Chromaffin Cells In Primary Culture Research Articles

Refractive Index of Cells Measured through a Microscope with Variable-Angle TIR

The refractive index in the interior of single cells is interesting because it both indirectly senses the concentration of solutes in the cytoplasm, and also directly affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence. However, only a few studies have directly measured the intracellular refractive index, notably that of Bereiter-Hahn et al (1979) using quantitative interference reflection contrast (IRC) microscopy. IRC microscopy produces sharp images but does contain some inherent (but partly surmountable) ambiguities, particularly in distinguishing refractive index effects from distance -to-substrate effects. We present an alternative: direct microscopic imaging of a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance -to-substrate can be separately determined. We show how this non-fluorescence technique can be implemented in a modified objective-based TIR setup and we present results on both calibration samples and on chromaffin cells in primary culture. The optical technique suffers from rather poor lateral resolution, so subcellular organelles cannot be seen easily. However, the technique as combined with appropriate theory and calibration provides reasonable results for both refractive index (∼1.375) and for distance-to-substrate (∼100 nm) for the cells. Supported by NIH RO1-GM110289 to RWH and DA.

O2‐04‐01: Beta‐amyloid peptides undergo activity‐dependent secretion with neurotransmitters

Secretion and extracellular accumulation of neurotoxic beta-amyloid peptides (Abeta) represent a major factor in the development of Alzheimer's disease that results in memory deficit. Knowledge of the secretory pathway(s) utilized by Abeta peptides can enhance understanding of the neurobiology of Abeta. Because neurons undergo activity-dependent secretion of Abeta and neurotransmitters, this study sought to investigate the hypothesis of that Abeta undergoes co-secretion with neurotransmitters via the regulated secretory pathway that provides activity-dependent secretion of neurotansmitters. Neuronal-like chromaffin cells in primary culture were subjected to activity-dependent secretion, induced by KCl depolarization (50 mM, 30 min), and stimulated secretion of Abeta was measured by ELISA. Selected neuropeptides and catecholamines (dopamine, norepinephrie, and epinephrine) were measured in the secretion media. In addition, dense core secretory vesicles were purified from chromaffin cells and their content of Abeta peptides with neurotransmitters was measured. APP processing components (beta- and gamma-secretases) were assessed by western blots. Neuronal immunofluorescence confocal microscopy assessed localization of Abeta peptides with neurotransmitters. The data show that Abeta peptides undergo activity-dependent secretion from neuronal chromaffin cells (induced by KCl depolarization). Furthermore, Abeta undergos cosecretion with the peptide neurotransmitters (neuropeptides) and catecholamines. Investigation of the secretory vesicles that provide regulated secretion of signaling molecules from these cells showed that isolated dense core secretory vesicles (DCSV) of neuronal-like chromaffin cells contain Abeta peptides with multiple peptide neurotransmitters of enkephalin, NPY, galanin, VIP, and somatostatin. Furthermore, these secretory vesicles contain the catecholamine neurotransmitters dopamine, norepipnephrine, and epinephrine that are co-secreted with Abeta and neuropeptides. This organelle contains full-length APP, Abeta peptides, and components for beta- and gamma-secretases that produce Abeta. These APP processing components co-reside with processing proteases for neuropeptide biosynthesis, as well as with catecholamine biosynthetic enzymes. The colocalization of Abeta with neuropeptides in secretory vesicles was further illustrated by cellular immunofluorescence confocal microscopy. These findings indicate the biosynthesis and secretion of Abeta peptides in dense core secretory vesicles with neuropeptide and catecholamine neurotransmitters, suggesting that Abeta peptide forms function with multiple neurotransmitter systems.

Endopin Serpin Protease Inhibitors Localize with Neuropeptides in Secretory Vesicles and Neuroendocrine Tissues

Background/Aims: The endopin serpin protease inhibitors have been identified by molecular studies as components of secretory vesicles that produce neuropeptides. Endopin 1 inhibits trypsin-like serine proteases, and endopin 2 inhibits cathepsin L that produces neuropeptides in secretory vesicles. To assess the secretory vesicle and neuroendocrine tissue distribution of these endopins, the goal of this study was to define specific antisera for each endopin isoform and to examine their localization with neuropeptides and in neuroendocrine tissues. Methods: This study utilized methods consisting of Western blots, immunoelectron microscopy, and immunofluorescence microscopy for evaluation of the localization of endopin protease inhibitors in neuroendocrine tissues. Results: Immunoelectron microscopy with these selective antisera demonstrated the localization of endopins 1 and 2 within secretory vesicles of adrenal medulla (bovine). Cellular immunofluorescence confocal microscopy illustrated the high level of colocalization of endopins 1 and 2 with enkephalin and NPY neuropeptides that are present in secretory vesicles of adrenal medullary chromaffin cells in primary culture. Tissue distribution studies (by Western blots) showed the expression of endopins 1 and 2 in bovine brain, pituitary, adrenal medulla, and other neuroendocrine tissues. Conclusions: These results implicate endopins 1 and 2 as endogenous protease inhibitors in neuropeptide-containing secretory vesicles and neuroendocrine tissues.

Mechanisms of nitric oxide‐induced apoptosis in bovine chromaffin cells: Role of mitochondria and apoptotic proteins

The aim of this work was to establish the possible involvement of mitochondria in the apoptotic event triggered by nitric oxide (NO) in chromaffin cells. Using bovine chromaffin cells in primary culture and several NO donors (SNP, SNAP, and GSNO) at apoptotic concentrations (50 microM-1 mM), we have shown that NO induces a time-dependent decrease in the mitochondrial transmembrane potential (DeltaPsi(m)), which correlates with the appearance of hypodiploid cells. Disruption in DeltaPsi(m) is followed by cytochrome c release to the cytosol, which in turn precedes caspase 3 activation. In this mechanism participates the Bcl-2 protein family, because NO donors downregulate the expression of anti-apoptotic members of the family such as Bcl-2 and Bcl-XL, and increase the expression of pro-apoptotic members, Bax and Bcl-Xs, inductors of cytochrome c release to cytosol. Different cell signaling pathways seem to regulate Bax induction and Bcl-2 inhibition because decreased Bcl-2 levels are detected later than enhanced Bax expression. The tumour suppressor protein p53 is also upregulated in a very early phase (30 min) of the NO-induced apoptosis and may be responsible for the further induction of Bax expression. Finally, the translocation of NF-kappaB to the nucleus seems to be another early event in NO-induced apoptosis and it may be involved in the regulation of p53 expression. These results support strongly the participation of mitochondrial mechanisms in NO-induced apoptosis in chromaffin cells and suggest that these cells may be good models for the investigation of molecular basis of neurodegeneration and neuroprotection.

Molecular mechanisms of glutamate release by bovine chromaffin cells in primary culture

Previous work indicated that glutamate could be involved in the regulation of catecholamine secretion in bovine chromaffin cells. Thus, the question arises on the source of this putative regulatory glutamate. In this work we have examined the possibility that glutamate could be released from chromaffin cells. Data from this study indicate that chromaffin cells are able to release glutamate when they are stimulated by different depolarising agents such as 60 mM KCl, 1 mM 4-aminopyridine or 50 μM veratridine. The amount of glutamate released by these compounds was 0.32 nmol/10 6 cells (9.24% of cellular glutamate content), 0.275 (7.86%) and 0.158 (4.52%) for KCl, 4-AP and veratridine stimulation, respectively. All these catecholamine-secretagogues induced glutamate secretion by two mechanisms: 1) a Ca 2+-dependent, probably exocytotic, mechanism and 2) a Ca 2+-independent mechanism mediated by reversion of the electrogenic glutamate transporter. Analysis of Ca 2+-dependent and independent releases for different compounds carried out by several experimental approaches, indicate that Ca 2+-dependent release was the predominant mechanism for release induced by 4-aminopyridine (84% of total release) and high KCl (63%) whilst Ca 2+-independent release was predominant for veratridine (67%). The Ca 2+-dependent glutamate release evoked by depolarisation of chromaffin cells with high KCl and 4-AP could be split into both a fast and a slow kinetic component, which might correspond to the release of docked and mobilised chromaffin granules, respectively. On the other hand, depolarisation of cells with veratridine result in glutamate release with only the fast kinetic component. In the case of 60 mM KCl-evoked glutamate release, the fast component exhibited a decay time of <1 s and accounted for 0.63 nmol glu/6×10 6 cells (70% of total exocytotic release), whereas the slow component, which exhibited a decay time of 231 s, accounted for the release of 0.27 nmol glu/6×10 6 cells (30% of total exocytotic release). By contrast in the case of 4-aminopyridine the fast component of exocytosis only represents a 19% of total secretion and the slow a 81% with a decay time of 94 s. These data are very similar to those found in neurones and support the possible intracellular origin of glutamate having a role in the regulation of catecholamine secretion from chromaffin cells. In support of this, we have found that glutamate secretion could be evoked by stimulation of the nicotinic cholinergic receptors.

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles.

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.

Rab3 is present on endosomes from bovine chromaffin cells in primary culture.

Rab3a, a small GTP-binding protein, is believed to mediate Ca2+-dependent exocytosis. Consistent with such a role was the previously reported specific association of Rab3a with synaptic vesicles in neurons and secretory granules in adrenal chromaffin cells. Secretory vesicles are believed to be the final point of Rab3a membrane association, as it was shown by several groups that Rab3a dissociates from the secretory vesicle membrane during stimulated exocytosis. In chromaffin cells, Rab3a is not exclusively localized on secretory granules since a fraction is present on a previously unidentified subcellular compartment equilibrating at light sucrose density. This 'light' membraneous structure could be the starting point for reassociation of Rab3a with membranes involved in granule formation, or it could be a structure unrelated to granules. The present study used several subcellular fractionation techniques and immunomicroscopy to unravel the nature of the 'light' Rab3a-containing structures from bovine chromaffin cells in primary culture. After stimulation, amounts of both Rab3a-d and the granule marker dopamine-beta-hydroxylase (DbetaH) increase transiently in sucrose gradient fractions enriched in endosomal markers. A diaminobenzidine-induced density shift of endosomes alters the distribution of DbetaH and Rab3a-d. At the ultrastructural level, subplasmalemmal pleiomorphic organelles were detected by Rab3a-d-immunogold labelling. Taken together our data provide for the first time evidence that internalised secretory granule membranes go through an endosomal stage where Rab3a is present, resembling the neuronal synaptic vesicle cycle. This indicates that the endosome is an important trafficking route in the biogenesis/recycling of secretory vesicles in chromaffin cells, in which Rab3a could have an as yet unknown regulatory function, and could point to the existence of alternative recycling pathways for the chromaffin granule membrane.

Pharmacological protection against the cytotoxicity induced by 6-hydroxydopamine and H 2O 2 in chromaffin cells

We present in this report the characteristics of the damage induced by 6-hydroxydopamine and H 2O 2 on bovine chromaffin cells in primary culture. Cytotoxicity was quantified using catecholamine cell contents, lactate dehydrogenase (LDH) release, trypan blue exclusion and morphological appearance. An excellent correlation between these four parameters was found. The cytotoxic effects of 6-hydroxydopamine were Ca 2+-independent. In spite of this, the Ca 2+ channel antagonists R56865 ( N-[1-(4-(fluorophenoxy)butyl)]-4-piperidinyl- N-methyl-2-benzo-thiazolamine) and lidoflazine exhibited marked cytoprotective effects against both 6-hydroxydopamine and H 2O 2. The selective dopamine uptake blocker, bupropion, increased the viability of 6-hydroxydopamine and H 2O 2-treated cells from 20% to around 80%. Catalase drastically protected against the cytotoxic effects of 6-hydroxydopamine and H 2O 2. In contrast, desferrioxamine gave better protection against H 2O 2 cytotoxicity; glutathione and N-acetylcysteine only afforded substantial protection against 6-hydroxydopamine. Three main conclusions emerge from this study. (1st) 6-Hydroxydopamine causes chromaffin cell damage via a mechanism probably related to the production of free radicals, but unrelated to Ca 2+ ions. Cytoprotection afforded by R56865 and lidoflazine must be unrelated to their Ca 2+ antagonist properties. This suggests a novel component in the cytoprotective mechanism of action of these drugs. (2nd) The strong cytoprotective effects of bupropion seem to be unrelated to its ability to block the plasmalemmal dopamine carrier. (3rd) Bovine adrenal chromaffin cells in primary cultures are a suitable model for adult neurons to study the basic mechanism of cell damage, and to screen new drugs with putative neuroprotective properties.

Ca 2+] i oscillations in rat chromaffin cells: frequency and amplitude modulation by Ca 2+ and InsP 3

Rat chromaffin cells in primary culture exhibit oscillations of cytosolic Ca 2+ concentration, sustained by the rhythmic discharge of Ca 2+ from specialized intracellular stores. Each Ca 2+ spike starts from a discrete region of the cell (pacemaker), and then propagates across the entire cytosol. Spike initiation and propagation, governing the oscillation frequency and amplitude respectively, appeared to be controlled by different mechanisms. The pacemaker was found to be directly activated by increases of cytosolic Ca 2+ concentration obtained by either K + depolarization or nicotinic stimulation. On the other hand, muscarinic or B 2 stimulation was required for an efficient spreading to occur, thus suggesting a key role of InsP 3 in the signal propagation. The pacemaker displayed an autonomous activity, as documented by the presence of local Ca 2+ discharges, which were not necessarily accompanied by spreading to the rest of the cell. This uncoupling could be stimulated by the selective increase of the pacemaker firing rate, due to the rise of the intracellular Ca 2+ concentration. Modulation of Ca 2+ spike amplitude by treatments affecting either the pacemaker or the spreading phase might be related to quantal Ca 2+ release from functionally discrete stores.

Oscillations of Cytosolic Calcium in Rat Chromaffin Cells: Dual Modulation in Frequency and Amplitude

Rat chromaffin cells in primary culture have a high tendency to exhibit [Ca 2+] i oscillations, spontaneously or after moderate stimulation with treatments that induce polyphosphoinositide hydrolysis or mild depolarization. Previous studies in a variety of cell systems had shown that strengthening of the above treatments increases the frequency and not the amplitude of the oscillations. We now demontrate that in cultured chromaffin cells either one of these oscillation reinforcements can be elicited, depending on whether the Ca 2+ influx induced by the applied stimulus is asynchronous with or timed by the [Ca 2+] i spikes of the oscillations. In excitable cells the encoding of the oscillation activity appears therefore to operate according to not only digital (modulation in frequency) but also analog (modulation in amplitude) models.

Regulation of c-fos expression by IGF-I in bovine chromaffin cells: desensitization following cholinergic activation

Effects of insulin-like growth factor I on the expression of the immediate-early gene c-fos in adrenomedullary tissue were assessed in bovine chromaffin cells in primary culture. The expression of c-fos mRNA and FOS protein were studied by Northern blot and immunofluorescence microscopy. IGF-I induced c-fos mRNA expression in a dose-dependent manner, reaching a maximum at 10–100 nM after 30 min incubation. Expression of FOS protein exhibited a transient time course peaking after 60–90 min. Repeated exposures to IGF-I attenuated c-fos transcriptional activity with a temporal coincidence of 90% repression and maximal FOS protein levels. Exposure to the cholinergic agonist carbachol or the protein tyrosine phosphatase inhibitor orthovandate also induced c-fos transcription. Both agents caused repression in c-fos gene activation induced by either IGF-I, carbachol or orthovanadate. This suggested autoinhibition of c-fos transcription by FOS protein synthesized during the initial stimulation period. These data are consistent with IGF-I stimulating c-fos gene activation in the adrenal medulla and show that c-fos expression can be negatively regulated by temporal neural and hormonal stimulation.

Tumor necrosis factor alpha inhibits contractions to sympathetic nerve stimulation by a nitric oxide-dependent mechanism.

Gram-negative sepsis and administration of tumor necrosis factor alpha (TNF alpha) are associated with hypotension and peripheral neuropathies suggestive of impaired sympathetic neurotransmission. We examined the effect of TNF alpha on the responses of the bovine pulmonary artery (BPA) to transmural sympathetic nerve stimulation (SNS). BPA contracted to SNS (0.5-32 Hz, 5-10 V, 2-msec duration, 2-msec delay) in a frequency-dependent manner. The contractions of the BPA to SNS were mediated by norepinephrine and activation of postsynaptic alpha 1-adrenoceptors, since they were attenuated by prazosin. Maximum contraction of the BPA to SNS was significantly enhanced (148 +/- 37% increase, n = 6) after inhibition of nitric oxide synthase with L-NG-monomethylarginine (LNMMA, 500 microM), an effect abrogated by L-arginine (1 mM). TNF alpha (0.0042, 0.042, and 0.42 micrograms/ml) selectively inhibited contractions of the BPA to SNS without affecting the contraction of the BPA to exogenous norepinephrine. In BPA incubated with LNMMA (5-500 microM), TNF alpha facilitated rather than inhibited SNS. TNF alpha increased the formation of amperiometrically measured free nitric oxide in bovine adrenal chromaffin cells in primary culture. The data show that in the absence of LNMMA, TNF alpha releases free nitric oxide from a sympathetic neuron and selectively inhibits the contractions of the BPA to SNS. In BPA in which nitric oxide synthase I is inhibited by LNMMA, TNF alpha amplifies the contractions to SNS, even in the absence of endothelium. Thus, TNF alpha can modify vascular smooth muscle tone by affecting SNS. TNF alpha inhibits SNS at the level of the neuron by a mechanism involving the L-arginine-nitric oxide pathway. TNF alpha-induced suppression of SNS and neurotransmission may contribute to the hypotension and peripheral neuropathy of sepsis.

IGF-I-induced c-fos and c-jun expression in bovine chromaffin cells in primary culture.

IGF-I-induced c-fos and c-jun expression in bovine chromaffin cells in primary culture.

Comparison of the intracellular effects of clostridial neurotoxins on exocytosis from streptolysin O-permeabilized rat pheochromocytoma (PC 12) and bovine adrenal chromaffin cells

The inhibitory effects of tetanus toxin, botulinum toxin A, their constituent light chains, and botulinum toxin B were compared using streptolysin O-permeabilized rat pheochromocytoma (PC 12) and bovine adrenal chromaffin cells in primary culture. In both types of chromaffin cells exocytosis can be triggered by micromolar amounts of free Ca 2+, bovine adrenal chromaffin cells in addition require ATP. In PC 12 cells the isolated tetanus toxin light chain alone blocks exocytosis without any additive. The time-course of the inhibitory action of tetanus toxin light chain in permeabilized PC 12 cells in the absence of ATP is similar to the one obtained with permeabilized bovine adrenal chromaffin cells, in the presence of ATP. Thus, ATP does not seem to be crucial for tetanus toxin (two-chain form) poisoning. Botulinum toxin B (two-chain form), if preactivated by dithiothreitol, also inhibits exocytosis from permeabilized PC 12 cells up to 90% in the absence of ATP. By contrast, botulinum toxin A (two-chain form) or its isolated light chain, which are highly potent in permeabilized bovine adrenal chromaffin cells, causes only a weak inhibition in PC 12 cells. In streptolysin O-permeabilized bovine adrenal chromaffin cells omission of ATP during the incubation with the toxin increases the potency of botulinum toxin A light chain. Under the same conditions the effect of tetanus toxin light chain remains unchanged. Tetanus toxin and botulinum toxin B (two-chain forms) probably block a step which occurs during exocytosis from both PC 12 cells and adrenal chromaffin cells and which could be closely related to the final fusion event. Botulinum toxin A (two-chain form) affects a more distal step almost completed in PC 12 cells. The botulinum toxin A (two-chain form)-sensitive event might be an ATP-dependent step, which is necessary for the priming of chromaffin vesicles.

Enkephalin biosynthesis is coupled to secretory activity via transcription of the proenkephalin A gene

The molecular mechanisms regulating neuropeptide and secretory protein biosynthesis in neuroendocrine cells were examined using the prototype neuropeptide and secretory proteins enkephalin and chromogranin A (CGA). Treatment with the secretogogue nicotine results in the calcium-dependent secretion of enkephalin peptides from bovine chromaffin cells in primary culture and a concomitant increase in enkephalin peptide biosynthesis. Both secretion and biosynthesis are also stimulated by cell depolarization with elevated potassium. Elevation of intracellular cyclic AMP, on the other hand, results in stimulation of enkephalin biosynthesis and long-term, but not acute, secretion of enkephalin peptides. Coupling of enkephalin biosynthesis to calcium influx occurs at the level of transcription of the enkephalin gene. Thus, potassium depolarization causes a calcium-dependent elevation of enkephalin mRNA which is preceded by an increase in the rate of transcription of the enkephalin gene in the chromaffin cell. The accumulation of enkephalin message or peptide by potassium depolarization or treatment with nicotine is prevented by D600 or hexamethonium respectively, added 1 h after addition of nicotine or KCl and following acute release, suggesting that calcium acts as a continuous rather than triggering stimulus for enkephalin biosynthesis. Sequence analysis of the bovine enkephalin promoter identified sequence conservation of three enhancers previously reported in the human gene which are required for regulation of the gene by calcium, cAMP, and phorbol ester in vitro. In contrast to the regulation of the enkephalin system, no increase in either CGA or CGB mRNA or gene transcription attended depolarization-induced secretion from chromaffin cells. Since enkephalin and CGA are co-stored in and co-released from the same secretory vesicles in these cells, the results imply that a surplus of CGA is constitutively synthesized in chromaffin cells such that compensatory up-regulation during changes in the secretory state of the cell, such as occurs for enkephalin, is not required for the secretory proteins.

Catecholamine release from bovine adrenal chromaffin cells during anoxia or metabolic inhibition.

A significant release of catecholamines within the heart has been observed during myocardial ischemia. Because this can be markedly inhibited by amine-uptake-blocking agents, it has been suggested that its mechanism is a carrier-mediated efflux from neurons, which is not operative under normal conditions. The present work examined this release process in chromaffin cells isolated from the bovine adrenal medulla, a model system for studying the sympathetic nervous system. Chromaffin cells in primary culture retained normal secretory responses for up to 7 days. Conditions designed to mimic ischemia, that is, anoxia or metabolic inhibition, resulted in a significant release of catecholamines. This release was shown to be independent of extracellular calcium but, in contrast to the release observed in ischemic hearts, was not inhibited by amine-uptake blockers. Electrophoresis with immunoblotting demonstrated that significant levels of the chromaffin granule protein, chromogranin A, were released during metabolic inhibition, indicative of an exocytotic mechanism. However, there was no release of the cytosolic protein, lactate dehydrogenase, indicating that there was no concomitant breakdown of the cell membrane. These results provide evidence for an exocytotic release of catecholamines mediated by the direct action of conditions of metabolic inhibition.

Binding of insulin and insulin-like growth factor I in bovine chromaffin cells in primary culture

1. 1. Insulin and insulin-like growth factor I (IGF-I) receptors were studied in bovine chromaffin cells isolated from the medulla by collagenase digestion and kept in primary culture. 2. 2. Specific 125I-labelled insulin binding increased with time in culture with no significant change in the dissociation constant, K d ~0.3nM. Insulin was nearly 100-fold more potent than IGF-I in displacing 125I-labelled insulin. 3. 3. Affinity crosslinking and SDS gel electrophoresis revealed increased binding of 125I-labelled insulin and 125I-IGF-I with time in culture, the densities of the labelling indicating relatively a much higher expression of IGF-I than insulin receptors in the cells. The apparent molecular weight of both the hormone binding subunits were 135,000, suggesting that the insulin and IGF-I receptors in the adrenal medulla are of the peripheral types. 4. 4. Both receptors thus appeared to be affected by the collagenase treatment but with a subsequent recovery when cells were kept in culture.

Galanin Gene Expression in Chromaffin Cells Is Controlled by Calcium and Protein Kinase Signaling Pathways

The neuropeptide galanin (GAL) is widely distributed throughout the diffuse neuroendocrine system, and is coexpressed with acetylcholine, norepinephrine, prolactin, and a variety of other messenger substances in different cell types. Bovine chromaffin cells in primary culture synthesize and store GAL along with catecholamines, chromogranin A, and enkephalin peptides, as well as other neurosecretory products, and secrete all these molecules in response to nicotinic stimulation. The regulation of GAL biosynthesis and secretion were studied by measuring changes in messenger RNA (mRNA(GAL], and peptide immunoreactivity, 24-72 h after stimulation of secretion (40 mM potassium or 10 microM veratridine), or exposure to stimulators of protein kinase C (100 nM phorbol myristate acetate) and protein kinase A (25 microM forskolin). Depolarization-induced stimulation of GAL biosynthesis, like that of enkephalin and other neuropeptides, was calcium dependent, suggesting that calcium generally mediates both exocytotic release and new peptide synthesis thus coordinating maintenance of neuropeptide levels in chromaffin cells. GAL and mRNA(GAL) were also upregulated by stimulation of protein kinase A with forskolin. Treatment with PMA increased GAL and mRNA(GAL) to an even greater extent than depolarization. Thus GAL expression can be regulated by three distinct signal transduction systems in chromaffin cells: depolarization-stimulated calcium influx, activation of protein kinase C and activation of protein kinase A, which in addition differentially up- and down-regulate the expression of several other neurosecretory proteins and peptides resulting in different patterns of GAL/neuropeptide coexistence in bovine chromaffin cells. GAL coexistence with diverse neuroendocrine substances may reflect the relative activity of these three signalling systems in other neuroendocrine cell types as well.

Mechanisms of [3H] γ‐aminobutyric acid release by chromaffin cells in primary culture

The basal and evoked [3H] gamma-aminobutyric acid (GABA) release from chromaffin cells in primary cultures was studied and compared with that of [3H]NA. [3H]GABA was found to be released, in a dose-dependent fashion, by different secretagogues known to induce noradrenaline (NA) release, that is, the cholinergic agonist nicotine, high-potassium chloride, veratridine, and calcium ionophores. In general, there was a parallelism between percentages of release of both [3H]GABA and [3H]NA, although in all circumstances the former were lower. The nicotine- and high-potassium-evoked [3H]GABA release was absolutely calcium dependent, thus indicating the existence of a exocytotic-like mechanism, whereas in the veratridine-induced release, a calcium-independent component was also detected. This latter component was sodium dependent, as it showed an absolute requirement for extracellular sodium and was enhanced by ouabain. Moreover, it was inhibited by known GABA uptake inhibitors, which indicate that this component of [3H]GABA release induced by veratridine could be due to GABA outflow through the membrane carrier. The above results, together with that obtained from studies about subcellular localization of [3H]GABA taken up by chromaffin cells, seem to support the existence of two mechanisms for [3H]GABA release by chromaffin cells: one calcium-dependent, exocytotic-like, and another calcium-independent and sodium-dependent, possibly mediated by the GABA carrier. Both processes could have a functional role on the regulation of extracellular GABA levels and so in the control of catecholamine release by chromaffin cells.

Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture

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