. Under moderate hypertonic conditions (630 mosM), individual vesicular events were decreased in frequency, and lower amounts were secreted per event. Furthermore, the events were temporally broadened relative to those observed during release in isotonic conditions. At 970 mosM, exposure to 5 mM Ba2+ evoked even smaller secretory events that resemble the prespike feature that has been attributed to the initial opening of the fusion pore. The lack of large spikes is not due to failure of Ba2+ entry because fura-2 fluorescence reveals an increase in intracellular divalent ions. After exposure to Ba2+ in hypertonic solution, spikes could be induced with isotonic solution transiently directed onto the cell, but this process was not accompanied by a change in the concentration of intracellular divalent ions. Thus, this procedure provides an unique opportunity to temporally separate exocytotic secretion from entry of divalent ions.

Histamine-Induced Cortisol Secretion from Bovine Adrenocortical Cells: Co-incubated with Bovine Adrenal Medullary Cells

Histamine at concentrations higher than 10–9 M significantly elicited cortisol secretion from bovine adrenocortical (BAC) cells co-incubated with bovine adrenal medullary (BAM) cells, suggesting that BAM cells are responsible for histamine-induced cortisol secretion. Cortisol secretion from BAC cells co-incubated with BAM cells was also elicited by both an H1 agonist, 2-methylhistamine, and an H2 agonist, 4-methylhistamine. However, 4-methylhistamine was much less effective than 2-methylhistamine. Histamine-induced cortisol secretion was inhibited not only by H1 antagonists (pyrilamine and diphenhydramine) but also by H2 antagonists (cimetidine and ranitidine). Histamine effectively increased 45Ca uptake and IP3 production in BAM cells. These responses were antagonized by the H1 antagonist but not by the H2 antagonist. Histamine-induced cortisol secretion from BAC cells co-incubated with BAM cells was inhibited by β-adrenoceptor antagonists, propranolol and timolol, as well as an NK1-receptor antagonist, d-Arg1-d-Trp7,9-Leu11-substance P. These results indicate that histamine can induce cortisol secretion from BAC cells at physiological concentrations through H1 receptors on BAM cells, and catecholamine and substance P may participate in histamine-induced cortisol secretion.

Effects of External Osmotic Pressure on Vesicular Secretion from Bovine Adrenal Medullary Cells

Effects of External Osmotic Pressure on Vesicular Secretion from Bovine Adrenal Medullary Cells

Antibacterial Activity of Glycosylated and Phosphorylated Chromogranin A-derived Peptide 173-194 from Bovine Adrenal Medullary Chromaffin Granules

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).

Evidence for cholecystokininA receptors in bovine adrenal chromaffin cells.

We have studied a possible role of cholecystokinin (CCK) in regulating adrenal medullary function. Caerulein (10(-10)-10(-7) M), a CCK receptor agonist, increased formation of inositol phosphates in primary cultured bovine adrenal medullary (BAM) chromaffin cells in a concentration-dependent manner. The effect of caerulein was antagonized by devazepide, a selective CCKA-receptor antagonist, but not by L-365.260, a selective CCKB-receptor antagonist. These results suggest that BAM cells possess functional CCK receptors of the CCKA-subtype. Stimulation of these receptors with caerulein activates a signal transduction pathway via phospholipase C. CCK may regulate catecholamine release in BAM cells.

Phosphorylation of Chromogranin A and Catecholamine Secretion Stimulated by Elevation of Intracellular Ca2+ in Cultured Bovine Adrenal Medullary Cells

We have recently isolated a new endogenous substrate of 70 kDa for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from bovine adrenal medullary cells (Yanagihara, N., Toyohira, Y., Yamamoto, H., Ohta, Y., Tsutsui, M., Miyamoto, E., and Izumi, F. (1994) Mol. Pharmacol. 46, 423-430). Here we report the sequence analysis of the 70-kDa protein and examine its phosphorylation by various protein kinases in vitro and by depolarization of the cultured cells. Protein sequencing and immunoblotting revealed that the 70-kDa protein is chromogranin A (CgA) or a closely related protein. Partially purified CgA was phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C as well as CaM kinase II. Tryptic phosphopeptide mapping patterns of CgA differed among these protein kinases. In 32P-labeled bovine adrenal medullary cells, 56 mM K+ increased the phosphorylation of CgA and catecholamine secretion in similar time- and concentration-dependent manners, both of which were inhibited by 20 mM MgSO4, an inhibitor of voltage-dependent Ca2+ channels. These findings suggest that CgA serves as a substrate for several multifunctional protein kinases and that the elevation of the intracellular Ca2+ stimulates the phosphorylation of CgA associated with catecholamine secretion in cultured adrenal medullary cells.

Simultaneous amperometric measurement of ascorbate and catecholamine secretion from individual bovine adrenal medullary cells.

Secretion of ascorbate and catecholamines from single bovine adrenal medullary cells has been detected with amperometry at carbon-fiber microelectrodes. Two carbon-fiber electrodes were employed. One was beveled, and voltammograms at this electrode showed overlap of the responses for ascorbate and the catecholamines. The other was beveled and electrochemically oxidized to shift the ascorbate oxidation wave to potentials more negative than that of the catecholamines. Thus, at an applied potential of 0.05 V vs SSCE, ascorbate was selectively oxidized at the treated electrode and both catecholamines and ascorbate were oxidized at an applied potential of 0.65 V at the untreated electrode. Exocytotic release from the cell was stimulated with K+, nicotine, and digitonin. Nicotine and K+ depolarize the cell membrane and elicit vesicular release. Digitonin is a detergent that reacts with cholesterol in the plasma membrane and causes the formation of pores. Ascorbate efflux from individual cells could be induced by digitonin but not by K+ or nicotine and was observed as a single peak with a full width at half-maximum of 4 s. In contrast, catecholamine release was observed as many rapid, sequential current spikes when the cell was exposed to either digitonin, K+, or nicotine. The two different types of release show that ascorbate and catecholamines are being released from two different cellular compartments. The calcium independence of the digitonin-induced ascorbate release provides additional evidence that ascorbate is released by a nonexocytotic process.

Expression of the proenkephalin A gene and [Met5]-enkephalin secretion induced by arachidonic acid in bovine adrenal medullary chromaffin cells: involvement of second messengers.

We have previously reported that arachidonic acid (AA) increases the long-term secretion of [Met5]-enkephalin (ME) and the expression of proenkephalin A (proENK) mRNA in bovine adrenal medullary chromaffin (BAMC) cells. To characterize the underlying signal transductional mechanisms for the AA-induced responses, the interactions of AA with several second messenger systems were studied. Long-term (24-h) treatment with AA (100 microM) increased both the secretion of ME and the expression of proENK mRNA. Pretreatment of BAMC cells with nimodipine (1 microM), but not with omega-conotoxin GVIA (1 microM), inhibited the secretion of ME and the expression of proENK mRNA induced by AA. Calmidazolium (1 microM), a calmodulin antagonist, also significantly inhibited AA-induced responses. However, a protein kinase C (PKC) inhibitor, sphingosine (36 microM), was ineffective in blocking AA-induced responses. In addition, the down-regulation of PKC by phorbol 12-myristate 13-acetate (0.1 microM) for 48 h did not inhibit the AA-induced responses. Forskolin (5 microM), an adenyl cyclase activator, alone increased the secretion of ME as well as proENK mRNA levels and, when coincubated with AA, showed an additive effect on the secretion of ME and the levels of proENK mRNA. The results suggest that the Ca2+/calmodulin pathway, but not the protein kinase A or PKC pathway, is partially involved in mediating the AA-induced increases of the long-term secretion of ME and the levels of proENK mRNA.

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.