Stimulated Catecholamine Release Research Articles

Continuous positive airway pressure therapy reduces the levels of catecholamines and blood pressure in pseudophaeochromocytoma with coexisting obstructive sleep apnoea.

BackgroundStress from obstructive sleep apnoea (OSA) stimulates catecholamine release and consequently can exacerbate hypertension, even in the absence of a catecholamine-producing tumour (phaeochromocytoma). As such, a positive screening test for suspected phaeochromocytoma may be misleading. There exists only a handful case reports, and no controlled trials, how continuous positive airway pressure (CPAP) to treat OSA influences catecholamine levels. We examined changes to levels of urinary catecholamine and blood pressure in response to CPAP treatment.MethodsWe conducted a meta-analysis of data aggregated from published case reports of individual patient data up to April 2020. The quality of the reports was evaluated using the risk of bias in non-randomized studies of interventions (ROBINS-I) tool.ResultsA total of 13 cases (seven men and six women) from seven reports met our search criteria. Patients had mean age of 49.1 years (range = 36–62) and body mass index of 37.4 kg/m2 (range = 27–56). Most had moderate to severe OSA with CPAP treatment. Nine cases had 24-hour urinary noradrenaline assessment before and after CPAP treatment. CPAP treatment led to a 21% reduction (104 nmol/24-hours, 95% credible interval =59 to 148) in 24-hour urinary noradrenaline to within reference ranges, and 25% reduction (from 131 to 100 mmHg) in mean arterial pressure. The risk of overall bias evaluated by the ROBINS-I tool was found to be low in the majority of reports.ConclusionsInvestigations of patients suspected of phaeochromocytoma, particularly obese individuals, should exclude OSA and treat this condition if present before performing screening tests to assess for catecholamine levels.

Meta-analysis of changes in the levels of catecholamines and blood pressure with continuous positive airway pressure therapy in obstructive sleep apnea.

Stress from obstructive sleep apnea (OSA) stimulates catecholamine release consequently exacerbating hypertension. However, different studies have shown a conflicting impact of continuous positive airway pressure (CPAP) treatment in patients with OSA on catecholamine levels and blood pressure. We aimed to examine changes to catecholamine levels and blood pressure in response to CPAP treatment. We conducted a meta‐analysis of data published up to May 2020. The quality of the studies was evaluated using standard tools for assessing the risk of bias. Meta‐analysis was conducted using RevMan (v5.3) and expressed in standardized mean difference (SMD) for catecholamines and mean difference (MD) for systolic (SBP) and diastolic blood pressure (DBP). A total of 38 studies met our search criteria; they consisted of 14 randomized control trials (RCT) totaling 576 participants and 24 prospective cohort studies (PCS) of 547 participants. Mean age ranged between 41 and 62 year and body mass index between 27.2 and 35.1 kg/m2. CPAP treatment reduced 24‐hour urinary noradrenaline levels both in RCT (SMD = −1.1; 95% confidence interval (CI): −1.63 to − 0.56) and in PCS (SMD = 0.38 (CI: 0.24 to 0.53). SBP was also reduced by CPAP treatment in RCT (4.8 mmHg; CI: 2.0‐7.7) and in PCS (7.5 mmHg; CI: 3.3‐11.7). DBP was similarly reduced (3.0 mmHg; CI: 1.4‐4.6) and in PCS (5.1 mmHg; CI: 2.3‐8.0). In conclusion, CPAP treatment in patients with OSA reduces catecholamine levels and blood pressure. This suggests that sympathetic activity plays an intermediary role in hypertension associated with OSA‐related stress.

Monitoring the Secretory Behavior of the Rat Adrenal Medulla by High-Performance Liquid Chromatography-Based Catecholamine Assay from Slice Supernatants.

Catecholamine (CA) secretion from the adrenal medullary tissue is a key step of the adaptive response triggered by an organism to cope with stress. Whereas molecular and cellular secretory processes have been extensively studied at the single chromaffin cell level, data available for the whole gland level are much scarcer. We tackled this issue in rat by developing an easy to implement experimental strategy combining the adrenal acute slice supernatant collection with a high-performance liquid chromatography-based epinephrine and norepinephrine (NE) assay. This technique affords a convenient method for measuring basal and stimulated CA release from single acute slices, allowing thus to individually address the secretory function of the left and right glands. Our data point that the two glands are equally competent to secrete epinephrine and NE, exhibiting an equivalent epinephrine:NE ratio, both at rest and in response to a cholinergic stimulation. Nicotine is, however, more efficient than acetylcholine to evoke NE release. A pharmacological challenge with hexamethonium, an α3-containing nicotinic acetylcholine receptor antagonist, disclosed that epinephrine- and NE-secreting chromaffin cells distinctly expressed α3 nicotinic receptors, with a dominant contribution in NE cells. As such, beyond the novelty of CA assays from acute slice supernatants, our study contributes at refining the secretory behavior of the rat adrenal medullary tissue, and opens new perspectives for monitoring the release of other hormones and transmitters, especially those involved in the stress response.

Identification of novel loci affecting circulating chromogranins and related peptides

Chromogranins are pro-hormone secretory proteins released from neuroendocrine cells, with effects on control of blood pressure. We conducted a genome-wide association study for plasma catestatin, the catecholamine release inhibitory peptide derived from chromogranin A (CHGA), and other CHGA- or chromogranin B (CHGB)-related peptides, in 545 US and 1252 Australian subjects. This identified loci on chromosomes 4q35 and 5q34 affecting catestatin concentration (P = 3.40 × 10-30 for rs4253311 and 1.85 × 10-19 for rs2731672, respectively). Genes in these regions include the proteolytic enzymes kallikrein (KLKB1) and Factor XII (F12). In chromaffin cells, CHGA and KLKB1 proteins co-localized in catecholamine storage granules. In vitro, kallikrein cleaved recombinant human CHGA to catestatin, verified by mass spectrometry. The peptide identified from this digestion (CHGA360-373) selectively inhibited nicotinic cholinergic stimulated catecholamine release from chromaffin cells. A proteolytic cascade involving kallikrein and Factor XII cleaves chromogranins to active compounds both in vivo and in vitro.

Using Single‐Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis

AbstractThe pretreatment of cultured pheochromocytoma (PC12) cells with cis‐diamminedichloroplatinum (cisplatin), an anti‐cancer drug, influences the exocytotic ability of the cells in a dose‐dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single‐cell amperometry reflects that 2 μm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 μm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.

Using Single‐Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis

The pretreatment of cultured pheochromocytoma (PC12) cells with cis-diamminedichloroplatinum (cisplatin), an anti-cancer drug, influences the exocytotic ability of the cells in a dose-dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single-cell amperometry reflects that 2 μm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 μm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.

Dual action of leptin on rest-firing and stimulated catecholamine release via phosphoinositide 3-kinase-driven BK channel up-regulation in mouse chromaffin cells.

Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect and, as such, exerts a relevant action on the adipo-adrenal axis. Leptin has a dual action on adrenal mouse chromaffin cells both at rest and during stimulation. At rest, the adipokine inhibits the spontaneous firing of most cells by enhancing the probability of BK channel opening through the phosphoinositide 3-kinase signalling cascade. This inhibitory effect is absent in db(-) /db(-) mice deprived of Ob receptors. During sustained stimulation, leptin preserves cell excitability by generating well-adapted action potential (AP) trains of lower frequency and broader width and increases catecholamine secretion by increasing the size of the ready-releasable pool and the rate of vesicle release. In conclusion, leptin dampens AP firing at rest but preserves AP firing and enhances catecholamine release during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release. Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect. Besides being expressed in the hypothalamus and hippocampus, leptin receptors (ObRs) are also present in chromaffin cells of the adrenal medulla. In the present study, we report the effect of leptin on mouse chromaffin cell (MCC) functionality, focusing on cell excitability and catecholamine secretion. Acute application of leptin (1nm) on spontaneously firing MCCs caused a slowly developing membrane hyperpolarization followed by complete blockade of action potential (AP) firing. This inhibitory effect at rest was abolished by the BK channel blocker paxilline (1μm), suggesting the involvement of BK potassium channels. Single-channel recordings in 'perforated microvesicles' confirmed that leptin increased BK channel open probability without altering its unitary conductance. BK channel up-regulation was associated with the phosphoinositide 3-kinase (PI3K) signalling cascade because the PI3K specific inhibitor wortmannin (100nm) fully prevented BK current increase. We also tested the effect of leptin on evoked AP firing and Ca(2+) -driven exocytosis. Although leptin preserves well-adapted AP trains of lower frequency, APs are broader and depolarization-evoked exocytosis is increased as a result of the larger size of the ready-releasable pool and higher frequency of vesicle release. The kinetics and quantal size of single secretory events remained unaltered. Leptin had no effect on firing and secretion in db(-) /db(-) mice lacking the ObR gene, confirming its specificity. In conclusion, leptin exhibits a dual action on MCC activity. It dampens AP firing at rest but preserves AP firing and increases catecholamine secretion during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release.

A case of ST elevation myocardial infarction precipitated by methylphenidate therapy for gait freeze

AbstractBackgroundParkinson's disease affects 1% of the population older than 60 years. Motor symptoms can be difficult to treat in end‐stage disease, leading to the use of therapies with less favourable adverse effect profiles.AimTo promote awareness of the risks associated with the off‐label use of methylphenidate for hypokinesia in Parkinson's disease patients with cardiovascular risk factors.Clinical detailsA 70‐year‐old Caucasian non‐smoking male presented to the hospital with chest pain and inferior ST segment elevation 2 months after commencing methylphenidate for gait hypokinesia. His past medical history was significant for end‐stage Parkinson's disease with associated psychosis, and ischaemic heart disease with previous myocardial infarction. Relevant therapies included levodopa, benserazide, amantadine, pramipexole, quetiapine, atorvastatin and perindopril.OutcomesThe patient received a drug eluting stent to the circumflex artery, followed by a staged percutaneous coronary intervention to the left anterior descending artery. Methylphenidate was discontinued on day 2 post myocardial infarction in light of published data indicating an increased risk of myocardial infarction with methylphenidate use. Methylphenidate is known to stimulate catecholamine release, thereby increasing the risk of ischaemic cardiovascular events by increasing myocardial oxygen demand.ConclusionThis is the first reported case to describe methylphenidate use in Parkinson's disease complicated by a myocardial infarction. This case, in conjunction with other published literature, suggests that the use of methylphenidate for this novel indication must be carefully considered for each individual, and avoided in those with cardiovascular risk factors or known ischaemic heart disease.

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.

Pituitary adenylate cyclase-activating peptide enhances electrical coupling in the mouse adrenal medulla

Neuroendocrine adrenal medullary chromaffin cells receive synaptic excitation through the sympathetic splanchnic nerve to elicit catecholamine release into the circulation. Under basal sympathetic tone, splanchnic-released acetylcholine evokes chromaffin cells to fire action potentials, leading to synchronous phasic catecholamine release. Under elevated splanchnic firing, experienced under the sympathoadrenal stress response, chromaffin cells undergo desensitization to cholinergic excitation. Yet, stress evokes a persistent and elevated adrenal catecholamine release. This sustained stress-evoked release has been shown to depend on splanchnic release of a peptide transmitter, pituitary adenylate cyclase-activating peptide (PACAP). PACAP stimulates catecholamine release through a PKC-dependent pathway that is mechanistically independent of cholinergic excitation. Moreover, it has also been reported that shorter term phospho-regulation of existing gap junction channels acts to increase junctional conductance. In this study, we test if PACAP-mediated excitation upregulates cell-cell electrical coupling to enhance chromaffin cell excitability. We utilize electrophysiological recordings conducted in adrenal tissue slices to measure the effects of PACAP stimulation on cell coupling. We report that PACAP excitation increases electrical coupling and the spread of electrical excitation between adrenal chromaffin cells. Thus PACAP acts not only as a secretagogue but also evokes an electrical remodeling of the medulla, presumably to adapt to the organism's needs during acute sympathetic stress.

Divergent roles of reactive oxygen species in the responses of perinatal adrenal chromaffin cells to hypoxic challenges

The fetus and neonate experience variable patterns of low P(O)₂(hypoxia) ranging from acute, sustained, and intermittent. Adaptation to hypoxia involves activation of key transcription factors, known as hypoxia-inducible factors (e.g. HIF-1α, HIF-2α), which regulate a number of genes in different cell types. This review focuses on the signaling pathways that mediate proper physiological responses of perinatal adrenomedullary chromaffin cells (AMC) to varying patterns of hypoxic challenges, and particularly on the controversial role of reactive oxygen species (ROS). At birth, acute hypoxia (seconds to minutes) directly stimulates catecholamine release from AMC via K+ channel inhibition, mediated by a decrease in mitochondrial-derived ROS. By contrast, exposure to chronic sustained hypoxia (CSH) induces HIF-2α in a fetal-derived chromaffin cell line independently of changes in ROS. Exposure to chronic intermittent hypoxia (CIH) activates antioxidant responses via the regulator Nrf-2, in association with an increase in ROS and the induction of HIF-1α. We propose that the physiological responses of perinatal AMC to hypoxia and the ensuing directional changes in ROS are dependent on the pattern and duration of the hypoxic exposure.

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.

Reinvestigation of the effect of orexin A on catecholamine release from adrenal chromaffin cells

Orexins have been shown to be implicated in the regulation of adrenal medulla functions. However, there are still inconsistent investigations on the effects of orexins on catecholamine release from chromaffin cells in varying species. In the present study, using the carbon-fiber amperometry, we investigated whether orexin A would stimulate catecholamine release from rat and mouse adrenal chromffin cells. Puff application of orexin A dose-dependently induced amperometric currents in the cultured rat chromaffin cells, which was completely blocked by the selective OX1R antagonist SB-334867 or by the removal of extracellular calcium. Likewise, in the mouse adrenal medulla slices, orexin A also induced catecholamine release mainly through the activation of OX1R. These results gain insight into our understanding of the pharmacological relevance of orexin system in modulating neuroendocrine functions.

CRF Facilitates Calcium Release from Intracellular Stores in Midbrain Dopamine Neurons

Changes in cytosolic calcium are crucial for numerous processes including neuronal plasticity. This study investigates the regulation of cytosolic calcium by corticotropin-releasing factor (CRF) in midbrain dopamine neurons. The results demonstrate that CRF stimulates the release of intracellular calcium from stores through activation of adenylyl cyclase and PKA. Imaging and photolysis experiments showed that the calcium originated from dendrites and required both functional IP3 and ryanodine receptor channels. The elevation in cytosolic calcium potentiated calcium-sensitive potassium channels (sK) activated by action potentials and metabotropic Gq-coupled receptors for glutamate and acetylcholine. This increase in cytosolic calcium activated by postsynaptic Gs-coupled CRF receptors may represent a fundamental mechanism by which stress peptides and hormones can shape Gq-coupled receptor-mediated regulation of neuronal excitability and synaptic plasticity in dopamine neurons.

Intracellular signaling mechanisms mediating catecholamine release upon activation of NPY Y1 receptors in mouse chromaffin cells

The adrenal chromaffin cells synthesize and release catecholamine (mostly epinephrine and norepinephrine) and different peptides, such as the neuropeptide Y (NPY). NPY stimulates catecholamine release through NPY Y1 receptor in mouse chromaffin cells. The aim of our study was to determine the intracellular signaling events coupled to NPY Y1 receptor activation that lead to stimulation of catecholamine release from mouse chromaffin cells. The stimulatory effect of NPY mediated by NPY Y1 receptor activation was lost in the absence of extracellular Ca2+. On the other hand, inhibition of nitric oxide synthase and guanylyl cyclase also decreased the stimulatory effect of NPY. Moreover, catecholamine release stimulated by NPY or by the nitric oxide donor (NOC-18) was inhibited by mitogen-activated protein kinase (MAPK) and protein kinase C inhibitors. In summary, in mouse chromaffin cells, NPY evokes catecholamine release by the activation the NPY Y1 receptor, in a Ca2+-dependent manner, by activating mitogen-activated protein kinase and promoting nitric oxide production, which in turn regulates protein kinase C and guanylyl cyclase activation.

Intraperitoneal administration of CDP-choline and its cholinergic and pyrimidinergic metabolites induce hyperglycemia in rats: involvement of the sympathoadrenal system

CDP-choline is an endogenous metabolite in phosphatidylcholine biosynthesis. Exogenous administration of CDP-choline has been shown to affect brain metabolism and to exhibit neuroprotective actions. On the other hand, little is known regarding its peripheral actions. Intraperitoneal administration of CDP-choline (200 – 600 μmol/kg) induced a dose- and time-dependent hyperglycemia in rats. Hyperglycemic response to CDP-choline was associated with several-fold elevations in serum concentrations of CDP-choline and its metabolites. Intraperitoneal administration of phosphocholine, choline, cytidine, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, uridine, uridine monophosphate, uridine diphosphate and uridine triphosphate also produced significant hyperglycemia. Pretreatment with atropine methyl nitrate failed to alter the hyperglycemic responses to CDP-choline and its metabolites whereas hexamethonium, the ganglionic nicotinic receptor antagonist which blocks nicotinic cholinergic neurotransmission at the autonomic ganglionic level, blocked completely the hyperglycemia induced by CDP-choline, phosphocholine and choline, and attenuated the hyperglycemic response to cytidine monophosphate and cytidine. Increased blood glucose following CDP-choline, phosphocholine and choline was accompanied by elevated plasma catecholamine concentrations. Hyperglycemia elicited by CDP-choline and its metabolites was entirely blocked either by pretreatment with a nonselective α-adrenoceptor antagonist phentolamine or by the α2-adrenoceptor antagonist, yohimbine. Hyperglycemic responses to CDP-choline, choline, cytidine monophosphate and cytidine were not affected by chemical sympathectomy, but were prevented by bilateral adrenalectomy. Phosphocholine-induced hyperglycemia was attenuated by bilateral adrenalectomy or by chemical sympathectomy. These data show that CDP-choline and its metabolites induce hyperglycemia which is mediated by activation of ganglionic nicotinic receptors and stimulation of catecholamine release that subsequently activates α2-adrenoceptors.

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

Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca2+-evoked release of catecholamine

Synaptotagmin (syt) I is a Ca2+-binding protein that is well accepted as a major sensor for Ca2+-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca2+ sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca2+ and stimulated Ca2+ influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca2+ sensor for regulated release of transmitter.

Stimulation of catecholamine biosynthesis via the PKC pathway by prolactin-releasing peptide in PC12 rat pheochromocytoma cells

We have previously shown that prolactin-releasing peptide (PrRP) stimulates catecholamine release from PC12 cells (rat pheochromocytoma cell line). However, it is not known whether PrRP also affects catecholamine biosynthesis. Thus, we examined the effect of PrRP on catecholamine biosynthesis in PC12 cells. PrRP31 (>10 nM) and PrRP20 (>100 nM) significantly increased the activity and expression level of tyrosine hydroxylase (TH), a rate-limiting enzyme, in catecholamine biosynthesis. However, the PrRP20-stimulated TH activity was markedly weaker than that of PrRP31. PrRP31 (>1 nM) and PrRP20 (>10 nM) significantly induced an increase in the level of PKC activity. Both Ro 32-0432 (a protein kinase C inhibitor) and H89 (a protein kinase A inhibitor) effectively suppressed the PrRP31 (100 nM)-induced TH mRNA level. Next, we examined the effect of PrRP on mitogen-activated protein kinases (MAPKs). PrRP31 (1 microM) significantly induced an increase in the activity of extracellular signal-related kinases (ERKs) and the stress-activated protein kinase/c-jun N terminal kinase (SAPK/JNK). In contrast to ERKs and JNK, PrRP31 did not affect P38 MAPK activity. Consistent with these findings, pretreatment of cells with the MEK-1-inhibitor, PD-98059 (50 microM), significantly inhibited the PrRP31 (100 nM)-induced increase in TH mRNA. These results indicate that PrRP stimulates catecholamine synthesis through both the PKC and PKA pathways in PC12 cells.

Differential effects of FK506 and cyclosporin A on catecholamine release from bovine adrenal chromaffin cells

1 The effects of the immunosuppressants, tacrolimus (FK506) and cyclosporin A (CsA), on catecholamine (CA) release were examined in cultured bovine adrenal chromaffin cells. 2 In intact cells, FK506 (1-30 microM) inhibited CA release stimulated by acetylcholine (ACh; 100 microM), 1,1-dimethyl-4-phenyl-piperazinium (DMPP, 10 microM) or high K+ (40 mM). CsA (1-30 microM) had a little inhibitory effect on the ACh- or DMPP-stimulated CA release, whereas it enhanced the high K(+)-stimulated CA release. 3 In beta-escin-permeabilized cells, FK506 inhibited CA release stimulated by Ca2+ (1 and 10 microM) in the presence and absence of MgATP (2 mM). CsA induced CA release under Ca(2+)-free condition and enhanced the Ca(2+)-stimulated CA release in the presence and absence of MgATP. 4 It is known that the Ca(2+)-dependent exocytosis involves at least two distinct steps, ATP-requiring priming stage and ATP-independent fusion step in adrenal chromaffin cells. Therefore, it is suggested that FK506 inhibits the Ca(2+)-dependent exocytosis probably at the fusion step whereas CsA induces CA release from bovine adrenal chromaffin cells.