Determination of norepinephrine apparent release rate and clearance in humans

A method for estimating the rate of entry of norepinephrine into plasma (norepinephrine apparent release rate) and clearance of norepinephrine from plasma in humans is presented. The procedure involves the intravenous infusion of tritiated ?-norepinephrine, of sufficiently high specific activity to avoid elevating blood pressure, until plateau concentration is reached in plasma, and measurement of norepinephrine specific activity under steady state conditions. In ten normal subjects at rest, the apparent release rate of norepinephrine was 0.54 ± 0.20 μg/m²/min. (mean ± standard deviation). It was significantly lower in four patients with idiopathic peripheral autonomic insufficiency, 0.19 ± 0.12 μg/m²/min., but in the latter, despite reduced norepinephrine release, plasma norepinephrine concentration was near normal because of slowed clearance of norepinephrine from the circulation, 1.69 ± 0.44 ?/min. compared with 2.80 ± 0.73 ?/min. in normal subjects (p<0.05). In four normal subjects given the norepinephrine uptake inhibitor, desipramine, to slow removal of norepinephrine from the circulation, again the plasma concentration of neurotransmitter was higher than would be expected from the existing apparent release rate of norepinephrine. The findings suggest that methods which measure the dynamic processes of norepinephrine release and removal quantify sympathetic nervous activity better than steady state plasma norepinephrine measurements alone.     

Support for a role for feedback regulation of norepinephrine release

There is abundant evidence that norepinephrine (NE) and other sympathomimetic amines with alpha-adrenoceptor activity inhibit the electrically evoked release of NE, whereas phenoxybenzamine and other alpha-adrenergic blocking agents enhance the electrically evoked release of NE. The physiological relevance of these observations, however, is disputed. The intent of this paper is to show that alpha-adrenergic blocking agents generally enhance transmitter output on nerve stimulation, but that some are more selective for presynaptic than for postsynaptic alpha receptors. Suggestions are made to account for the modulation of NE release as evoked by a single pulse.     

The median eminence as a model to study presynaptic regulation of neural peptide release

Presynaptic receptors in peptidergic neurons within the brain should be considered as an important target upon which different neurotransmitters or neuromodulators can act to affect peptide release. Evidence reviewed in this paper indicates that the median eminence (ME) of the hypothalamus is an area where many such interactions at the presynaptic level take place. Release of LHRH, somatostatin and vasopressin is affected by a variety of neurotransmitters or neuromodulators, such as norepinephrine, dopamine, epinephrine, histamine, cholinergic and opioid agonists, and peptides such as angiotensin II. The actions of these agents were prevented by the use of specific receptor blockers, indicating the specificity of the response evoked. Furthermore, with the use of classical pharmacological approaches, the type and affinity of the receptor involved is well defined. Other agents, such as prostaglandins (PGE2) or steroids (estradiol) were found to affect the activity of the peptidergic neuron at the synaptic terminal by stimulating directly peptide release (as seems to be the case for the PGE2/LHRH interaction) or by changing the sensitivity of the terminal to other transmitters, as shown for estradiol. In conclusion, the evidence presented indicates that the ME is an excellent model to study presynaptic regulation of neural peptide release. A set of criteria was defined within the text to establish the physiological significance of the in vitro studies. Several of the substances tested, and particularly norepinephrine and dopamine, seem to meet all the requirements to be considered physiological presynaptic regulators of neural peptide release at the level of the ME.     

Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP

Neuronal presynaptic terminals contain hundreds of neurotransmitter‐filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon feature decreased SV release competence and increased resting pool of SVs as assessed by imaging of SV release in cultured neurons. CDK5/calcineurin and cAMP/PKA presynaptic signalling are dysregulated, resulting in an aberrant phosphorylation of their downstream effectors synapsin1 and SNAP25, well‐known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalises the SV pools in neurons lacking bassoon. Finally, we demonstrate that CDK5‐dependent regulation of PDE4 activity interacts with cAMP/PKA signalling and thereby controls SV release competence. These data reveal that bassoon organises SV pools in glutamatergic synapses via regulation of presynaptic phosphorylation and cAMP homeostasis and indicate a role of CDK5/PDE4/cAMP axis in the control of neurotransmitter release.     

Pharmacological evidence for a dual GABAergic regulation of growth hormone release in humans

Effects of sodium valproate, which is believed to act via a gamma-aminobutyric acid mechanism, on basal and exercise-induced rise of growth hormone release have been tested in eighteen healthy volunteers. The exercise test consisted of using a stationary bicycle ergometer at 450 kg/min for 20 min. 600 mg per os of the drug resulted in a significant enhancement in plasma hormonal concentrations, whereas no effects were induced by placebo (p less than 0.005). Conversely, the growth hormone rise stimulated by exercise was markedly inhibited by sodium valproate (p less than 0.001 and p less than 0.01 at time 20, 40 and 60 min respectively). The results of this study are consistent with the hypothesis that a dual GABAergic control of growth hormone secretion is present in man.     

Alpha-2 adrenergic regulation of norepinephrine release in the rat submandibular gland as measured by HPLC-EC

In many tissues, norepinephrine appears to inhibit its own release through an interaction at alpha adrenergic receptors. We have developed an assay for measuring the release of endogenous norepinephrine based on HPLC and have studied the regulation of release in the rat submandibular gland by alpha adrenergic antagonists. The method uses electrochemical detection to quantitate norepinephrine released from tissue slices and does not require preloading of the tissue with [3H]norepinephrine. Yohimbine, an alpha-2 adrenergic antagonist, potentiates by 50% the release caused by potassium induced depolarization with an EC50 of 0.14 microM. Prazosin, an alpha-1 antagonist, has a similar effect, but is less potent with an EC50 of 0.77 microM. Thus, the alpha adrenergic receptor mediating the regulation of norepinephrine release is of the alpha-2 subtype. The observed equal efficacies and lack of additivity of release potentiation by yohimbine and prazosin at maximal doses suggest that both drugs act at the same receptor. The five-fold difference in potency between prazosin and yohimbine is consistent with the recent observations indicating species differences between rodent and non-rodent alpha-2 adrenergic receptors.     

The adrenoreceptor regulation of ureteral contractile function in the guinea pig]

The effects of specific agonists and antagonists of adrenoceptors and inhibitors of cAMP phosphodiesterase on electrostimulated phasic contractions in the ureter of guinea pig were studied. It has been shown that there mainly excitatory alpha 1-adrenoceptors in this object, the density of beta-adrenoceptors is slight and functional alpha 2-adrenoceptors are probably absent. Some aspects of adrenergic regulation of the contractile function of guinea pig ureter are discussed.     

Developmental regulation of nicotinic acetylcholine receptor-mediated [3H]norepinephrine release from rat cerebellum

Presynaptic modulation of synaptic transmission is the primary function of central nicotinic acetylcholine receptors (nAChRs) in developing and adult brain. nAChR activation regulates release of various neurotransmitters, including norepinephrine (NA). Given evidence that NA may serve a critical functional role in cerebellar development, we have undertaken studies to determine whether nAChRs modulate NA release in developing cerebellum. In vitro experiments using cerebellar slices examined the effects of nAChR stimulation on release of radiolabeled NA ([3H]NA). Our data indicate the presence of functional nAChRs on NA terminals in immature cerebellum and subsequent developmental regulation of receptor properties. During postnatal week one, the maximally effective dose of nicotine released 35.0 +/- 1.2% of cerebellar [3H]NA stores. There was a subsequent decline in maximal nicotine-stimulated NA release until postnatal day 30, when Emax values were statistically indistinguishable from adult. Although the efficacy of nicotine changed substantially throughout development, EC50 values did not differ significantly (EC50 = 4.4-12.0 micro m). Pharmacological analysis indicated that this developmental shift in maximum nicotine effect reflects a change in the properties of the nAChRs. These data support recent findings of a possible functional role of nAChRs in regulating cerebellar ontogeny, and provides further support for the role of NA as a neurotrophic factor during development.     

Calcium channel regulation and presynaptic plasticity

Voltage-gated calcium (Ca(2+)) channels initiate release of neurotransmitters at synapses, and regulation of presynaptic Ca(2+) channels has a powerful influence on synaptic strength. Presynaptic Ca(2+) channels form a large signaling complex, which targets synaptic vesicles to Ca(2+) channels for efficient release and mediates Ca(2+) channel regulation. Presynaptic plasticity regulates synaptic function on the timescale of milliseconds to minutes in response to neurotransmitters and the frequency of action potentials. This article reviews the regulation of presynaptic Ca(2+) channels by effectors and regulators of Ca(2+) signaling and describes the emerging evidence for a critical role of Ca(2+) channel regulation in control of neurotransmission and in presynaptic plasticity. Failure of function and regulation of presynaptic Ca(2+) channels leads to migraine, ataxia, and potentially other forms of neurological disease. We propose that presynaptic Ca(2+) channels serve as the regulatory node in a dynamic, multilayered signaling network that exerts short-term control of neurotransmission in response to synaptic activity.     

Comparative regulation of potassium and amphetamine induced release of 3H-norepinephrine from rat brain via presynaptic mechanisms.

This study examined the ability of various drugs to modify the potassium (K) or d-amphetamine (d-A) induced release of ³H-norepinephrine ³HNE) from chopped rat cortical tissue. The K induced release of the transmitter, which occurs from reserpine sensitive sites of cortical tissue, was significantly reduced by the beta receptor antagonist propranolol, the alpha receptor agonist clonidine and also by PGE₂. Pretreatment with eicosatetrynoic acid, an inhibitor of prostaglandin synthesis, did not influence the effect of clonidine on ³HNE release; thus this latter effect appears to be independent of enhanced prostaglandin formation. The proposed alpha receptor mediated negative feedback exhibits stereospecificity since addition of exogenous 1-, but not d-, NE decreased release of the transmitter. Blockade of alpha receptors by phentolamine or stimulation of beta receptors by isoproterenol significantly enhanced the K induced release of ³HNE from cortical tissue. By contrast, the d-A induced release of ³HNE which occurs from reserpine-insensitive sites, was reduced by propranolol and clonidine; and was not altered by phentolamine, isoproterenol or PGE₂. These data indicate that the K, but no d-A, induced release of ³HNE from cortical tissue is modified in accordance with postulated presynaptic negative and positive feedback mechanisms.     

The role of presynaptic ryanodine receptors in regulation of the kinetics of the acetylcholine quantal release in the mouse neuromuscular junction

To determine the role of presynaptic ryanodine receptors in the regulation of the kinetics of neurotransmitter quantum secretion caused by a nerve impulse in the experiments on the mouse neuromuscular junction, temporal parameters of phase synchronous and asynchronous delayed release of acetylcholine under the conditions of ryanodine receptors block and rhythmic stimulation were examined. The analysis of histograms of synaptic delays of the uni-quantal end-plate currents registered within 50 ms after the onset of the presynaptic action potential showed that ryanodine receptor blockers ryanodine, TMB-8 and dantrolene reduced the intensity of both phase synchronous and delayed asynchronous release of the mediator. The proportion of quanta released synchronously increased at the expense of the reduction of quantum numbers forming the delayed asynchronous release, i.e., there was a redistribution of quanta between synchronous and asynchronous phases of secretion. A block of ryanodine receptors also reduced the fluorescence intensity of the specific fluorescent calcium-sensitive dye Fluo-3 AM, which indicates a decrease in the intracellular calcium ion concentration. Thus, the presynaptic ryanodine receptors control the intracellular content of calcium ions under repetitive stimulation of the nerve endings and contribute to the modulation of the time parameters of the evoked release of the neurotransmitter quanta by increasing the intensity of the delayed asynchronous release of neurotransmitters.     

Suppression of norepinephrine appearance rate in plasma by diazepam in humans

Studies demonstrating benzodiazepine-induced reductions in plasma norepinephrine (NE) have assumed that changes in circulating plasma NE closely parallel changes in sympathetic nervous system (SNS) activity and that benzodiazepines suppress SNS outflow. However, decreases in plasma NE could also result from increased removal of NE from plasma via neuronal uptake or tissue metabolism. This study used a tritiated norepinephrine ([3H] NE) isotope dilution technique for measurement of plasma NE kinetics to determine if the fall in plasma NE induced by a single dose of diazepam orally administered to eight psychiatrically-healthy volunteers was due to a fall in plasma NE appearance rate or an increase in plasma NE removal. Diazepam decreased plasma NE appearance, but not clearance, and also decreased plasma epinephrine and mean arterial pressure, memory performance and alertness. Plasma levels of diazepam were correlated with drug effects on memory and alertness but not cardiovascular or SNS effects.     

The norepinephrine transporter and its regulation

For many years, the norepinephrine transporter (NET) was considered a 'static' protein that contributed to the termination of the action of norepinephrine in the synapse of noradrenergic neurons. The concept that the NET is dynamically regulated, adjusting noradrenergic transmission by changing its function and/or expression, was considered initially in the mid 1980s. Since that time, a plethora of studies demonstrate that the NET is regulated by several intracellular and extracellular signaling molecules, and that phosphorylation of the NET is a major pathway regulating its cell surface expression and thereby its function. The NET is a target of action of a number of drugs that are used long-term therapeutically or abused chronically. This has driven numerous investigations of how the NET and its function are regulated by long-term exposure to drugs. While repeated exposure to many drugs has been shown to affect NET function and expression, the intracellular mechanisms for these effects remains elusive.     

Direct presynaptic regulation of GABA/glycine release by kainate receptors in the dorsal horn: an ionotropic mechanism.

In the spinal cord dorsal horn, excitatory sensory fibers terminate adjacent to interneuron terminals. Here, we show that kainate (KA) receptor activation triggered action potential-independent release of GABA and glycine from dorsal horn interneurons. This release was transient, because KA receptors desensitized, and it required Na+ entry and Ca2+ channel activation. KA modulated evoked inhibitory transmission in a dose-dependent, biphasic manner, with suppression being more prominent. In recordings from isolated neuron pairs, this suppression required GABA(B) receptor activation, suggesting that KA-triggered GABA release activated presynaptic GABA(B) autoreceptors. Finally, glutamate released from sensory fibers caused a KA and GABA(B) receptor-dependent suppression of inhibitory transmission in spinal slices. Thus, we show how presynaptic KA receptors are linked to changes in GABA/glycine release and highlight a novel role for these receptors in regulating sensory transmission.     

Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-alpha as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-alpha complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.     

Adenosine mediation of presynaptic feedback inhibition of glutamate release

Conditions of increased metabolic demand relative to metabolite availability are associated with increased extracellular adenosine in CNS tissue. Synaptic activation of postsynaptic NMDA receptors on neurons of the cholinergic brainstem arousal center can increase sufficient extracellular adenosine to act on presynaptic A1 adenosine receptors (A1ADRs) of glutamate terminals, reducing release from the readily releasable pool. The time course of the adenosine response to an increase in glutamate release is slow (tau > 10 min), consistent with the role of adenosine as a fatigue factor that inhibits the activity of cholinergic arousal centers to reduce arousal.     

Genetics of HDL regulation in humans

PURPOSE OF REVIEW: To review gene regulation of HDL-cholesterol and discuss molecular abnormalities in HDL candidate genes that may lead to human pathologic states. RECENT FINDINGS: The inverse association between HDL-cholesterol and vascular disease, especially coronary heart disease, has long been recognized, but understanding gene regulation of HDL in humans gained considerable momentum following the identification of ABCA1 as playing a pivotal role in reverse cholesterol transport. Recent data suggest that potentially important targets for upregulating HDL in humans include upregulators of ABCA1 and APOA1 (e.g. peroxisome proliferator activated receptor and liver X receptor agonists) and downregulators of CETP (e.g. JTT-705). A host of other nuclear receptors under investigation in animal models may advance to human testing in the near future. SUMMARY: Disorders affecting HDL metabolism are complex because monogenic disorders causing low HDL do not necessarily correlate with premature vascular disease. To date, pathologic phenotypes have only been deduced among several HDL candidate genes. Understanding the genetic underpinnings associated with variant HDL and reverse cholesterol transport provides an exceptional opportunity to identify novel agents that may optimize this process and reduce vascular event rates beyond currently available LDL lowering therapies.