Sympathetic Nerve Varicosities Research Articles

CaV2.3 Mediate ATP Release from Rat Tail Artery Sympathetic Nerves

Peripheral sympathetic nerves are essential for regulating homeostatic control of target organs, such as the tone of blood vessels, and many details of the molecular organization of peripheral sympathetic nerve varicosities remain to be defined. Unlike central synapses, peripheral varicosities typically exhibit co-transmission of multiple neurotransmitters. Several lines of evidence show co-transmission of the sympathetic transmitters norepinephrine (NE) or ATP is mediated by the preferential association of separate pools of vesicles with CaV2.1 and CaV2.2 isoforms that provide the Ca2+ entry for vesicle release. CaV2.3 is a closely related channel that has been studied in several central synapses, but its role in peripheral sympathetic nerves is largely unknown. Using confocal microscopy to study perivascular nerves, we previously found that ATP and NE are stored in segregated pools of vesicles. Further, we found that CaV2.3 are expressed in rat tail perivascular sympathetic nerves and that they co-localize with vesicles expressing the vesicular nucleotide transporter (VNUT) that loads ATP into vesicles rather than vesicle harbouring the NE-transporting vesicular monoamine translocase 2 (VMAT2). We now report one of the first functional characterizations of CaV2.3 in perivascular nerves. We assessed the functional role of CaV2.3 in field-stimulated isometric force generated by rat tail arteries in response to pulse-number ramps (3-100 pulses, 20 Hz). Inhibition of vasoconstriction with the CaV2.3 blocker SNX-482 (250 nM) mimicked the effect of blocking smooth muscle P2X receptors with suramin while blocking α-adrenergic receptors with phentolamine productive a distinct inhibitory profile. These functional tests were corroborated by immuno-fluorescence microscopy showing that CaV2.3 immunoreactivity correlated with the sensitivity of individual blood vessels to suramin and SNX-482.

Chemistry and biology of radiotracers that target changes in sympathetic and parasympathetic nervous systems in heart disease.

Following the discovery of the sympathetic and parasympathetic nervous system, numerous adrenoceptor drugs were radiolabeled and potent radioligands were prepared in order to image the β-adrenergic and the muscarinic systems. But the greatest effort has been in preparing noradrenaline analogs, such as norepinephrine, (11)C-metahydroxyephedrine, and (123)I-metaiodobenzylguanidine that measure cardiac sympathetic nerve varicosities. Given the technical and clinical challenges in designing and validating targeted adrenoceptor-binding radiotracers, namely the heavily weighted flow dependence and relatively low target-to-background ratio, both requiring complicated mathematic analysis, and the inability of targeted adrenoceptor radioligands to have an impact on clinical care of heart disease, the emphasis has been on radioligands monitoring the norepinephrine pathway. The chemistry and biology of such radiotracers, and the clinical and prognostic impact of these innervation imaging studies in patients with heart disease, are examined.

Sympathetic Nervous System Moves Toward Center Stage in Cardiovascular Medicine

In 1664, the first anatomically correct depiction of the sympathetic nervous system came from Thomas Willis and his circle of London anatomists,1 included in The Anatomy of the Brain and Nerves , 1664 (Figure 1). This, the first work dedicated completely to the nervous system, also described the arterial loops at the base of the brain, which we now know as the Circle of Willis.1 Christopher Wren, an anatomist member of the group, was the principal illustrator1 before being asked by the City Fathers to turn his talents to town planning, architecture, and cathedral building after the 1666 Great Fire of London. Figure 1. An illustration of the human sympathetic nerves of the neck and thorax, from The Anatomy of the Brain and Sympathetic Nerves , published in 1664 by Thomas Willis and reproduced in Soul Made Flesh .1 Almost 2 centuries later, subsequent microscopic examination demonstrated that blood vessel walls were densely innervated, leading Stelling in 18402 to correctly conclude that these vasomotor fibers were in fact sympathetic nerves that were carried from the central nervous system to the blood vessels. In the mid-19th century, celebrated European physiologists, including Brown-Sequard, Waller, and Bernard,2 built on these observations, demonstrating vasoconstriction with electrical stimulation of the cut nerves and vasodilatation on nerve section, which indicated that the sympathetic fibers exerted a tonic, vasoconstrictor influence. The pressor nerves had gained recognition. Identification of the sympathetic neurotransmitter proved to be difficult. Claims for epinephrine3,4 and the hypothetical sympathins I and E confused the picture. Ulf von Euler compared bioassay responses of epinephrine, norepinephrine, and dihydroxy norephedrine with those of cattle splenic nerve extract, by testing blood pressure (BP) responses in the anesthetized cat and contractile responses in the isolated pregnant rabbit uterus, to definitively demonstrate the …

Sympathetic Nerve Varicosities in Close Apposition to Basolateral Membranes of Collecting Duct Epithelial Cells of Rat Kidney

Background: There are reports of sympathetic innervation of the nephron and of P2 purinergic receptors on epithelial cells. Since ATP is a cotransmitter with noradrenaline in sympathetic nerves, the objective of the present study was to re-investigate basolateral innervation of rat renal collecting duct epithelial cells by sympathetic nerves in the context of recent data on the effects of ATP on this nephron segment. Methods: Kidney sections were processed for electron immunocytochemistry, using tyrosine hydroxylase rabbit polyclonal antibody, with a second layer of biotinylated donkey anti-rabbit antibody and finally extravidin-horseradish peroxidase. Immunoreactivity was visualised with 3,3′-diaminobenzidine and examined with a Philips CM120 transmission electron microscope. Results: Electron microscopic evidence is presented for close apposition of sympathetic nerve varicosities immunolabelled with tyrosine hydroxylase to principal and intercalated type epithelial cells of the collecting duct of the cortical region. Conclusions: It is suggested that ATP is released as a cotransmitter from sympathetic nerve varicosities to act on basolateral P2 purinoceptors to influence sodium and water (and potentially acid-base) transport, in conjunction with the known (typically inhibitory) actions of autocrine and/or paracrine release of ATP from collecting duct epithelial cells acting via luminal P2 receptors. It is suggested that while luminal responses may dominate under normal physiological conditions, in pathophysiological states, such as stress and dehydration, sympathetic nerves might also be involved in modulating collecting duct fluid and electrolyte transport.

Endothelin receptors, localized in sympathetic nerve terminals of the heart, modulate norepinephrine release and reperfusion arrhythmias

Endothelin (ET)-1 is an endogenous vasoconstrictor which modulates norepinephrine (NE) release in myocardial ischemia reperfusion. Recent studies have demonstrated the pro- or anti-arrhythmic effects in reperfusion. The present studies were undertaken to test the hypothesis that ET receptors located in sympathetic nerve terminals modulate NE release associated with reperfusion arrhythmias (ventricular fibrillation; VF). Immunohistochemical studies showed that both ETA and ETB receptors exist in the sympathetic nerve varicosities, which were stained positive for tyrosine hydroxylase (TH) in the left ventricular wall in guinea pigs. Isolated guinea pig hearts were subjected to 20 min of normothermic global ischemia followed by 30 min reperfusion. Exogenously applied ET-1 (0.1 and 1 nM) dose-dependently increased NE release and the duration of VF, but these responses were significantly suppressed with the Na(+)/H(+) exchanger inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (10 microM). The ETA receptor antagonist (BQ123, 1 microM) and nonselective ET receptor antagonist (PD142893, 1 microM) significantly attenuated NE release and VF, whereas the ETB receptor antagonist (BQ788,300 nM) markedly elevated NE release but did not affect VF. These studies provide the first evidence that both ETA and ETB receptors, located in the sympathetic nerve varicosities, modulate NE release, at least in part, in association with reperfusion arrhythmias.

Disruption of Lipid Rafts Inhibits P2X1 Receptor-mediated Currents and Arterial Vasoconstriction

P2X1 receptors for ATP are ligand-gated cation channels expressed on a range of smooth muscle preparations and blood platelets. The receptors appear to be clustered close to sympathetic nerve varicosities and mediate the underlying membrane potential changes and constriction following nerve stimulation in a range of arteries and resistance arterioles. In this study we have used discontinuous sucrose density gradients, Western blot analysis, and cholesterol measurements to show that recombinant and smooth muscle (rat tail artery, vas deferens, and bladder) P2X1 receptors are present in cholesterol-rich lipid rafts and co-localize with the lipid raft markers flotillin-1 and -2. Lipid rafts are specialized lipid membrane microdomains involved in signaling and trafficking. To determine whether lipid raft association was essential for P2X1 receptor channel function we used the cholesterol-depleting agent methyl-beta-cyclodextrin (10 mm for 1 h). This led to a redistribution of the P2X1 receptor throughout the sucrose gradient and reduced P2X1 receptor-mediated (alpha,beta-methylene ATP, 10 microm) currents in HEK293 cells by >90% and contractions of the rat tail artery by approximately 50%. However contractions evoked by potassium chloride (60 mm) were unaffected by methyl-beta-cyclodextrin and the inactive analogue alpha-cyclodextrin had no effect on P2X1 receptor-mediated currents or contractions. P2X1 receptors are subject to ongoing regulation by receptors and kinases, and the present results suggest that lipid rafts are an essential component in the maintenance of these localized signaling domains and play an important role in P2X1 receptor-mediated control of arteries.

Responses of the isolated sympathetic nerve‐ductus deferens preparation of the guinea‐pig

Responses of the isolated sympathetic nerve‐ductus deferens preparation of the guinea‐pig

Incorporation in and release from sympathetic nerve varicosities of adrenaline intravenously injected

Incorporation in and release from sympathetic nerve varicosities of adrenaline intravenously injected

Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens.

1. The effects of calcium on the secretion of quanta recorded from single varicosities on the surface of the mouse vas deferens has been determined. 2. If recordings were made from two adjacent varicosities and the [Ca2+]o increased from a low value (1 mM), then the increase in the mean quantal content of the endplate potential (m(e)) was almost entirely due to an increase in the binomial probability for secretion (pe). At higher [Ca2+]o there was an increasing tendency for the binomial parameter (ne) to increase from 1 to 2. When ne increased there was very little change in pe, indicating that the new release site recruited from the other varicosity has a relatively low probability for secretion. 3. If recordings were restricted to single varicosities and the [Ca2+]o increased in the range from 1 to 4 mM then the increases in m(e) were almost always due to an increase in pe. The gradient relating the log of m(e) to the log of [Ca2+]o for [Ca2+]o of 1.0-1.5 mM was in the range 3.2-5.4 (mean 4.2). 4. Test impulses gave a similar proportional increase in m(e) following a conditioning impulse at all varicosities from which recordings were made. 5. Facilitation of m(e) declined exponentially with an increase in the test-conditioning interval from 0.5 to 4 s. The time constant for this decline was about 6 s. 6. The results indicate that single release sites show a similar fourth power dependency on [Ca2+]o and facilitate to about the same degree following a conditioning impulse.

Intermittent release of noradrenaline by single pulses and release during short trains at high frequencies from sympathetic nerves in rat tail artery

As shown by electrophysiological analysis, the release of the sympathetic co-transmitter adenosine 5′-triphosphate (ATP) from individual release sites is monoquantal and intermittent; the average release probability may be as low as 0.01. 1–3 Indirect evidence from biochemical studies of noradrenaline overflow is compatible with a similar monoquantal, low probability release of noradrenaline as well. 10 In the present study our first aim was to address this issue more directly in rat tail artery, using continuous amperometry to monitor in real time the release of noradrenaline from a relatively small number of sympathetic nerve varicosities. The results seem to provide the first direct evidence that noradrenaline, similarly to ATP, may be released intermittently during nerve stimulation at low frequency. Our second aim was to use the same technique to study the release of noradrenaline caused by nerve stimulation with single pulses or short trains (two to eight pulses) at high frequencies. The results show that during stimulation at 20 Hz the peak amplitude of the noradrenaline oxidation current response grew linearly with the train length, but at 50 Hz the curve describing this growth was sigmoid in shape.

Quantal Secretion at Visualized Sympathetic Nerve Varicosities

It has recently been shown that varicosities of single sympathetic nerve terminals possess a heterogeneous distribution of secretion probabilities. This discovery was made possible by the introduction of techniques that allow the visualization of a single live varicosity and the recording of the electrical signs of its secretion of quanta.

Probabilistic secretion of quanta from visualized sympathetic nerve varicosities in mouse vas deferens.

1. Sympathetic varicosities on the surface of smooth muscle cells of the mouse vas deferens were visualized with the fluorescent dye 3-3 Diethyloxardicarbocyanine iodide (DiOC2(5)) and quantal secretion recorded from these with both small diameter (4-6 microns) and large diameter (20-50 microns) microelectrodes. Small diameter electrodes were placed over one to three varicosities and large diameter electrodes over three to seven varicosities. 2. The size and distribution of varicosities along individual terminal branches was about the same when these were fluoresced with DiOC2(5) (length 1.09 +/- 0.40 microns (mean +/- S.D.); intervaricosity distance 5.53 +/- 2.68 microns) as when they were stained for catecholamines using Faglu fluorescence (length 1.05 +/- 0.43 microns; intervaricosity distance 5.12 +/- 2.79 microns) suggesting that DiOC2(5) does allow for identification of the catecholamine-containing varicosities. 3. The spontaneous excitatory junctional currents (EJCs) recorded from visualized varicosities with small diameter electrodes (amplitudes 59-67 microV) were much larger than those recorded with large diameter electrodes (amplitudes 25-29 microV). The frequency of evoked EJCs as well as the amplitude-frequency distribution of these EJCs varied greatly between sets of visualized varicosities recorded along individual branches, either with a small or large diameter electrode. These amplitude-frequency distributions typically followed Poisson statistics, in which the mean quantal content of the EJC (m) varied by over threefold for different sets of varicosities on the same branch (m was 0.07-0.21 for small electrodes whereas m was 1-3 for large electrodes). 4. Although m varied considerably for a constant number of varicosities beneath the electrode at different sites along a single branch, there was an overall correlation between m and the number of varicosities, m increasing on average 0.25 for each additional varicosity in a [Ca2+]o of 4.0 mM. 5. The frequency of evoked EJCs at visualized sets of varicosities along some branches was sufficiently high to allow binomial statistics to predict the amplitude-frequency distributions of evoked EJCs. In these cases m was again shown to vary considerably along single terminal branches, and this was primarily due to variation in the probability of secretion (p) between sets of varicosities and not to variation in binomial parameter n. 6. In one case a relatively isolated varicosity, over 3 microns from adjacent varicosities, was recorded for 30 min with a 4 microns diameter electrode. The mean and variance of the evoked EJC was similar to that of the spontaneous EJCs suggesting that this varicosity secreted at most one quantum on arrival of the nerve impulse.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of desipramine on sympathetic nerve firing and norepinephrine spillover to plasma in humans

In isolated organs, or when given in low dose intra-arterially, tricyclic antidepressant drugs are known to block reuptake of norepinephrine into sympathetic nerve varicosities, with a resultant increased norepinephrine washout. On the other hand, systemic administration of such drugs in humans reduces norepinephrine spillover to plasma. To clarify these seemingly contradictory findings, we have measured concurrently muscle sympathetic activity in the peroneal nerve (microneurography) and rates of norepinephrine spillover to plasma for the body as a whole and for the heart, the kidneys, and the forearm (radiotracer technique), both before and after intravenous infusion of desipramine, 0.5 mg/kg. Desipramine lowered the overflow of norepinephrine to plasma for the body as a whole and from the forearm and the kidneys (by 30-50%) but increased cardiac norepinephrine spillover by 25%. Both the number of sympathetic bursts per min in the peroneal nerve and their mean voltage amplitudes were markedly reduced after desipramine; total activity (bursts/min x mean burst amplitude) fell by approximately 90%. The effects of desipramine on norepinephrine spillover are explicable in terms of inhibition of central sympathetic outflow, balanced against the local blockade of transmitter reuptake. In most sites, the predominant effect is a reduction of norepinephrine overflow. For the heart, where reuptake is so important in transmitter disposition, the net effect is increased overflow.

“Upstream” regulation of the release probability in sympathetic nerve varicosities

The results appear to support the following tentative working hypothesis. (1) Nerve impulse-induced transmitter release from sympathetic nerve varicosities is monoquantal and highly intermittent (probability range: 0-0.03). (2) Nerve impulses invade varicosities as all-or-none, Na+ channel-dependent action potentials; invasion failure may be rare. (3) The release probability is not controlled by properties (amplitude or duration) of the invading action potential or the resulting Ca2+ current, but by the availability of an as yet unidentified permissive factor. (4) The permissive factor is actively transported intra-axonally, probably in association with organelles (LDVs?). (5) The activation and/or transport of the permissive factor are controlled "upstream" of the varicosity; they depend on Ca2+ influx through channels insensitive to nifedipine (hence, not of L-type) but blocked by Cd2+ and apparently opened by slight depolarization of the resting membrane, in this respect behaving more as T- than N-type channels. (6) A high resting K+ efflux "upstream" of the varicosity restricts the availability of the permissive factor; it is the main mechanism maintaining the (economically necessary) low release probability. (7) Prejunctional agonists do not inhibit transmitter secretion by causing a conduction block or by reducing the action potential-induced Ca2+ influx into the varicosity itself, but by depressing the Ca2(+)-dependent activation and/or transport of the permissive factor; they act at least in part via receptors "upstream" of the varicosity. (8) This hypothesis for regulation of the release probability in sympathetic nerves may apply, at least in part, to other neurons as well.

On the secretory activity of single varicosities in the sympathetic nerves innervating the rat tail artery.

1. Nerve terminal impulses (NTIs) and spontaneous or stimulus-evoked excitatory junction currents (SEJCs or EJCs), reflecting secretion of transmitter quanta from release sites in the sympathetic nerves of rat tail artery, were recorded by extracellular electrodes. 2. The release of transmitter quanta from single varicosities was analysed on a pulse-by-pulse basis. 3. Since the SEJCs were tetrodotoxin-resistant, and hence probably caused by single quanta, they were employed to analyse the quantal content of EJCs. 4. In the majority of recordings, EJCs were large compared to SEJCs from the same attachment, and preceded by prominent NTIs. This type of activity appeared to reflect simultaneous activation of several nerve fibres and numerous varicosities. 5. By focal stimulation, it was usually possible to improve the resolution by examining spots in which a large proportion of the suprathreshold stimuli failed to cause EJCs. Here, averaged NTIs preceding large EJCs were indistinguishable from averaged NTIs not followed by EJCs. Thus, failure of invasion by the nerve impulse was not a cause of the frequent secretory failure. 6. In these attachments the amplitude distribution of nerve stimulus-evoked EJCs was similar to that of the SEJCs and many individual EJCs could be matched in amplitude and time course by SEJCs. Thus, transmitter secretion from these sympathetic nerve varicosities seems to be basically monoquantal. 7. Under conditions when all EJCs were smaller than or equal to the largest SEJCs some characteristic EJC profiles appeared only a few times in response to several hundred suprathreshold stimuli at low frequency (0.5-1 Hz). Using tentatively these EJCs as 'fingerprints' of single quanta from particular release sites, the probability for activation of individual release sites ranges from 0.002 to 0.02.

The functional significance of the sympathetic innervation of mucous glands in the bronchi of man.

1. Pieces of human bronchi, from lung resected for carcinoma of the bronchus, were mounted in Ussing chambers and given [35S]sulphate as radiolabelled precursor of mucous glycoproteins (mucins). The release of 35S, bound to macromolecules, into the luminal half-chamber was used as an index of mucin secretion. 2. Noradrenaline, at concentrations of 1, 10 and 100 microM, was given into both halves of the Ussing chamber. At the lowest concentration, noradrenaline failed to change mucin output, but at the two higher concentrations it stimulated output. 3. In other experiments the sympathetic nerves in the bronchial wall were labelled with 5-hydroxydopamine and examined under the electron microscope. The distances between adrenergic nerve varicosities and submucosal glands were measured; some sympathetic nerve varicosities were seen within 1 microns of gland cells. 4. A simple mathematical model for the diffusion of noradrenaline was used to predict the concentrations of the transmitter likely to result at different distances from a nerve if one or more vesicles of noradrenaline were released. 5. The model predicts that the release of a single large vesicle of noradrenaline is likely to generate an effective concentration of transmitter provided that the nerve is within 1 micron of the target cell.

New paradigm: sympathetic transmission by multiple messengers and lateral interaction between monoquantal release sites?

Evidence is accumulating that noradrenaline (NA) is not the only transmitter substance in sympathetic nerves, and that individual sympathetic nerve varicosities are only activated intermittently by impulses in the parent axon and may then secrete only a single ‘mixed’ transmitter quantum. Hence_sympathetic neurotransmission may not be based on accumulation of NA in each junctional cleft, as currently widely believed, but rather on lateral interaction between scattered sites of release of single quanta composed of NA and one or more co-transmitters.