Cultured Chromaffin Cells Research Articles

Genetic analysis of Parkinson's disease-linked leucine-rich repeat kinase 2

Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common genetic cause of PD (Parkinson's disease). To investigate how mutations in LRRK2 cause PD, we generated LRRK2 mutant mice either lacking its expression or expressing the R1441C mutant form. Homozygous R1441C knockin mice exhibit no dopaminergic neurodegeneration or alterations in steady-state levels of striatal dopamine, but they show impaired dopamine neurotransmission, as was evident from reductions in amphetamine-induced locomotor activity and stimulated catecholamine release in cultured chromaffin cells as well as impaired dopamine D2 receptor-mediated functions. Whereas LRRK2-/- brains are normal, LRRK2-/- kidneys at 20 months of age develop striking accumulation and aggregation of α-synuclein and ubiquitinated proteins, impairment of the autophagy-lysosomal pathway, and increases in apoptotic cell death, inflammatory responses and oxidative damage. Our further analysis of LRRK2-/- kidneys at multiple ages revealed unique age-dependent biphasic alterations of the autophagic activity, which is unchanged at 1 month of age, enhanced at 7 months, but reduced at 20 months. Levels of α-synuclein and protein carbonyls, a general oxidative damage marker, are also decreased in LRRK2-/- kidneys at 7 months of age. Interestingly, this biphasic alteration is associated with increased levels of lysosomal proteins and proteases as well as progressive accumulation of autolysosomes and lipofuscin granules. We conclude that pathogenic mutations in LRRK2 impair the nigrostriatal dopaminergic pathway, and LRRK2 plays an essential role in the dynamic regulation of autophagy function in vivo.

Blockade of β-adrenoceptors restores the GRK2-mediated adrenal α(2) -adrenoceptor-catecholamine production axis in heart failure.

Sympathetic nervous system (SNS) hyperactivity is characteristic of chronic heart failure (HF) and significantly worsens prognosis. The success of β-adrenoceptor antagonist (β-blockers) therapy in HF is primarily attributed to protection of the heart from the noxious effects of augmented catecholamine levels. β-Blockers have been shown to reduce SNS hyperactivity in HF, but the underlying molecular mechanisms are not understood. The GPCR kinase-2 (GRK2)-α(2) adrenoceptor-catecholamine production axis is up-regulated in the adrenal medulla during HF causing α(2) -adrenoceptor dysfunction and elevated catecholamine levels. Here, we sought to investigate if β-blocker treatment in HF could lower SNS activation by directly altering adrenal GRK2 levels. Four weeks after myocardial infarction-induced HF, adult rats were randomized to 10-week treatment with vehicle (HF/C) or bisoprolol (HF/B). Cardiac function and dimensions were measured. In heart and adrenal gland, GRK2 levels were assessed by RT-PCR and Western blotting and adrenoceptors studied with radioligand binding. Catecholamines and α(2) adrenoceptors in adrenal medulla chromaffin cell cultures were also measured. Bisoprolol treatment ameliorated HF-related adverse cardiac remodelling and reduced plasma catecholamine levels, compared with HF/C rats. Bisoprolol also attenuated adrenal GRK2 overexpression as observed in HF/C rats and increased α(2) adrenoceptor density. In cultures of adrenal medulla chromaffin cells from all study groups, bisoprolol reversed HF-related α(2) adrenoceptor dysfunction. This effect was reversed by GRK2 overexpression. Blockade of β-adrenoceptors normalized the adrenal α(2) adrenoceptor-catecholamine production axis by reducing GRK2 levels. This effect may contribute significantly to the decrease of HF-related sympathetic overdrive by β-blockers.

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.

Gabapentin Inhibits Catecholamine Release from Adrenal Chromaffin Cells

Gabapentin is most commonly prescribed for chronic pain, but acute perioperative effects, including preemptive analgesia and hemodynamic stabilization, have been reported. Adrenal chromaffin cells are a widely used model to investigate neurosecretion, and adrenal catecholamines play important physiologic roles and contribute to the acute stress response. However, the effects of gabapentin on adrenal catecholamine release have never been tested. Primary cultures of bovine adrenal chromaffin cells were treated with gabapentin or vehicle for 18-24 h. The authors quantified catecholamine secretion from dishes of cells using high-performance liquid chromatography and resolved exocytosis of individual secretory vesicles from single cells using carbon fiber amperometry. Voltage-gated calcium channel currents were recorded using patch clamp electrophysiology and intracellular [Ca2+] using fluorescent imaging. Gabapentin produced statistically significant reductions in catecholamine secretion evoked by cholinergic agonists (24 ± 3%, n = 12) or KCl (16 ± 4%, n = 8) (mean ± SEM) but did not inhibit Ca2+ entry or calcium channel currents. Amperometry (n = 51 cells) revealed that gabapentin inhibited the number of vesicles released upon stimulation, with no change in quantal size or kinetics of these unitary events. The authors show Ca2+ entry was not inhibited by gabapentin but was less effective at triggering vesicle fusion. The work also demonstrates that chromaffin cells are a useful model for additional investigation of the cellular mechanism(s) by which gabapentin controls neurosecretion. In addition, it identifies altered adrenal catecholamine release as a potential contributor to some of the beneficial perioperative effects of gabapentin.

Regulation of Neuropeptide Processing Enzymes by Catecholamines in Endocrine Cells

Treatment of cultured bovine adrenal chromaffin cells with the catecholamine transport blocker reserpine was shown previously to increase enkephalin levels severalfold. To explore the biochemical mechanism of this effect, we examined the effect of reserpine treatment on the activities of three different peptide precursor processing enzymes: carboxypeptidase E (CPE) and the prohormone convertases (PCs) PC1/3 and PC2. Reserpine treatment increased both CPE and PC activity in extracts of cultured chromaffin cells; total protein levels were unaltered for any enzyme. Further analysis showed that the increase in CPE activity was due to an elevated V(max), with no change in the K(m) for substrate hydrolysis or the levels of CPE mRNA. Reserpine activation of endogenous processing enzymes was also observed in extracts prepared from PC12 cells stably expressing PC1/3 or PC2. In vitro experiments using purified enzymes showed that catecholamines inhibited CPE, PC1/3, and PC2, with dopamine quinone the most potent inhibitor (IC(50) values of ∼50-500 μM); dopamine, norepinephrine, and epinephrine exhibited inhibition in the micromolar range. The inhibition of purified CPE with catecholamines was time-dependent and, for dopamine quinone, dilution-independent, suggesting covalent modification of the protein by the catecholamine. Because the catecholamine concentrations found to be inhibitory to PC1/3, PC2, and CPE are well within the physiological range found in chromaffin granules, we conclude that catecholaminergic transmitter systems have the potential to exert considerable dynamic influence over peptidergic transmitter synthesis by altering the activity of peptide processing enzymes.

Calcium entry through slow-inactivating L-type calcium channels preferentially triggers endocytosis rather than exocytosis in bovine chromaffin cells

Calcium (Ca(2+))-dependent endocytosis has been linked to preferential Ca(2+) entry through the L-type (α(1D), Ca(V)1.3) of voltage-dependent Ca(2+) channels (VDCCs). Considering that the Ca(2+)-dependent exocytotic release of neurotransmitters is mostly triggered by Ca(2+) entry through N-(α(1B), Ca(V)2.2) or PQ-VDCCs (α(1A), Ca(V)2.1) and that exocytosis and endocytosis are coupled, the supposition that the different channel subtypes are specialized to control different cell functions is attractive. Here we have explored this hypothesis in primary cultures of bovine adrenal chromaffin cells where PQ channels account for 50% of Ca(2+) current (I(Ca)), 30% for N channels, and 20% for L channels. We used patch-clamp and fluorescence techniques to measure the exo-endocytotic responses triggered by long depolarizing stimuli, in 1, 2, or 10 mM concentrations of extracellular Ca(2+) ([Ca(2+)](e)). Exo-endocytotic responses were little affected by ω-conotoxin GVIA (N channel blocker), whereas ω-agatoxin IVA (PQ channel blocker) caused 80% blockade of exocytosis as well as endocytosis. In contrast, nifedipine (L channel blocker) only caused 20% inhibition of exocytosis but as much as 90% inhibition of endocytosis. Conversely, FPL67146 (an activator of L VDCCs) notably augmented endocytosis. Photoreleased caged Ca(2+) caused substantially smaller endocytotic responses compared with those produced by K(+) depolarization. Using fluorescence antibodies, no colocalization between L, N, or PQ channels with clathrin was found; a 20-30% colocalization was found between dynamin and all three channel antibodies. This is incompatible with the view that L channels are coupled to the endocytotic machine. Data rather support a mechanism implying the different inactivation rates of L (slow-inactivating) and N/PQ channels (fast-inactivating). Thus a slow but more sustained Ca(2+) entry through L channels could be a requirement to trigger endocytosis efficiently, at least in bovine chromaffin cells.

Altered excitability of cultured chromaffin cells following exposure to multi-walled carbon nanotubes

We studied the effects of multi-walled carbon nanotubes (MWCNTs) on the electrophysiological properties of cultured mouse chromaffin cells, a model of spontaneously firing cells. The exposure of chromaffin cells to MWCNTs at increasing concentrations (30–263 μg/ml) for 24 h reduced, in a dose-dependent way, both the cell membrane input resistance and the number of spontaneously active cells (from 80–52%). Active cells that survived from the toxic effects of MWCNTs exhibited more positive resting potentials, higher firing frequencies and unaltered voltage-gated Ca2+, Na+ and K+ current amplitudes. MWCNTs slowed down the inactivation kinetics of Ca2+-dependent BK channels. These electrophysiological effects were accompanied by MWCNTs internalization, as confirmed by transmission electron microscopy, indicating that most of the toxic effects derive from a dose-dependent MWCNTs-cell interaction that damages the spontaneous cell activity.

Modulation by endogenously released ATP and opioids of chromaffin cell calcium channels in mouse adrenal slices

Modulation of high-threshold voltage-dependent calcium channels by neurotransmitters has been the subject of numerous studies in cultures of neurons and chromaffin cells. However, no studies on such modulation exist in chromaffin cells in their natural environment, the intact adrenal medullary tissue. Here we performed such a study in voltage-clamped chromaffin cells of freshly prepared mouse adrenal slices under the whole cell configuration of the patch-clamp technique. The subcomponents of the whole cell inward Ca(2+) current (I(Ca)) accounted for 49% for L-, 28% for N-, and 36% for P/Q-type channels. T-type Ca(2+) channels or residual R-type Ca(2+) currents were not seen. However, under the perforated-patch configuration, 20% of I(Ca) accounted for a toxin-resistant R-type Ca(2+) current. Exogenously applied ATP and methionine-enkephalin (Met-enk) inhibited I(Ca) by 33%. Stop-flow and Ca(2+) replacement by Ba(2+), which favored the release of endogenous ATP and opioids, also inhibited I(Ca), with no changes in activation or inactivation kinetics. This inhibition was partially voltage independent and insensitive to prepulse facilitation. Furthermore, in about half of the cells, suramin and naloxone augmented I(Ca) in the absence of exogenous application of ATP/Met-enk. No additional modulation of I(Ca) was obtained after bath application of exogenous ATP and opioids to these already inhibited cells. Augmentation of I(Ca) was also seen upon intracellular dialysis of guanosine 5'-[β-thio]diphosphate (GDPβS), indicating the existence in the intact slice of a tonic inhibition of I(Ca) in resting conditions. These results suggest that in the intact adrenal tissue a tonic inhibition of I(Ca) exists, mediated by purinergic and opiate receptors.

Calcium signalling mediated through α7 and non-α7 nAChR stimulation is differentially regulated in bovine chromaffin cells to induce catecholamine release.

Ca(2+) signalling and exocytosis mediated by nicotinic receptor (nAChR) subtypes, especially the α7 nAChR, in bovine chromaffin cells are still matters of debate. We have used chromaffin cell cultures loaded with Fluo-4 or transfected with aequorins directed to the cytosol or mitochondria, several nAChR agonists (nicotine, 5-iodo-A-85380, PNU282987 and choline), and the α7 nAChR allosteric modulator PNU120596. Minimal [Ca(2+) ](c) transients, induced by low concentrations of selective α7 nAChR agonists and nicotine, were markedly increased by the α7 nAChR allosteric modulator PNU120596. These potentiated responses were completely blocked by the α7 nAChR antagonist α-bungarotoxin (α7-modulated-response). Conversely, high concentrations of the α7 nAChR agonists, nicotine or 5-iodo-A-85380 induced larger [Ca(2+) ](c) transients, that were blocked by mecamylamine but were unaffected by α-bungarotoxin (non-α7 response). [Ca(2+) ](c) increases mediated by α7 nAChR were related to Ca(2+) entry through non-L-type Ca(2+) channels, whereas non-α7 nAChR-mediated signals were related to L-type Ca(2+) channels; Ca(2+) -induced Ca(2+) -release contributed to both responses. Mitochondrial involvement in the control of [Ca(2+) ](c) transients, mediated by either receptor, was minimal. Catecholamine release coupled to α7 nAChRs was more efficient in terms of catecholamine released/[Ca(2+) ](c) . [Ca(2+) ](c) and catecholamine release mediated by α7 nAChRs required an allosteric modulator and low doses of the agonist. At higher agonist concentrations, the α7 nAChR response was lost and the non-α7 nAChRs were activated. Catecholamine release might therefore be regulated by different nAChR subtypes, depending on agonist concentrations and the presence of allosteric modulators of α7 nAChRs.

INTRATHECAL TRANSPLANTATION OF CULTURED CALF CHROMAFFIN CELLS ATTENUATE SENSORY MOTOR DYSFUNCTION IN A RAT MODEL OF NEUROPATHIC PAIN

The potential usefulness of chromaffin cells as a source of neuroactive agents for transplantation in the CNS is based on several promising features, including the diversity of biologically active neurotransmitters, neuropeptides and trophic factors produced by the cells. The purpose of this study was to test the possibility that motor as well as sensory dysfunction is reduced by cultured chromaffin cells. For this reason chromaffin cells were isolated from calf adrenal gland and were kept in DMEM-F12 at 37? C for two weeks. One week before transplantation, peripheral neuropathy was induced by a chronic constriction injury of the sciatic nerve of rats. At the time of transplantation, each rat received 100000-150000 cells. Behavioral tests included radiant heat, paw pinch, and, acetone. Motor function was tested by grasping reflex. The experiments illustrate that chromaffin cells can alleviate sensory and motor dysfunction consequence to peripheral nerve injury. These suggest that adrenal medullary chromaffin cells function as a local long-lasting agent at the spinal segment and further support the potential use of this approach for the treating of chronic pain.

Selective Recapture of Secretory Granule Components After Full Collapse Exocytosis in Neuroendocrine Chromaffin Cells

In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-and-run, cavicapture and full-collapse fusion. During kiss-and-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain. Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-β-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane.

Prevention in Health Care Reform: The Time Has Come

Treatment of cultured bovine adrenal chromaffin cells with the catecholamine transport blocker reserpine was shown previously to increase enkephalin levels severalfold. To explore the biochemical mechanism of this effect, we examined the effect of reserpine treatment on the activities of three different peptide precursor processing enzymes: carboxypeptidase E (CPE) and the prohormone convertases (PCs) PC1/3 and PC2. Reserpine treatment increased both CPE and PC activity in extracts of cultured chromaffin cells; total protein levels were unaltered for any enzyme. Further analysis showed that the increase in CPE activity was due to an elevated *V*~max~, with no change in the *K*~m~ for substrate hydrolysis or the levels of CPE mRNA. Reserpine activation of endogenous processing enzymes was also observed in extracts prepared from PC12 cells stably expressing PC1/3 or PC2. In vitro experiments using purified enzymes showed that catecholamines inhibited CPE, PC1/3, and PC2, with dopamine quinone the most potent inhibitor (IC~50~ values of ∼50--500 μM); dopamine, norepinephrine, and epinephrine exhibited inhibition in the micromolar range. The inhibition of purified CPE with catecholamines was time-dependent and, for dopamine quinone, dilution-independent, suggesting covalent modification of the protein by the catecholamine. Because the catecholamine concentrations found to be inhibitory to PC1/3, PC2, and CPE are well within the physiological range found in chromaffin granules, we conclude that catecholaminergic transmitter systems have the potential to exert considerable dynamic influence over peptidergic transmitter synthesis by altering the activity of peptide processing enzymes.

Measuring Exocytosis At Single Cells and in Intact Tissue

Exocytosis is a fundamental mechanism of intercellular communication. It involves the release of small packets of molecules that are packaged within the cell in vesicles that are released upon receipt of an appropriate stimulus. Because individual exocytotic events at most cells consist of release of a few attomoles or less, these events have been difficult to directly measure. Almost two decades ago we circumvented this problem by placing a carbon-fiber microelectrode adjacent to a single chromaffin cell in primary culture. With an appropriate potential applied to the electrode, catecholamines were oxidized by the electrode following exocytosis. The current arising from these events was shown to be due to the release of the catecholamines contained within a single vesicle. The detailed view of exocytosis provided by this approach provided new insights into this fundamental process. Subsequent research showed that this approach could be extended to exocytosis at neurons whose vesicles contain only zeptomole amounts of catecholamines. Research today is exploring exocytotic release events in more intact tissue to understand the competing events of release, uptake and diffusion.

Neurite Outgrowth on Chromaffin Cells Applying Extremely Low Frequency Magnetic Fields by Permanent Magnets

There is an increasing interest about the effects of electromagnetic fields on health and clinical applications. Electromagnetic fields have been shown to promote differentiation and regeneration of many tissues. The purpose of the present study was to evaluate if a magnetic field (MF) varying in time is able to induce neurite outgrowth in cultured chromaffin cells. For this reason, a stimulation system was developed in order to generate a magnetic field, using permanent magnets as a supply. In this investigation we used a pair of permanent ferrite magnets. These were mounted in a mechanical system in which both magnets rotate around a culture Petri dish. The stimulation device was designed at Centro de Investigación y de Estudios Avanzados, Avanzados del IPN, Mexico City. Primary cultures of chromaffin cells were stimulated with a magnetic field of 6.4 mT and 4, 7, 10 or 12Hz (2h daily, during a 7-day period). After treatment, percentage of neurite outgrowth was calculated. Our results show that the magnetic fields produced by rotating permanent magnets induced neurite outgrowth on chromaffin cells at 7 and 10Hz. The present study provides evidence that MFs varying in time (7 and 10Hz) induce neurite outgrowth in chromaffin cells. These studies will contribute to elucidate the effect of noninvasive MF stimulus in order to apply it in future regeneration therapies. Also, the device designed could be used for different kind of cells and may work as a model for future clinical devices.

R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice.

Dominantly inherited mutations in leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson's disease (PD). The importance of the R1441 residue in the pathogenesis is highlighted by the identification of three distinct missense mutations. To investigate the pathogenic mechanism underlying LRRK2 dysfunction, we generated a knockin (KI) mouse in which the R1441C mutation is expressed under the control of the endogenous regulatory elements. Homozygous R1441C KI mice appear grossly normal and exhibit no dopaminergic (DA) neurodegeneration or alterations in steady-state levels of striatal dopamine up to 2 years of age. However, these KI mice show reductions in amphetamine (AMPH)-induced locomotor activity and stimulated catecholamine release in cultured chromaffin cells. The introduction of the R1441C mutation also impairs dopamine D2 receptor function, as suggested by decreased responses of KI mice in locomotor activity to the inhibitory effect of a D2 receptor agonist, quinpirole. Furthermore, the firing of nigral neurons in R1441C KI mice show reduced sensitivity to suppression induced by quinpirole, dopamine, or AMPH. Together, our data suggest that the R1441C mutation in LRRK2 impairs stimulated dopamine neurotransmission and D2 receptor function, which may represent pathogenic precursors preceding dopaminergic degeneration in PD brains.

PACAP-cytokine interactions govern adrenal neuropeptide biosynthesis after systemic administration of LPS

We have examined induction of neuropeptide expression in adrenal medulla after treatment of mice with lipopolysaccharide (LPS), a model for septic shock, which activates both immune and stress responses in vivo. Messenger RNAs encoding vasoactive intestinal polypeptide (VIP) and galanin, both modulators of steroidogenesis in neighboring adrenal cortex, are up-regulated at 24 h (eight-fold for VIP and two-fold for galanin) after LPS injection, and remain elevated for the following 24 h. Up-regulation of VIP and galanin by LPS is abrogated in pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice, suggesting an interaction between LPS, or LPS-induced cytokines, and PACAP released in adrenal medulla from the splanchnic nerve. Treatment of cultured chromaffin cells with 100 nM PACAP and 10 nM tumor necrosis factor-alpha (TNF-α), a cytokine whose production is elevated by LPS, results in long-term synergistic up-regulation of VIP and galanin mRNA. PACAP blocks the earlier induction by TNF-α of mRNA encoding inhibitor of NF-κB alpha (IκBα), normally a negative autoregulator of TNF-α signaling through nuclear factor-κB (NF-κB), without affecting the induction of TNF-α-induced protein 3 (TNFAIP3), another NF-κB-dependent gene induced by TNF-α in chromaffin cells. By acting downstream of NF-κB to inhibit IκBα gene induction by TNF-α, PACAP may block IκBα-dependent negative autoregulation of TNF-α signaling through NF-κB, prolonging TNF-α-dependent signaling to neuropeptide-encoding genes in chromaffin cells. This mechanism may also underlie PACAP-dependent neuropeptide gene induction by LPS in vivo.

Enhanced cAMP production mediates the stimulatory action of pituitary adenylate cyclase activating polypeptide (PACAP) on in vitro catecholamine secretion from bovine adrenal chromaffin cells.

The 38 amino acid peptide pituitary adenylate cyclase activating polypeptide (PACAP) induced a dose dependent increase of catecholamine secretion in cultures of bovine chromaffin cells. This secretagogue activity of PACAP was strictly dependent on the presence of calcium in the culture medium. If calcium was omitted from the medium no effect of PACAP on catecholamine secretion could be detected during an incubation of 20 min. Preincubation of cells with 1 nM PACAP for 5 min facilitated the subsequent nicotine stimulated catecholamine secretion during a 20 min incubation without addition of the peptide. PACAP induced catecholamine secretion was clearly accompanied by a dose dependent increase of intracellular cAMP concentrations. The percentage of cells responding to PACAP with increased catecholamine secretion was assessed by immunocytochemistry of the transient appearance of dopamine-beta-hydroxylase, associated with the membranes of the chromaffin granules on the cell surface during the secretory process. About 70% of adrenal medullary cells responded to 100 nM PACAP with enhanced secretory activity. Though PACAP stimulated catecholamine secretion, we did not observed major effects on intracellular free calcium concentrations ([Ca2+]i) as determined with fura-2 by single cell fluorescence microscopy. In maximally 20% of the cells a rise in [Ca2+]i in response to a challenge with 500 nM PACAP was observed. Lower concentrations of PACAP were without an effect on [Ca2+]i. These data indicate that the stimulatory action of PACAP on in vitro catecholamine secretion from bovine chromaffin cells is linked to a rise of intracellular cAMP.

RET Expression and Neuron-like Differentiation of Pheochromocytoma and Normal Chromaffin Cells

Receptor tyrosine kinase RET is normally expressed at low levels in chromaffin cells and high levels in sympathetic neurons. Paradoxically, it is overexpressed in subsets of pheochromocytomas. The overexpressed protein is usually wild-type, except in multiple endocrine neoplasia type 2. Possible explanations for overexpression include tumor origin from RET-expressing sympathoadrenal progenitors that escape developmental culling during embryogenesis, or reactivation of signaling pathways related to neuronal differentiation. Normal adult chromaffin and pheochromocytoma cells can undergo neuron-like differentiation in cell culture. In this investigation, cultured cells from two normal human adrenal medullas, two of three human pheochromocytomas, and one extra-adrenal paraganglioma showed RET induction corresponding with extensive nerve growth factor-induced outgrowth of neurite-like processes, while one pheochromocytoma showed neither processes nor RET induction. RET was similarly upregulated in parallel with process outgrowth in cultures of normal rat chromaffin cells and PC12 rat pheochromocytoma cells. In contrast, mouse pheochromocytoma cells that constitutively express high levels of wild-type RET together with other neuronal progenitor markers showed no further RET increase after cyclic AMP-induced process outgrowth. The RET-activating ligand GDNF was anti-apoptotic for mouse pheochromocytoma but not for PC12 cells. The findings suggest that overexpression of RET in pheochromocytomas could result either from a secondary event that activates signaling pathways mediating adult chromaffin cell plasticity or as a component of a persistent sympathoadrenal progenitor phenotype. Whether wild-type RET contributes to tumor development or is merely a lineage marker for cells at various stages of neuronal differentiation may vary, with other tumor characteristics.

Regulation of catecholamine release and tyrosine hydroxylase in human adrenal chromaffin cells by interleukin‐1β: role of neuropeptide Y and nitric oxide

Adrenal chromaffin cells synthesize and secrete catecholamines and neuropeptides that may regulate hormonal and paracrine signaling in stress and also during inflammation. The aim of our work was to study the role of the cytokine interleukin-1beta (IL-1beta) on catecholamine release and synthesis from primary cell cultures of human adrenal chromaffin cells. The effect of IL-1beta on neuropeptide Y (NPY) release and the intracellular pathways involved in catecholamine release evoked by IL-1beta and NPY were also investigated. We observed that IL-1beta increases the release of NPY, norepinephrine (NE), and epinephrine (EP) from human chromaffin cells. Moreover, the immunoneutralization of released NPY inhibits catecholamine release evoked by IL-1beta. Moreover, IL-1beta regulates catecholamine synthesis as the inhibition of tyrosine hydroxylase decreases IL-1beta-evoked catecholamine release and the cytokine induces tyrosine hydroxylase Ser40 phosphorylation. Moreover, IL-1beta induces catecholamine release by a mitogen-activated protein kinase (MAPK)-dependent mechanism, and by nitric oxide synthase activation. Furthermore, MAPK, protein kinase C (PKC), protein kinase A (PKA), and nitric oxide (NO) production are involved in catecholamine release evoked by NPY. Using human chromaffin cells, our data suggest that IL-1beta, NPY, and nitric oxide (NO) may contribute to a regulatory loop between the immune and the adrenal systems, and this is relevant in pathological conditions such as infection, trauma, stress, or in hypertension.

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.