Catecholamine Molecules Research Articles

Increased Catecholamine Secretion from Single Adrenal Chromaffin Cells in DOCA-Salt Hypertension Is Associated with Potassium Channel Dysfunction

The mechanism of catecholamine release from single adrenal chromaffin cells isolated from normotensive and DOCA-salt hypertensive rats was investigated. These cells were used as a model for sympathetic nerves to better understand how exocytotic release of catecholamines is altered in this model of hypertension. Catecholamine secretion was evoked by local application of acetylcholine (1 mM) or high K+ (70 mM), and continuous amperometry was used to monitor catecholamine secretion as an oxidative current. The total number of catecholamine molecules secreted from a vesicle, the total number of vesicles fusing and secreting, and the duration of secretion in response to a stimulus were all significantly greater for chromaffin cells from hypertensive rats as compared to normotensive controls. The greater catecholamine secretion from DOCA-salt cells results, at least in part, from functionally impaired large conductance, Ca2+-activated (BK) and ATP-sensitive K+ channels. This work reveals that there is altered vesicular release of catecholamines from these cells (and possibly from perivascular sympathetic nerves) and this may contribute to increased vasomotor tone in DOCA-salt hypertension.

Chaos evidence in catecholamine secretion at chromaffin cells

Chromaffin cells secrete catecholamine molecules via exocytosis process. Each exocytotic event is characterized by a current spike, which corresponds to the amount of released catecholamine from secretory vesicles after fusing to plasma membrane. The current spike might be measured by the oxidation of catecholamine molecules and can be experimentally detected through amperometry technique. In this contribution, the secretion of catecholamine, namely adrenaline, of a set of bovine chromaffin cells is measured individually at each single cell. The aim is to study quantitative results of chaotic behavior in catecholamine secretion. For analysis, time series were obtained from amperometric measurements of each single chromaffin cell. Three analysis techniques were exploited: (i) A low-order attractor was generated by means of phase space reconstruction, Average Mutual Information (AMI) and False Nearest Neighbors (FNN) were used to compute embedding lag and embedding dimension, respectively. (ii) The properties of power spectrum density of time series were studied by Fast Fourier Transform (FFT) looking for possible dominant frequencies in power spectrum. (iii) Maximun Lyapunov Exponent (MLE) analysis was done to study the divergence of trajectories of the time series. Nevertheless, in order to dismiss the possibility of positiveness of MLE are due to the inherent noise in experiments, seven surrogate data sets computed using the Amplitude Adjusted Fourier Transform (AAFT) algorithm was computed. The phase space reconstruction showed that, in all cases, the trajectories lie in an embedding subspace suggesting oscillatory nature. The FFT analysis showed high dispersion of the power spectrum without any predominant frequency range. MLE analysis showed that the MLE values are positive for a given orbit time and a defined range of maximum scale values. Moreover, the trajectory of the MLE evolution of all the surrogate data are asymptotic and hold positive along the maximum scale range. These findings are preliminary evidence on detecting chaotic behavior in catecholamine secretion and, in general, their provide a first step towards a deeply understanding of nonlinear behavior of protein releasing dynamics.

Calcein Inhibits Vesicle Release

Charged fluorescent dyes are frequently used as fluid phase makers to detect vesicle fusion events with the cell membrane by means of transient omega-like indentations. We used calcein (300 μM) to image vesicle fusion from nerve growth factor (NGF)-treated PC12 cells, a model secretory cell line. We stimulated the cells with either high K+ saline or 100 μM nicotine to evoke calcium-dependent vesicle secretion. However, when the cells were stimulated in the presence of calcein, they showed a reduction in the number of vesicles that were released and/or endocytosed compared to cells that were stimulated in the absence of calcein. This observation from imaging experiments led us to hypothesize that calcein was inhibiting vesicle release from NGF-treated PC12 cells. In order to understand whether calcein was having an inhibitory affect on vesicle secretion and/or reuptake of vesicle membranes, we utilized amperometry to analyze vesicle release of catecholamine transmitter content from NGF-treated PC12 cells. Amperometry is an electrochemical detection method used routinely to measure release of vesicle contents from individual cells with a carbon fiber electrode. Analysis of amperometric spikes provides information as to the number of catecholamine molecules released and the kinetics of release. Our results show that calcein caused a reduction in the number of released amperometric spike events from 106 ± 13 events measured from control nicotine-stimulated cells, to 60 ± 11 events (p<0.05) in the presence of calcein. Peak amplitude, half-width, quantal content, or kinetics of rising or falling phase were unaffected by calcein. These data indicate that calcein has an inhibitory effect on vesicle fusion with the cellular membrane resulting in reduced amperometry events and a subsequent reduction in uptake of the fluid phase marker when used to track vesicles while imaging.

β‐blockers as endocrine disruptors: the potential effects of human β‐blockers on aquatic organisms

β-Adrenergic blockers or β-blockers have been used therapeutically to treat human hypertension since the late 1960s. The global market value and prescription rates of β-blockers keep rising substantially each year, and over the past decade the number of prescriptions has doubled. The widespread use of β-blockers has resulted in their appearance in the aquatic environment originating primarily from sewage effluents. The objective of this review is to analyze the literature as a means to determine the endocrine-disrupting potential of β-blockers in aquatic organisms. The mammalian adrenergic system is compared with the adrenergic system of fish and the homologous octopaminergic system in aquatic invertebrates, in particular mollusks. The structure and functions of these systems are linked to the molecular similarities between adrenoceptors and the octopaminergic/tyraminergic receptors, the various catecholamine molecules (epinephrine, norepinephrine, octopamine, and tyramine), and the processes controlled. Knowledge of these similarities as well as the effects of β-blockers, mainly in humans, is then used to create a broad picture of the endocrine-disrupting potential of β-blockers, particularly during the stress response. The main conclusion is that β-blockers have endocrine-disrupting effects.

Centimeter-Long and Large-Scale Micropatterns of Reduced Graphene Oxide Films: Fabrication and Sensing Applications

Recently, the field-effect transistors (FETs) with graphene as the conducting channels have been used as a promising chemical and biological sensors. However, the lack of low cost and reliable and large-scale preparation of graphene films limits their applications. In this contribution, we report the fabrication of centimeter-long, ultrathin (1-3 nm), and electrically continuous micropatterns of highly uniform parallel arrays of reduced graphene oxide (rGO) films on various substrates including the flexible polyethylene terephthalate (PET) films by using the micromolding in capillary method. Compared to other methods for the fabrication of graphene patterns, our method is fast, facile, and substrate independent. In addition, we demonstrate that the nanoelectronic FETs based on our rGO patterns are able to label-freely detect the hormonal catecholamine molecules and their dynamic secretion from living cells.

Transparent diamond microelectrodes for biochemical application

In this investigation a technology has been developed to use diamond electrodes in Micro Electrode Arrays (MEAs) on a transparent sapphire substrate, thus combining the outstanding electrochemical properties of boron-doped diamond electrodes (BDD) with the transparency needed for simultaneous fluorescence analysis. Nanodiamond films were grown on double side polished sapphire substrates by hot filament CVD (HFCVD) and Bias Enhanced Nucleation (BEN). A simple four microelectrode array (quadrupole) has been fabricated, fully characterized in optical and electrochemical properties, and tested with adrenal chromaffin cells, identifying amperometric spikes by the four recording diamond electrodes, corresponding to the oxidation current of catecholamine molecules.

Pain, stress and relaxation: Involvement of basic biological principles and healthy autoregulation

Trauma and stress often involve the expression of pain. In the course of traumatic and stressful events, an ‘orchestra’ of neurobiological mechanisms and signaling molecules get activated with the primary goal of ensuring survival and fighting against the perturbating event, i.e., stressor. Pain can be a major process in this phenomenon, directing attention to the initial trigger situation. It is now known that pain processes also involve local and/or systemic immune as well as cardiovascular and neuronal pathways, including proinflammtory endogenous opioid peptides (e.g., endorphins) and down-regulatory opiate alkaloids (e.g., morphine). Usually, relaxation is a result of the systemic down-regulation that occurs after stress response pathways get shut-down, following their initial induction. As pain occurs in the primary phase of this process, it gets an inhibitory push-back under the influence of endorphins and other analgetic endogenous signaling molecules in this still early, i.e., stress response-associated stage. The rationale of this phase would be that the ‘stressed’ organism chiefly fights against the stressor and then, subsequently, cares about the possible wounds and traumas acquired. These secondary processes include endogenous morphine signaling, allowing the pain to come back again, yet in an altered and more ‘distant’ way, enabling the organism to face it and decide upon necessary actions for recovery and learning. This secondary phase also reduces over-stimulation, e.g., of the immune system, involving anti-inflammation. Hence, down-regulation serves recovery, i.e., systemic back-up. Stress induction and subsequent termination follow each other automatically and naturally, i.e., ideally, therefore activating, among others, endogenous limbic reward and motivation circuitries. However, little is known about the interplay between pain perception and its relationship with catecholamine molecules other than dopamine serving as an endogenous morphine precursor. We believe that the perception of pain and the body's self-attempt to alleviate it, utilizing conventional homeostatic mechanisms, is mediated by key catecholamines, and that this effect is further modulated by nitric oxide. We further propose a paradigm which biologically links pain, autoregulation, endogenous morphine and the catecholamines together, demonstrating a complex symbiotic signalling system. Integrative, complementary and mind–body medicine would, by their nature, include this paradigm in their underlying concepts and therapeutic strategies.

Pain and relaxation (Review)

The modern notion of pain and its clinical management, along with its physiological origins, is of exceeding interest to both clinicians and basic science researchers. While much is known about the control of pain via non-steroidal anti-inflammatory medications or comparative exogenous analgesics, little is known about the interplay between pain perception and its relationship with catecholamine molecules. We believe that the perception of pain and the body's self-attempt to alleviate it utilizing conventional homeostatic mechanisms via endogenous opiate release is mediated by key catecholamines, and that this effect is further modulated by nitric oxide. We further propose a new paradigm which links pain, endogenous opiates, and the catecholamines in a unique robust fashion demonstrating a complex symbiotic signaling system.

Electrochemical imaging of fusion pore openings by electrochemical detector arrays

Opening of individual exocytotic fusion pores in chromaffin cells was imaged electrochemically with high time resolution. Electrochemical detector arrays that consist of four platinum microelectrodes were microfabricated on a glass coverslip. Exocytosis of single vesicles containing catecholamines from a cell positioned on top of the array is detected by the individual electrodes as a time-resolved oxidation current, reflecting the time course of arrival of catecholamine molecules at the electrode surfaces. The signals exhibit low noise and reveal foot signals indicating fusion pore formation and expansion. The position of individual release events is determined from the fraction of catecholamines recorded by the individual electrodes. Simultaneous fluorescence imaging of release of acridine orange from individual vesicles confirmed the electrochemical position assignments. This electrochemical camera provides very high time resolution, spatiotemporal localization of individual fusion pore openings and quantitative data on the flux of transmitter from individual vesicles. Analysis of the amperometric currents employing random walk simulations indicates that the time course of amperometric spikes measured near the cell surface is due to a low apparent diffusion coefficient of catecholamines near the cell surface and not due to slow dissociation from the granular matrix.

Different Structural Requirements for the Constitutive and the Agonist-induced Activities of the β2-Adrenergic Receptor

We converted Ser-207, located in helix 5 of the beta2-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to G(s) activation, we expressed the mutants in a G alpha(s)-tethered form. GTP exchange in such constructs is restricted to the fused alpha-subunit and is a linear function of the receptor concentration. Except S207R, all other mutants were expressed to a suitable level for investigation. All mutations reduced the binding affinities of the catechol agonists, epinephrine and isoproterenol, and the extent of reduction was unrelated to the residue ability to form hydrogen bonds. Instead, both enhancements and reductions of affinity were observed for the partial agonist halostachin and the antagonist pindolol. The mutations also enhanced and diminished ligand-induced receptor activation, but the effects were strictly ligand-specific. Polar residues such as Asp and His exalted the activation by full agonists but suppressed that induced by the partial agonists halostachin and dichloroisoproterenol. In contrast, hydrophobic residues such as Ile and Val augmented partial agonist activation. Only Ile and Lys produced a significant increase of constitutive activity. The effects on binding and activity were not correlated, nor did such parameters show any clear correlation with up to 78 descriptors of amino acid physicochemical properties. Our data question the idea that Ser-207 is exposed to the polar crevice in the unbound receptor. They also suggest that the active receptor form induced by a full agonist might be substantially different from that caused by constitutive activation.

1H nuclear magnetic resonance spectroscopy analysis for simultaneous determination of levodopa, carbidopa and methyldopa in human serum and pharmaceutical formulations

A method utilizing NMR spectroscopy has been developed to confirm the identity and quantity of levodopa, carbidopa and methyldopa in human serum and pharmaceutical preparations. The method is based on 500 MHz proton NMR spectra of individual catecholamine molecules. Qualitative and quantitative analyses are based on resonance characteristics of the functional groups present in their structures and the integral ratio of selected signals belonging to different compounds with respect to those of an internal standard, respectively. Experiments are performed to validate the quantitative NMR method, and the linearity and reproducibility of the proposed method are verified. The detection limit of the proposed method was estimated as 4.2, 1.7 and 1.6 μg ml −1 for levodopa, carbidopa and methyldopa, respectively. The recovery studies performed on human serum samples ranged from about 82–96% with relative standard deviations of <4%. The method was also applied successfully to the determination of each active compound in real pharmaceutical samples, and compared with the results obtained by the reference methods. The method is rapid, precise, accurate, and suitable for routine analyses.

Secretory responses of intact glomus cells in thin slices of rat carotid body to hypoxia and tetraethylammonium.

We have developed a thin-slice preparation of whole rat carotid body that allows us to perform patch-clamp recording of membrane ionic currents and to monitor catecholamine secretion by amperometry in single glomus cells under direct visual control. In normoxic conditions (P(O(2)) approximately 140 mmHg; 1 mmHg = 133 Pa), most glomus cells did not have measurable secretory activity, but exposure to hypoxia (P(O(2)) approximately 20 mmHg) elicited the appearance of a large number of spike-like exocytotic events. This neurosecretory response to hypoxia was fully reversible and required extracellular Ca(2+) influx. The average charge of single quantal events was 46 +/- 25 fC (n = 218), which yields an estimate of approximately 140,000 catecholamine molecules per vesicle. Addition of tetraethylammonium (TEA; 2-5 mM) to the extracellular solution induced in most (>95%) cells tested (n = 32) a secretory response similar to that elicited by low P(O(2)). Cells nonresponsive to hypoxia but activated by exposure to high external K(+) were also stimulated by TEA. A secretory response similar to the responses to hypoxia and TEA was also observed after treatment of the cells with iberiotoxin to block selectively Ca(2+)- and voltage-activated maxi-K(+) channels. Our data further show that membrane ion channels are critically involved in sensory transduction in the carotid body. We also show that in intact glomus cells inhibition of voltage-dependent K(+) channels can contribute to initiation of the secretory response to low P(O(2)).

Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells.

In synapses, a rise in presynaptic intracellular calcium leads to secretory vesicle fusion in less than a millisecond, as indicated by the short delay from excitation to postsynaptic signal. In nonsynaptic secretory cells, studies at high time resolution have been limited by the lack of a detector as fast and sensitive as the postsynaptic membrane. Electrochemical methods may be sensitive enough to detect catecholamines released from single vesicles. Here, we show that under voltage-clamp conditions, stochastically occurring signals can be recorded from adrenal chromaffin cells using a carbon-fibre electrode as an electrochemical detector. These signals obey statistics characteristic for quantal release; however, in contrast to neuronal transmitter release, secretion occurs with a significant delay after short step depolarizations. Furthermore, we identify a pedestal or 'foot' at the onset of unitary events which may represent the slow leak of catecholamine molecules out of a narrow 'fusion pore' before the pore dilates for complete exocytosis.

Effect of catecholamines and metal chelating agents on the brain and brown adipose tissue Na,K-ATPase

1. 1. Catecholamines stimulate Na.K-ATPase activity in the microsomal membranes of the brain and brown adipose tissue. This stimulation is apparent in the absence of soluble, cytosolic inhibitors and exhibits the same characteristics in both tissues: 1.1. (a) it occurs at high concentrations (10 −6-10 −4m) only; 1.2. (b) there is no difference in potency between isoprenaline, norepinephrine and epinephrine ( ec 50 = 1−2 × 10 −5 M) ; 1.3. (c) the D-stereoisomer of isoprenaline is equally as effective as the L-form; 1.4. (d) stimulation of Na,K-ATPase may also be achieved by the metal chelators EDTA, EGTA and desferal; 1.5. (e) the hydrophobic beta-blockers, propranolol and alprenolol, inhibit both the norepinephrine-stimulated and basal levels of enzyme activity at concentrations of 10 −5-10 −3 M; 1.6. (f) phenoxybenzamine, an irreversible α-adrenergic blocker, inhibits basal Na,K-ATPase as well as norepinephrine-stimulated enzyme activity ( ec 50= 2.5 × 10 −5 M) . 2. 2. Because none of these observations can be related to the properties of the stereospecific adrenergic receptor (α or β), it may be concluded that the catecholamine-Na,K-ATPase interaction is not mediated by the receptor. 3. 3. More probably, catecholamines may antagonize the Na,K-ATPase inhibition caused by some tightly membrane-bound metals (but not vanadium) via the ortho -catechol moiety of the catecholamine molecule. 4. 4. The stimulation of brown fat Na,K-ATPase by catecholamines does not have much relevance to the norepinephrine-stimulated thermogenesis in this tissue.

A proton NMR study of the interactions of catecholamines with phospholipids from chicken erythrocyte membranes

High-resolution NMR spectroscopy has been applied to the study of the interaction of catecholamines, norepinephrine, epinephrine, isoproterenol and their antagonists propranolol and alprenolol with sonicated phospholipids extracted from chicken erythrocyte membranes (CEM). The catecholamine molecules are immobilized by the phospholipids of CEM and the magnitude of the effect seems to depend on the alkyl substitution of their amino group. Upon introduction of alprenolol and propranolol into phospholipid vesicles a broadening of the resonances of theN-methyl alkyl chain and the terminal methyl protons was observed. The results present evidence about the specificity of the interactions of catecholamines with phospholipids from CEM.

Activating effect of chlorpromazine on the peroxidase-catalysed oxidation of catecholamines

1. Chlorpromazine was found to have an activating effect on the horseradish peroxidase-catalysed oxidation of catecholamines (noradrenaline, dopamine and dopa) to aminochrome, an intermediate in the melanin synthesis. 2. Chlorpromazine, a good electron donor, is enzymatically oxidized by peroxidase to a radical, the rate of chlorpromazine oxidation being much higher than the rate of catecholamine oxidation. 3. The present study suggests that chlorpromazine enhances the catecholamine oxidation by functioning as a cycling (redox) intermediate between peroxidase and catecholamine molecule.

Preface

In 1964 Lands reported that he could produce an adrenergic agent with considerably greater bronchial activity than cardiac activity by changing the N-alkyl substituent of the catecholamine molecule. With this discovery the concept of selective beta1 and beta2 adrenoreceptor function was established, and with it a new class of drugs for the treatment of asthma emerged. Since then several such agents have been synthesized, tested in the laboratory, and are either currently available or undergoing clinical trials. While these agents have not yet proven to be a panacea, progress in this realm of therapeutics is in a sense only beginning, and the future appears to hold great promise for the development of even more selective agents with greater therapeutic potential and fewer side effects.

ChemInform Abstract: STEREOSELECTIVE SYNTHESIS OF CIS‐ AND TRANS‐3‐AMINO‐4‐CHROMANOLS

AbstractAuf den im Formelschema skizzierten Wegen werden stereoisomere Aminochromanole dargestellt.

Trans-synaptic induction of adrenal tyrosine hydroxylase following amphetamine treatment in the rat

Daily injections of d-amphetamine sulfate (5 mg/kg), given in combination with the hepatic microsomal inhibitor iprindole, increased the tyrosine hydroxylase activity in rat adrenals by 41% after 4 days. Choline acetyl-transferase activity also increased significantly. Neither change was observed in animals whose adrenals had been denervated (by transection of the splanchic nerve), thus indicating that the increases were mediated by the central nervous system and by trans-synaptic mechanisms. The chronic administration of methamphetamine increases tyrosine hydroxylase activity in the adrenal glands of the chicken (MANDELL and MORGAN, 1970) and the rat (BUENING and GIBB, 1974). BREESE, COOPER and MUELLER (1974) hove noted similar increases in adrenal tyrosine hydroxylase activity in animals receiving 5 mg/kg of d-amphetamine twice daily for 3 days. The mechanisms by which amphet-amines modify adrenal tyrosine hydroxylase activity could involve direct actions on chromaffin cells (e.g., to release catecholamine molecules, thereby activating feedback mechanisms coupled to the enzyme), or central actions (to activate the flow of impulses along the preganglionic cholinergic nerves innervating the adrenal, thereby increasing enzyme activity via trans-synaptic mechanisms (COSTA, GUIDOTTI and HANBAUER, 1974). To explore the latter possibility, the extent to which treatment with d-amphetamine changes tyrosine hydroxylase activity in adrenals previously subjected to surgical denervation was determined.

Stereoselective synthesis of cis- and trans-3-amino-4-chromanols.

3-Alkylamino-(cis-1a-c) and trans-1a-c), 7-hydroxy-3-isopropylamino-(cis-1d and trans-1d), 7, 8-dihydroxy-3-isopropylamino-(cis-1e and trans-1e) and 6, 7-dihydroxy-3-isopropylamino-4-chromanol (cis-1f) were synthesized. By the use of lithium cyanoborohydride (LiBH3CN), the reductive N-alkylation of 3-amino-4-chromanones with acetone proceeded smoothly to produce 3-isopropylamino-4-chromanones (12e and 12f) in good yields. Stereoselective hydrogenation of 4-chromanones in a basic or an acidic medium to give trans- or cis-aminoalcohols was discussed on the basis of the thermodynamic stability of the end products. Among the compounds synthesized, 7, 8-dihydroxy derivatives showed a strong β2-stimulating activity, whereas the 6, 7-dihydroxy congener was devoid of the activity. The results suggest an important role of the spatial arrangement of functional groups in these catecholamine molecules with regard to their pharmacological properties.