Bovine Adrenal Medullary Chromaffin Cells Research Articles

Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles.

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. Graphical Abstract ᅟ.

Design, Characterization, and Optimization of a Broadband Mini Exposure Chamber for Studying Catecholamine Release From Chromaffin Cells Exposed to Microwave Radiation: Finite-Difference Time-Domain Technique

A free-space in vitro exposure system for identifying specific microwave (MW) parameters in the frequency range of 1-6 GHz that can induce nonthermal effects on exocytosis, which is the process by which neurotransmitter release occurs, has been designed, constructed, characterized, and optimized. The exposure system is placed within an anechoic chamber and incorporates continuous online monitoring of basal and stimulated catecholamine release from cultured bovine adrenal-medullary chromaffin cells, which are well-established models of neural-type cells. The cells are immobilized inside a cell-perfusion apparatus (CPA) and are continuously superfused with temperature-controlled balanced salt solution, with the entire CPA placed within a mini exposure chamber (MEC) constructed out of a MW-absorbing material. All relevant equipment for carrying out the experiments is shielded from the MW field by being housed in an aluminum conductor box located behind the MEC. Detailed distributions of the electric field and the specific absorption rate (SAR) at the location of the cells within the MEC were computed using the finite-difference time-domain (FDTD) method. FDTD computations were also used for optimizing the exposure system, so that the highest intensity of electric field could be delivered under dynamic temperature control and with an acceptable degree of field homogeneity (to within 30%) over the entire frequency range of 1-6 GHz. A major finding is that maintaining an acceptable level of homogeneity of the electric field and SAR for exposing cells to 1-6 GHz MW fields requires a different distribution of the cells within the CPA for exposures carried out at the lower versus the higher end of the frequency range of interest

The Tyrosine Phosphorylation and Cytoskeletal Translocation of Phospholipase C 1 in Bovine Adrenal Medullary Chromaffin Cells

The tyrosine phosphatase inhibitor BpV(phen) stimulated a concentration-dependent increase of phospholipase C (PLC) activity in bovine adrenal medullary chromaffin cells. This response was accompanied by an increase in PLCgamma1 tyrosine phosphorylation and its cytosketetal translocation. Insulin, at high concentrations, stimulated PLC activity to a similar extent as BpV(phen), a response that was also accompanied by an increase in PLCgamma1 translocation but not its tyrosine phosphorylation. BpV(phen) strongly enhanced the insulin-stimulated increase in PLC activity and caused a small rise in PLCgamma1 translocation above that seen with insulin alone. Despite the synergistic rise in activity PLCgamma1 tyrosine phosphorylation did not increase beyond that seen with BpV(phen) alone. These results indicate that PLCgamma1 activation in chromaffin cells may be more closely associated with its cytoskeletal translocation than its tyrosine phosphorylation although other factors may also be important for activation of enzyme activity.

A Novel Waveguide-Based Radio Frequency/Microwave Exposure System for Studying Nonthermal Effects on Neurotransmitter Release—Finite-Difference Time-Domain Modeling

A research effort is underway to identify specific radio frequency/microwave parameters in the frequency range 0.75-1.12 GHz that can produce nonthermal effects on the release of catecholamines from cultured bovine adrenal medullary chromaffin cells, an established in vitro model of neural-type cells. A well-characterized exposure system is crucial for interpreting the biological outcomes of experiments. This paper describes a novel waveguide-based exposure system that permits perfusion of the cells with a temperature-controlled balanced salt solution for online monitoring of catecholamine release from the cells during radio frequency/microwave exposure. The finite-difference time-domain method was used to optimize the exposure conditions, the goal being to achieve the maximum possible homogeneity in the distribution of the specific absorption rate at the location of the cells in the waveguide. At a frequency of 1 GHz, optimal coupling of the radio frequency/microwave field into the region containing the cells was obtained only when the cells were placed at the location of the electric field maximum of the standing wave pattern, with the electric field parallel to the plane containing the cells. In this case, the specific absorption rate distribution was found to be the highest as well as homogeneous to within 30%.

Microencapsulated Bovine Adrenal Medullary Chromaffin Cells Transplanted into Rat Spinal Cord Alleviated Cold Allodynia

Background: The intrathecal grafting of adrenal chromaffin cells as a potential analgesic source, to delivery analgesic substances such as catecholamines and opioid peptides, is known to be effective at treating acute and chronic pain in several animal pain models. We tested whether the intrathecal implantation of encapsulated bovine chromaffin cells reduces cold allodynia in a rat model of neuropathic pain induced by chronic constriction injury of the sciatic nerve. Methods: Bovine adrenal medullary chromaffin cells microencapsulated in sodium alginate-poly-I-lysin-alginate (APA) were implanted into the subarachnoid space of rats (n = 10) and foot cold sensitivity was investigated using an acetone test. At the end of the study, histology and capsule catecholamine production were evaluated. Results: A significant reduction in cold allodynia was observed in animals implanted with chromaffin cells. In addition, the suppression of cold allodynia was reversed by naloxone. Abundant clusters of viable chromaffin cells stained with neutral red, were observed in the retrieved implants and after nicotine stimulation, and catecholamine was quantified. An ultrastructural study showed no fibrotic reaction against capsules, or disorganised capsules. Conclusions: These results suggest that intrathecal encapsulated chromaffm cells act as mini pumps, which continuously deliver analgesic substances and produce analgesia in this chronic pain model of nerve injury-without immunosuppressant.

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells.

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.

SNX482 selectively blocks P/Q Ca 2+ channels and delays the inactivation of Na + channels of chromaffin cells

The effects of the toxin SXN482 on Ca 2+ channel currents ( I Ca), Na + currents ( I Na), and K + currents ( I K) have been studied in bovine adrenal medullary chromaffin cells voltage-clamped at −80 mV. Currents were elicited by depolarising pulses to 0–10 mV ( I Ca and I Na) or to +60 mV ( I K). SNX482 blocked I Ca in a concentration-dependent manner. The inhibition curve exhibited two phases. The first high-affinity phase comprised 28% of the whole-cell current and exhibited an IC 50 of 30.2 nM. The second low-affinity phase comprised over 70% of I Ca and had an IC 50 of 758.6 nM. Blockade was rapid and fully reversible upon washout of the toxin. Occlusion experiments showed additivity of blockade exerted by nifedipine plus SNX482 (0.3 μM) and by ω-conotoxin GVIA plus SNX482. In contrast, blockade exerted by combined ω-agatoxin IVA plus SNX482 (about 50% of the whole cell) did not show additivity. At 0.3 μM and higher concentrations, SNX482 delayed the inactivation of I Na. The time constant ( τ) for inactivation of I Na in control conditions doubled in the presence of 0.5 μM SNX482. At 0.3 μM, SNX482 did not affect I K. Our data demonstrate that: (i) SNX482 selectively blocks P/Q Ca 2+ channels at submicromolar concentrations; (ii) the toxin partially blocks Na + channels; (iii) SNX482 delays the inactivation of Na + channels. These results reveal novel properties of SNX482 and cast doubts on the claimed selectivity and specificity of the toxin to block the R-type Ca 2+ channel.

The involvement of specific phospholipase C isozymes in catecholamine release from digitonin permeabilized bovine adrenal medullary chromaffin cells

The role of phospholipase C (PLC) in exocytosis has been investigated using digitonin permeabilized, [ 3H]noradrenaline ([ 3H]NA) loaded, bovine adrenal medullary chromaffin cells. The PLC inhibitor U-73122 caused a concentration-dependent suppression of Ca 2+-evoked [ 3H]NA release but increased basal release (that occurring in the absence of Ca 2+). Preincubation with antibodies against PLCγ1 or PLCβ3 (but not PLCδ1, δ2, β1 and β2) also inhibited [ 3H]NA release evoked by Ca 2+ and increased basal release, indicating that only specific PLC isozymes are involved in these actions. Interestingly, PLCγ1 (but not PLCβ3) antibodies inhibited the ability of Ca 2+ to increase PLC activity in these permeabilized cells. These data therefore suggest that PLCγ1 activity may have a specific role in regulating the exocytotic response from the adrenal chromaffin cell.

Catecholamine release from cultured bovine adrenal medullary chromaffin cells in the presence of 60-Hz magnetic fields

Effects of powerline frequency (50/60 Hz) electric and magnetic fields on the central nervous system may involve altered neurotransmitter release. This possibility was addressed by determining whether 60-Hz linearly polarized sinusoidal magnetic fields (MFs) alter the release of catecholamines from cultured bovine adrenal chromaffin cells, a well-characterized model of neural-type cells. Dishes of cells were placed in the center of each of two four-coil Merritt exposure systems that were enclosed within mu-metal chambers in matched incubators for simultaneous sham and MF exposure. Following 15-min MF exposure of the cells to flux densities of 0.01, 0.1, 1.0 or 2 mT, norepinephrine and epinephrine release were quantified by high-performance liquid chromatography (HPLC) coupled with electrochemical detection. No significant differences in the release of either norepinephrine or epinephrine were detected between sham-exposed cells and cells exposed to MFs in either the absence or presence of Bay K-8644 (2 μM) or dimethylphenylpiperazinium (DMPP, 10 μM). Consistent with these null findings is the lack of effect of MF exposure on calcium influx. We conclude that catecholamine release from chromaffin cells is not sensitive to 60-Hz MFs at magnetic flux densities in the 0.01–2 mT range.

Do Microencapsulated Bovine Adrenal Medullary Chromaffin Cells Transplanted in Rats' Spinal Cord Produce Analgesic Effects?

Background: Pain remains the chief complaint that brings patients to physician's office, despite recent insights into underlying mechanism and the identification of potential new therapeutic targets. In recent years, however, with the development of molecular biology cell transplantation gives us a new chance for treating intractable chronic pain. The major purpose of the present study was to determine if the chromaffin cells that were encapsulated with 1.3% (w/v) sodium alginate-poly-l-lysine-alginate (APA) had robust analgesic effects in the spinal atlanto-occipital subarachnoid space even without nicotine stimulation. Methods: In order to determine whether microencapsulated bovine adrenal medullary chromaffin cells transplanted in the spinal cord can produce analgesic effects, we microencapsulated adrenal medullary chromaffin cells with APA and implanted them into the subarachnoid space of rats' (n = 10) spinal cord, and investigated the hot sensitivity of rats' hind-paw by a light-beam test. Results: It was found that compared with the control group, hot response latency of the group which received adrenal medullary chromaffin cells increased from the 12th day and the analgesic efficacy was maintained for at least 75 days. Conclusions: Microencapsulated bovine adrenal medullary chromaffin cells transplanted in the rats' spinal cord may provide a permanent and locally available source of neuropeptides for the relief of intractable pain. Furthermore, these kinds of analgesic effect were produced without any stimulation such as nicotine.

Angiotensin II causes calcium entry into bovine adrenal chromaffin cells via pathway(s) activated by depletion of intracellular calcium stores.

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to angiotensin 11 have been investigated. In fura-2 loaded cells exposure to a maximally effective concentration of angiotensin II (100 nM) caused a rapid, but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+ only the initial brief transient was observed. In cells previously treated with thapsigargin in Ca2+-free medium to deplete the internal Ca2+ stores, angiotensin II caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Reintroduction of external Ca2+ to thapsigargin-treated, store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was not further increased upon exposure to angiotensin II. Analysis of the data suggests that in bovine chromaffin cells angiotensin II causes Ca2+ entry via a pathway(s) activated as a consequence of internal store mobilization, and entry through this pathway(s) forms the majority of the sustained Ca2+ influx evoked by angiotensin II.

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.

Localization of the Secretory Granule Marker Protein Chromogranin B in the Nucleus: POTENTIAL ROLE IN TRANSCRIPTION CONTROL

Chromogranins A (CGA) and B (CGB) are two major Ca(2+) storage proteins of the secretory granules of neuroendocrine cells. Nevertheless, we found in the present study that CGB was also localized in the nucleus. In immunogold electron microscopy using bovine adrenal medullary chromaffin cells, it was found that the number of CGB-labeled gold particles localized per microm(2) of the nucleus was equivalent to 20% that of CGB-labeled gold particles localized per microm(2) of the secretory granules. Considering that CGB is estimated to exist in the 0.1-0.2-mm range in the secretory granules of bovine chromaffin cells, 20% of these amounts to 20-40 microm. In addition, transfection of CGA and CGB into nonneuroendocrine COS-7 and NIH3T3 cells repeatedly indicated the nuclear localization of CGB in addition to its usual localization in the cytoplasm. Moreover, immunoblot and immunogold electron microscopy analyses of neuroendocrine PC12 cells also showed the existence of endogenous CGB in both the cytosol and the nucleus. Nuclear routing of CGB did not appear to depend entirely upon the nuclear localization signal as some of the nuclear localization signal mutant CGB were still targeted to the nucleus. In gene array assay, CGB was shown to either induce or suppress transcription of many genes including those of transcription factors. Of these we have analyzed eight genes, four induced (zinc finger protein, MEF2C, hCRP2, abLIM) and four suppressed (hcKrox, T3-receptor, troponin C, integrin) using the quantitative reverse transcription-PCR method and spectrophotometry to determine the transcription levels of each mRNA. CGB was shown to increase the transcription of zinc finger protein, MEF2C, hCRP2, and abLIM by 2.5-5-fold while suppressing that of hcKrox, T3-receptor, troponin C, and integrin by 60-75%. Given that MEF2C and hcKrox genes are transcription factors, these results pointed to the transcription control role of CGB in the nucleus.

Localization of Three Types of the Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channel in the Secretory Granules and Coupling with the Ca2+ Storage Proteins Chromogranins A and B

Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca(2+) store and the presence of the IP(3) receptor (IP(3)R)/Ca(2+) channel on the secretory granule membrane have been established, the identity of the IP(3)R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP(3)R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP(3)R in the secretory granules. To determine whether these IP(3)Rs interact with CGA and CGB, each IP(3)R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP(3)R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP(3)R form complexes with CGA and CGB in the cells. To further confirm whether the IP(3)R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP(3)R and CGA and CGB was tested by co-immunoprecipitation experiments of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP(3)R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP(3)R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca(2+) binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca(2+)/mol with a dissociation constant (K(d)) of 1.5 mm at pH 5.5 but virtually no Ca(2+) at pH 7.5. The high capacity, low affinity Ca(2+)-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca(2+) storage protein in the secretory granules.

Blockade by agmatine of catecholamine release from chromaffin cells is unrelated to imidazoline receptors

The blockade of exocytosis induced by the putative endogenous ligand for imidazoline receptors, agmatine, was studied by using on-line measurement of catecholamine release in bovine adrenal medullary chromaffin cells. Agmatine inhibited the acetylcholine-evoked release of catecholamines in a concentration-dependent manner (IC 50=366 μM); the K +-evoked release of catecholamines was unaffected. Clonidine (100 μM) and moxonidine (100 μM) also inhibited by 75% and 50%, respectively, the acetylcholine-evoked response. In cells voltage-clamped at −80 mV, the intermittent application of acetylcholine pulses elicited whole-cell inward currents ( I ACh) that were blocked 63% by 1 mM agmatine. The onset of blockade was very fast ( τ on=31 ms); the recovery of the current after washout of agmatine also occurred very rapidly ( τ off=39 ms). Efaroxan (10 μM) did not affect the inhibition of I ACh elicited by 1 mM agmatine. I ACh was blocked 90% by 100 μM clonidine and 50% by 100 μM moxonidine. The concentration–response curve for acetylcholine to elicit inward currents was shifted to the right in a non-parallel manner by 300 μM agmatine. The blockade of I ACh caused by agmatine (100 μM) was similar at various holding potentials, around 50%. When intracellularly applied, agmatine did not block I ACh. At 1 mM, agmatine blocked I Na by 23%, I Ba by 14%, I K(Ca) by 16%, and I K(VD) by 18%. In conclusion, agmatine blocks exocytosis in chromaffin cells by blocking nicotinic acetylcholine receptor currents. In contrast to previous views, these effects seem to be unrelated to imidazoline receptors.

Ba 2+-induced chromaffin cell death: cytoprotection by Ca 2+ channel antagonists

Exposure of bovine adrenal medullary chromaffin cells to Ba 2+ ions (in the absence of Ca 2+ ions) caused their death, measured as lactate dehydrogenase (LDH) release. The concentration of Ba 2+ required to damage the cells by about 65% ranged between 1 and 10 mM (no Ca 2+ added); the required exposure time was rather brief (15 min–4 h). The simultaneous presence of Ca 2+, Mg 2+ or Zn 2+ together with Ba 2+ (2 mM, 4 h) afforded cyprotection (60–80%). Individual selective blockers of Ca 2+ channel subtypes afforded no protection. However, combined nifedipine (3 μM) plus ω-conotoxin MVIIC (3 μM) offered full protection. Substantial protection was also seen with the “wide-spectrum” Ca 2+ channel blockers penfluridol (0.3 μM), lubeluzole (3 μM), dotarizine (3 μM), flunarizine (3 μM), and mibefradil (3 μM). This protection was due to blockade of Ba 2+ entry through Ca 2+ channels because dotarizine (10 μM) inhibited the increase in cytosolic [Ba 2+] seen in fura-2-loaded chromaffin cells. Once Ba 2+ accumulated in the cytosol, it was not extruded by the Na +/Ca 2+ exchanger, as shown by the prolonged and sustained elevation of the fura-2 signal. This contrasts with the fast dissipation of the fura-2 signal generated by [Ca 2+] i elevation. Thus, Ba 2+ overload can cause cell death by mechanisms similar to those reported for Ca 2+ overload and might be used as a novel and convenient tool to search for new cytoprotective compounds.

Histamine-stimulated phospholipase C activity in bovine adrenal medullary chromaffin cells: the effect of chloride-channel antagonists and low extracellular chloride concentrations

Histamine activates phospholipase C (PLC) in a number of cell-types including those of neuronal and neuroendocrine origin. We report here that Cl−-channel antagonists of the niflumic acid-, but not stilbene disulphonic acid-class, produced a concentration-dependent inhibition of histamine-stimulated PLC activity in bovine adrenal medullary chromaffin cells. Low extracellular [Cl−] (10 mM) produced a similar degree of inhibition. While the mechanism(s) responsible for this inhibition are not resolved it may be significant that low extracellular Cl− also reduced the magnitude of the histamine-induced Ca2+ signal. Thus, PLC inhibition may be secondary to a reduction in Ca2+-inflow, a conclusion consistent with the known actions of niflumic acid-type compounds and the previously reported importance of Ca2+-influx in supporting histamine-stimulated PLC activity.

Neural regulation of phenylethanolamine N-methyltransferase (PNMT) gene expression in bovine chromaffin cells differs from other catecholamine enzyme genes.

Expression of the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) is regulated by hormonal and neural stimuli. Because the 5'-upstream regions of the PNMT do not contain sequences analogous to those demonstrated to convey neural regulation to the tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) catecholamine-synthesizing enzyme genes, functional and biochemical analyses have been utilized to characterize PNMT promoter responses to cholinergic and depolarizing agents. In primary cultures of bovine adrenal medullary chromaffin cells, reporter gene expression from transiently transfected 3- and 0.9-kb-containing PNMT promoter constructs is stimulated approximately twofold by nicotine and muscarine. Depolarizing concentrations of K+ produce fourfold increases in expression. These responses are not detected with constructs containing the proximal 0.3-kb promoter, indicating that the regions between -273 and -877 bp convey neural responsiveness for the PNMT gene in bovine chromaffin cells. Electrophoretic mobility shift assays (EMSAs) with oligonucleotides encoding these regions of the PNMT promoter revealed distinctions in migration of nuclear protein complexes formed following treatment of chromaffin cells with nicotine, muscarine, or 50 mM K+. Thus, the PNMT promoter between 0.3 and 0.9 kb contains sequences capable of responding to cholinergic and depolarization stimuli. Moreover, these treatments influence the interactions of specific nuclear proteins with this region of the PNMT promoter.

Histamine causes Ca 2+ entry via both a storemoperated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca 2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca 2+ enters the cell through a (capacitative) Ca 2+ entry pathway activated as a consequence of intracellular Ca 2+ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 μM) caused a rapid but transient increase in cytosolic [Ca 2+] followed by a lower plateau that was sustained as long as external Ca 2+ was present. In the absence of external Ca 2+ only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca 2+-free medium to deplete the internal Ca 2+ stores, histamine caused no increase in cytosolic [Ca 2+] when external Ca 2+ was absent. Re-introduction of external Ca 2+ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca 2+] that was further increased ( P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H 1-receptor antagonist, mepyramine (2 μM). A comparison was made between store-dependent Ca 2+ entry consequent upon store mobilization with histamine in Ca 2+-free medium and plateau phase Ca 2+ entry resulting from stimulation with histamine in Ca 2+-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P ⪡ 0.0001) at the same concentration of histamine (10 μM). It is concluded that histamine causes Ca 2+ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca 2+ entry through other pathways.

Synergism between toxin-gamma from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells.

Toxin-gamma (Tgamma) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tgamma was approximately 200-fold more potent than veratridine judged from EC50 values, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tgamma (1.2 vs. 12 microg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+ concentration ([Ca2+]o), when 30 microM veratridine plus 0.45 microM Tgamma were used. Tgamma (0.45 microM) doubled the basal uptake of 45Ca2+, whereas veratridine (100 microM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seen when Tgamma and veratridine were combined; this was particularly pronounced at 5 mM [Ca2+]o. Veratridine induced oscillations of cytosolic Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tgamma elevated basal [Ca2+]i levels, causing only small oscillations. When added together, Tgamma and veratridine elevated the basal levels of [Ca2+]i without causing large oscillations. Tgamma shifted the current-voltage (I-V) curve for Na+ channel current to the left. The combination of Tgamma with veratridine increased the shift of the I-V curve to the left, resulting in a greater recruitment of Na+ channels at more hyperpolarizing potentials. This led to enhanced and more rapid accumulation of Na+ in the cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, and Ca2+ entry and secretion.