Translatable mitochondria-targeted protection against programmed cardiovascular dysfunction.

The prenatal origins of heart disease in offspring have been established. However, research in species with developmental milestones comparable to humans is lacking, preventing translation of this knowledge to clinical contexts. Using sheep and chickens, two species with similar cardiovascular developmental milestones to humans, we combined in vivo experiments with in vitro studies at organ, cellular, mitochondrial, and molecular levels. We tested mitochondria-targeted antioxidant intervention with MitoQ against cardiovascular dysfunction programmed by developmental hypoxia, a common complication in human pregnancy. Experiments in sheep determined in vivo fetal and adult cardiovascular function through surgical techniques not possible in humans, while those in chicken embryos isolated effects independent of maternal or placental influences. We show that hypoxia generates mitochondria-derived oxidative stress during cardiovascular development, programming endothelial dysfunction and hypertension in adult offspring. MitoQ treatment during hypoxic development protects against this cardiovascular risk via enhanced nitric oxide signaling, offering a plausible intervention strategy.

Fetal in vivo continuous cardiovascular function during chronic hypoxia.

Although the fetal cardiovascular defence to acute hypoxia and the physiology underlying it have been established for decades, how the fetal cardiovascular system responds to chronic hypoxia has been comparatively understudied. We designed and created isobaric hypoxic chambers able to maintain pregnant sheep for prolonged periods of gestation under controlled significant (10% O2) hypoxia, yielding fetal mean P(aO2) levels (11.5 ± 0.6 mmHg) similar to those measured in human fetuses of hypoxic pregnancy. We also created a wireless data acquisition system able to record fetal blood flow signals in addition to fetal blood pressure and heart rate from free moving ewes as the hypoxic pregnancy is developing. We determined in vivo longitudinal changes in fetal cardiovascular function including parallel measurement of fetal carotid and femoral blood flow and oxygen and glucose delivery during the last third of gestation. The ratio of oxygen (from 2.7 ± 0.2 to 3.8 ± 0.8; P < 0.05) and of glucose (from 2.3 ± 0.1 to 3.3 ± 0.6; P < 0.05) delivery to the fetal carotid, relative to the fetal femoral circulation, increased during and shortly after the period of chronic hypoxia. In contrast, oxygen and glucose delivery remained unchanged from baseline in normoxic fetuses. Fetal plasma urate concentration increased significantly during chronic hypoxia but not during normoxia (Δ: 4.8 ± 1.6 vs. 0.5 ± 1.4 µmol l(-1), P<0.05). The data support the hypotheses tested and show persisting redistribution of substrate delivery away from peripheral and towards essential circulations in the chronically hypoxic fetus, associated with increases in xanthine oxidase-derived reactive oxygen species.

Prostaglandin E2 induces spontaneous rhythmic activity in mouse urinary bladder independently of efferent nerves.

BACKGROUND AND PURPOSE: The acute effects of PGE(2) on bladder smooth muscle and nerves were examined to determine the origin of PGE(2)-induced spontaneous rhythmic contractions. EXPERIMENTAL APPROACH: Contraction studies, confocal Ca(2+) imaging and electrophysiological recordings in strips of mouse urinary bladder were used to differentiate the effects of PGE(2) on bladder smooth muscle and efferent nerves. KEY RESULTS: PGE(2) (50 µM) increased the tone and caused phasic contractions of detrusor smooth muscle strips. Confocal Ca(2+) imaging showed that PGE(2) increased the frequency of whole-cell Ca(2+) transients (WCTs) (72 ± 5%) and intracellular recordings showed it increased the frequency of spontaneous depolarizations, from 0.31·s(-1) to 0.90·s(-1). Non-selective inhibition of EP receptors using SC-51322 and AH-6809 (10 µM), or the L-type Ca(2+) channel blocker nifedipine (1 µM), prevented these phasic contractions and WCTs, and reduced the tone (by 45 ± 7% and 59 ± 6%, respectively). Blocking P2X1 receptors with NF449 (10 µM) caused a small but significant reduction in the frequency of PGE(2)-induced phasic contractions (24 ± 9%) and WCTs (28 ± 17%) but had no significant effect on spontaneous depolarizations or tone. Inhibiting muscarinic receptors with cyclopentolate (1 µM) had no significant effect on these measures. Spontaneous WCTs became synchronous in PGE(2), implying enhanced functional coupling between neighbouring cells. However, the electrical input resistance was unchanged. CONCLUSIONS AND IMPLICATIONS: It was concluded that depolarization alone is sufficient to explain a functional increase in intercellular coupling and the ability of PGE(2) to increase detrusor spontaneous rhythmic activity does not require parasympathetic nerves.

Ion channel modulators and urinary tract function.

The membrane potential fulfils an important role in initiating smooth muscle contraction, through its depolarization and the subsequent influx of Ca(2+) through voltage-gated Ca(2+) channels. Changes in membrane potential can also coordinate contraction across great distances, utilizing the speed of electrical current flow through gap junctions. Hence, regulating membrane potential can greatly influence smooth muscle function. In this chapter, we will consider the influence of ion channels, as dynamic gatekeepers of membrane permeability, on urogenital function. Through their ability to act as key regulators of both the resting membrane potential and its dynamic changes, they provide important pharmacological targets for influencing urogenital function.Urogenital smooth muscle and urothelia contain a diverse range of molecularly and functionally distinct K(+) channels, which are key to regulating the resting membrane and for re-establishing the normal membrane potential following both active and passive changes. The voltage-gated Ca(2+) channels are key to initiating contraction and causing rapid depolarization, supplemented in some smooth muscles by rapid Na(+) conductances. The Cl(-) channels, often assumed to be passive, can actively change the membrane potential, and hence, cellular function, because Cl(-) is not usually at its equilibrium potential. The useful ways in which these ion channels can be targeted therapeutically in the ureter, bladder and urethra are discussed, focussing particularly on treatments for ureteric obstruction and detrusor overactivity. Current treatments for many urinary tract disorders, particularly the overactive bladder, are complicated by side effects. While ion channels have traditionally been considered as poor therapeutic targets by the pharmaceutical industry, our increasing knowledge of the molecular diversity of K(+) and Cl(-) channels gives new hope for more narrowly focused drug targeting, while the exciting discoveries of active currents in interstitial cells give us a new set of cellular targets for drugs.

Neuroeffector Ca2+ transients for the direct measurement of purine release and indirect measurement of cotransmitters in rodents.

Determining whether ATP and noradrenaline are released from the same vesicle at mature autonomic neuroeffector junctions is challenging because of the difficulty of simultaneously detecting the packeted release of these neurotransmitters. Contraction, overflow and electrophysiology experiments all show that both ATP and noradrenaline are released following field stimulation (although the ratio might vary) from autonomic nerves in tissues including the vas deferens, rat tail artery and mesenteric artery. The occurrence of purinergic neuroeffector Ca(2+) transients (NCTs) has been used to detect the packeted release of the neurotransmitter ATP acting on postjunctional P2X receptors to cause Ca(2+) influx. Neuroeffector Ca(2+) transients can also be used to detect the local effects of noradrenaline through its alpha(2)-adrenoceptor-mediated prejunctional autoinhibitory effects on nerve terminal Ca(2+) concentration and the probability of exocytosis (measured by counting NCTs). Evidence is presented that exocytosis from sympathetic varicosities does not occur in a manner independent of the history of that varicosity, but rather that the release of a packet of ATP transiently suppresses (or predicts the transient suppression of) subsequent release. This could arise by autoinhibition (by the prejunctional action of noradrenaline or purines) or due to a transient shortage of vesicles readily available for release. In summary, two high-resolution approaches are proposed to measure the intermittent release of packets of neurotransmitter: (1) local transient suppression of nerve terminal Ca(2+) transients; and (2) the local and transient inhibition of NCTs to infer local autoinhibition, hence transmitter release. Such approaches may allow the packeted corelease of ATP and noradrenaline to be investigated without the need to measure both neurotransmitters directly.

Bretylium abolishes neurotransmitter release without necessarily abolishing the nerve terminal action potential in sympathetic terminals.

BACKGROUND AND PURPOSE: The antidysrhythmic bretylium is useful experimentally because it selectively abolishes neurotransmitter release from sympathetic peripheral nerve terminals. Its mechanism of action seemed settled, but recent results from optical monitoring of single terminals now suggests a new interpretation. EXPERIMENTAL APPROACH: Orthograde transport of a dextran-conjugated Ca(2+) indicator to monitor Ca(2+) in nerve terminals of mouse isolated vas deferens with a confocal microscope. In some experiments, local neurotransmitter release was detected by monitoring neuroeffector Ca(2+) transients (NCTs) in adjacent smooth muscles, a local measure of purinergic transmission. Sympathetic terminals were identified with catecholamine fluorescence (UV excitation) or post-experiment immunohistochemistry. KEY RESULTS: Bretylium (10 microM) abolished NCTs at 60/61 junctions over the course of 2 h, indicating effective abolition of neurotransmitter release. However, bretylium did not abolish the field stimulus-induced Ca(2+) transient in most nerve terminals, but did increase both action potential delay (by 2+/-0.4 ms) and absolute refractory period (by 4+/-2 ms). Immunohistochemistry demonstrated that 85-96% of terminals orthogradely filled with a dextran-conjugated fluorescent probe contained Neuropeptide Y (NPY). A formaldehyde-glutaraldehyde-induced catecholamine fluorescence (FAGLU) technique was modified to allow sympathetic terminals to be identified with a Ca(2+) indicator present. Most terminals contained catecholamines (based on FAGLU) or secrete ATP (as NCTs in adjacent smooth muscle cells are abolished). CONCLUSIONS AND IMPLICATIONS: Bretylium can inhibit neurotransmitter release downstream of Ca(2+) influx without abolishing the nerve terminal action potential. Bretylium-induced increases in the absolute refractory period permit living sympathetic terminals to be identified.

Electrical and optical study of nerve impulse-evoked ATP-induced, P2X-receptor-mediated sympathetic neurotransmission at single smooth muscle cells in mouse isolated VAS deferens.

Simultaneous electrophysiology and confocal microscopy were used to investigate purinergic neurotransmission at single smooth muscle cells (SMCs) in mouse isolated vas deferens, and to explore the relationship between two high-resolution P2X-receptor-mediated measures of per pulse ATP release: transient peaks in the first time derivative of the rising phase of excitatory junction potentials (EJPs) recorded in single SMCs ('discrete events'; DEs) and neuroeffector Ca(2+) transients (NCTs) in the impaled SMCs. This study shows that discrete events represent neurotransmitter release onto the impaled cell. First, the median amplitude of the first derivative of the EJP was larger when there was a coincident NCT in the impaled cell, compared with instances when no coincident NCT occurred. Second, the time-to-peak amplitude of the first derivative was shorter if there was a coincident NCT in the impaled cell, compared with when no coincident NCT was observed within the field. Surprisingly, first derivative amplitude increased with the distance (of the corresponding NCT) from the microelectrode. The microelectrode did not locally inhibit the functional quantal size as there was no effect of distance on the normalized NCT amplitude. When the significant effect of distance (between the microelectrode and NCTs) on the first derivative amplitude was removed, there was no correlation between the unstandardized residual (of distance vs. first derivative amplitude) and NCT amplitude. The absence of a correlation between DE and NCT amplitudes suggests that the NCT amplitude is a poor measure of quantal size. The usefulness of NCTs hence lies primarily in locating neurotransmitter release and measuring changes in local release probability.

The effect of epibatidine on spontaneous and evoked neurotransmitter release in the mouse and guinea pig isolated vas deferens.

BACKGROUND AND PURPOSE: Nicotinic agonists increase sympathetic field-stimulus-evoked contraction of the rodent vas deferens, presumably by increasing evoked neurotransmitter release. This presumption was tested in two species. EXPERIMENTAL APPROACH: The effect of the nicotinic acetylcholine receptor (nAChR) agonist epibatidine on neurotransmitter release in mouse and guinea pig isolated vas deferens was investigated using contraction studies and conventional intracellular recording techniques. KEY RESULTS: In 12 of 14 mouse vasa deferentia, slow bath application of epibatidine (100 nM) had no significant effect on excitatory junction potential (EJP) amplitude and spontaneous EJP (SEJP) frequency. However, rapid application of epibatidine to the mouse vas deferens caused an increase in SEJP frequency (by 530%), with no effect on EJP amplitude. Despite the absence of an effect on EJPs, electrically-evoked contractions of the mouse vas deferens were significantly increased in the presence of epibatidine (by 50%). A transient contraction was reliably induced by a higher epibatidine concentration (1 microM). This contraction was significantly reduced in the presence of prazosin, tetrodotoxin, or alpha,beta-methyleneATP. Epibatidine did not induce a contraction in the presence of a combination of prazosin, alpha,beta-methyleneATP and cyclopentolate. In guinea pig vasa deferentia, bath-applied epibatidine potentiated EJP amplitude in a biphasic pattern, lasting for at least 30 minutes. CONCLUSION AND IMPLICATIONS: The nAChR-mediated augmentation of neurogenic contraction is indeed prejunctional, but in the mouse arises from an increase in spontaneous neurotransmitter release that primes smooth muscle for subsequent contraction, while in the guinea pig there is a direct augmentation of evoked neurotransmitter (ATP) release.

The origin of the skewed amplitude distribution of spontaneous excitatory junction potentials in poorly coupled smooth muscle cells.

The skewed amplitude distribution of spontaneous excitatory junction potentials (sEJPs) in the mouse vas deferens and other electrically-coupled smooth muscle syncytia has been attributed to electrically-attenuated depolarizations resulting from the spontaneous release of quantized packets of ATP acting on remote smooth muscle cells (SMCs). However, in the present investigation surface SMCs of the mouse isolated vas deferens were poorly electrically coupled, with input resistances (176+/-18 MOmega, range: 141-221 MOmega, n=4) similar to those of dissociated cells. Furthermore, the amplitude of evoked EJPs was more variable in surface compared with deeper SMCs (F test, F=17.4, P<0.0001). Using simultaneous electrophysiology and confocal microscopy to investigate these poorly-coupled cells, it is shown that alpha-latrotoxin-stimulated sEJPs correlate, in timing (median delay ranged from -30 to -57 ms, P<0.05 in all experiments, n=5) and amplitude (Pearson product moment correlation, rho>0.55 and P<0.001), with purinergic neuroeffector Ca2+ transients (NCTs) in SMCs. The temporal correlation between sEJPs of widely ranging amplitude with NCTs in the impaled SMC demonstrates that all sEJPs could arise from neurotransmitter action on the impaled cell and that the skewed distribution of sEJPs can be explained by the variable effect of packets of ATP on a single SMC. The amplitude correlation of sEJPs and NCTs argues against the attenuation of electrical signal amplitude along the length of a single SMC. The skewed sEJP amplitude distribution arising from neurotransmitter release on single SMCs is consistent with a broad neurotransmitter packet size distribution at sympathetic neuroeffector junctions.

Characterization of action potential-evoked calcium transients in mouse postganglionic sympathetic axon bundles.

1. Action potential-evoked Ca(2+) transients in postganglionic sympathetic axon bundles in mouse vas deferens have been characterized using confocal microscopy and Ca(2+) imaging. 2. Axonal Ca(2+) transients were tetrodotoxin sensitive. The amplitude depended on both the frequency of stimulation and the number of stimuli in a train. 3. Removal of extracellular Ca(2+) abolished the Ca(2+) transient. Cd(2+)(100 microM) inhibited the Ca(2+) transient by 78 +/- 10 %. The N-type Ca(2+) channel blocker omega-conotoxin GVIA (0.1 microM) reduced the amplitude by -35 +/-4 %, whereas nifedipine (10 microM; L-type) and omega-conotoxin MVIIC (0.1 microM; P/Q type) were ineffective. 4. Caffeine (10 mM), ryanodine (10 microM), cyclopiazonic acid (30 microM) or CCCP (10 microM) had no detectable effects. 5. Blockade of large and small conductance Ca(2+)-dependent K+ channels with iberiotoxin (0.1 microM) and apamin (1 microM), respectively, or Ca(2+)-dependent Cl(-) channels by niflumic acid (100 microM) did not alter Ca(2+) transients. 6. In contrast, the non-specific K+ channel blockers tetraethylammonium (10 mM) and 4-aminopyridine (10 mM) markedly increased the amplitude of the Ca(2+) transient. Blockade of delayed rectifiers and A-like K+ channels, by tityustoxin-K (alpha) (0.1 microM) and pandinustoxin-K (alpha) (10 nM), respectively, also increased the Ca(2+) transient amplitude. 7. Thus, Ca(2+) transients are evoked by Na(+)-dependent action potentials in axons. These transients originate mainly from Ca(2+) entry through voltage-dependent Ca(2+) channels (80 % Cd(2+) sensitive of which 40 % was attributable to N-type). Twenty per cent of the Ca(2+) transient was not due to Ca(2+) entry through voltage-gated Ca(2+) channels. Intracellular stores and mitochondria were not involved in the generation of the transient. Ca(2+) transients are modulated by A-like K+ channels and delayed rectifiers (possibly K(V)1.2) but not by Ca(2+)-activated ion channels.

Nicotine induces calcium spikes in single nerve terminal varicosities: a role for intracellular calcium stores.

While nicotine is known to act at neuronal nicotinic acetylcholine receptors (nAChRs) to facilitate neurotransmitter release, the mechanisms underlying this action are poorly understood. Some of its effects are known to be mediated by presynaptic receptors. In the mouse vas deferens nicotine (10-30 microM) transiently increased the force of neurogenic contraction by 135+/-25%, increased the amplitude of excitatory junction potentials by 74+/-6% and increased the frequency of spontaneous excitatory junction potentials in four out of six preparations. Confocal microscopy and the calcium indicator Oregon Green 488 BAPTA-1 dextran were used to measure calcium concentration changes in the nerve terminals. Nicotine did not affect the action potential-evoked calcium transient but instead triggered small, random fluctuations ("calcium spikes") in intra-varicosity calcium concentrations at an average frequency of 0.09+/-0.02 Hz. These were insensitive to tetrodotoxin at a concentration that blocked action-potential evoked calcium transients (300 nM). They were abolished by the nAChR blocker hexamethonium (100 microM) and by both ryanodine (100 microM) and caffeine (3 mM), agents that modify calcium release from intracellular stores. We propose a novel mechanism whereby nicotine's action at nAChRs triggers calcium-induced calcium release from a ryanodine-sensitive calcium store in nerve terminals. This primes neurotransmitter release mechanisms and enhances both spontaneous and action potential-evoked neurotransmitter release.

Individual sympathetic varicosities possess different sensitivities to alpha 2 and P2 receptor agonists and antagonists in mouse vas deferens.

1. The diversity of alpha(2) and purinergic autoreceptor actions on action potential evoked calcium transients in single varicosities has been investigated using the calcium indicator Oregon Green 488 BAPTA-1. 2. During long trains of impulses (10 Hz for 30 s), the change in calcium concentration in varicosities from its resting level (Delta[Ca(2+)](v)) increased in many varicosities during the first 3 s of stimulation before reaching a plateau. 3. The alpha(2) adrenoceptor agonist clonidine (1 microM) decreased Delta[Ca(2+)](v) by over 40% during short trains (five impulses at 5 Hz) in most varicosities, although some were unaffected. The alpha(2) adrenoceptor antagonist idazoxan (2 microM) increased the Delta[Ca(2+)](v) plateau following long trains in most varicosities. Hence, most varicosities possess alpha(2) adrenoceptors which are activated when noradrenaline accumulates extracellularly. 4. During long trains of impulses, the P(2y)-purinergic receptor agonist 2-methyl-thio-ATP (100 microM) decreased Delta[Ca(2+)](v) plateau by about 50% in most varicosities; alpha,beta-methylene ATP (100 microM) decreased it by about 50% in a minority of varicosities; adenosine (200 microM) had no significant effect. Suramin (100 microM) increased the Delta[Ca(2+)](v) during all stimulus protocols in most varicosities, suggesting that ambient ATP modulates Delta[Ca(2+)](v) responses. The P(2y) receptor antagonist reactive blue (100 microM) affected a minority of varicosities. Given that most varicosities respond to suramin, other P(2) receptor subtypes are probably present. 5. The ATP ectoenzyme antagonist ARL67157 (50 microM) decreased the plateau Delta[Ca(2+)](v) during long trains in complete strings of varicosities but not in others. 6. The present technique indicates that varicosities have diverse autoreceptor utilization.

Phosphorylation of proteins in chick ciliary ganglion under conditions that induce long-lasting changes in synaptic transmission: phosphoprotein targets for nitric oxide action.

Production of nitric oxide and the activation of protein kinases are required for long-term potentiation of synaptic transmission at the giant synapses in chicken ciliary ganglion. In the present study, we investigated the ability of nitric oxide to regulate the phosphorylation of endogenous proteins under conditions that induced long-term potentiation in intact ciliary ganglion and the protein kinases responsible for the phosphorylation of these proteins in lysed ciliary ganglion. Using Calcium Green-1 we showed that the nitric oxide donor sodium nitroprusside did not change the intraterminal Ca2+ dynamics in ciliary ganglion. Two dimensional phosphopeptide analysis of 32Pi-labelled intact ciliary ganglion showed that the sodium nitroprusside (300 microM) increased the phosphorylation of several phosphopeptides (P50a, P50b and P41) derived from proteins at 50,000 and 41,000 mol. wts which we have called nitric oxide-responsive phosphoproteins. A similar stimulation of phosphorylation was achieved by 8-bromo-cyclic AMP (100 microM), which also induced long-term potentiation, but not by phorbol dibutyrate (2 microM) that does not induce long-term potentiation in ciliary ganglion. When subcellular fractions from lysed ciliary ganglion were labelled in vitro by [gamma-32P]ATP in the presence of purified cGMP-dependent, cAMP-dependent or Ca2+-phospholipid-dependent protein kinases, we identified cyclic GMP-dependent protein kinase substrates that gave rise to phosphopeptides co-migrating with P50a, P50b and P41 from 32Pi-labelled intact ciliary ganglion. P50a and P41 were derived from soluble proteins while P50b was derived from a membrane-associated protein. The proteins giving rise to P50a, P50b and P41 were also substrates for cyclic AMP-dependent protein kinase, but not for calcium and phospholipid-dependent protein kinase in vitro, suggesting that nitric oxide-responsive phosphoproteins are convergence points in information processing in vivo and their phosphorylation might represent an important mechanism in nitric oxide-mediated synaptic plasticity in ciliary ganglion.

The self-mutilative nature of severe onychophagia: a comparison with self-cutting.

OBJECTIVE: To investigate the psychophysiological pattern associated with severe and mild onychophagia, and to compare this pattern with that demonstrated by previous research on self-cutting. METHOD: Comparisons between the psychophysiological responses accompanying 3 behaviours were made using a guided imagery methodology. Imagery of nail-related, skin-cutting, and neutral events were presented in 4 stages. RESULTS: Experiment I distinguished participants exhibiting severe and mild onychophagia by the severity and frequency of nail-biting and by the pattern of psychophysiological response across the stages. Experiment II indicated that the change in psychophysiological arousal accompanying severe onychophagia was not as dramatic as that demonstrated for skin-cutting. The behaviour seems to be less effective in reducing tension. CONCLUSION: Severe onychophagia appears to manage the level of tension experienced by an individual, instead of dramatically reducing it in times of crisis. Such a process is consistent with that demonstrated in individuals with obsessive-compulsive disorder.

Calcium in sympathetic boutons of rat superior cervical ganglion during facilitation, augmentation and potentiation.

The sympathetic preganglionic nerve terminals of the rat superior cervical ganglion were loaded with the calcium indicator oregon green 488 BAPTA-1 to measure the change in calcium concentration in the terminal boutons, (delta[Ca2+]b) following short (1 or 5 impulses) and long (200 impulses) trains at 30 Hz. The delta[Ca2+]b after a single action potential or a short train declined in two phases: a fast phase with a time constant of 530+/-30 ms and a moderate phase with a time constant of 4.0+/-0.2 s. The delta[Ca2+]b following a long train eventually declined with a time constant of 127+/-34 s (slow phase). The addition of either omega-agatoxin TK (100 nM), omega-conotoxin GVIA (100 nM) or nifedipine (20 microM) to block P-type, N-type or L-type calcium channels respectively showed that the rise in delta[Ca2+ ]b in boutons was predominantly mediated by an influx of calcium through P-type (53+/-7%) and N-type (46+/-4%) calcium channels. Experiments with caffeine, ryanodine and thapsigargin indicate that intracellular caffeine-sensitive calcium stores have a small but statistically significant effect on the fast and moderate phases. The mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 2 microM) significantly decreased the amplitude of the slow phase of delta[Ca2+]b relaxation, and sped its time course, suggesting that mitochondria normally dump calcium during this phase. Adenosine reduced the amplitude of delta[Ca2+]b in response to single action potentials by 30+/-6%, suggesting that adenosine-mediated autoinhibition in these boutons reduces Ca2+ influx. Spontaneous increases in delta[Ca2+]b demonstrated Ca2+ coupling between adjacent boutons. The delta[Ca2+]b kinetics are compared with F2 facilitation, augmentation and post-tetanic potentiation.

Sympathetic neuromuscular transmission at a varicosity in a syncytium.

The autonomic neuromuscular junction at a varicosity in the vas deferens is defined by the localization of the vesicle-associated protein syntaxin in high concentrations in the axolemma and a high density of P2x1 receptors in a cluster beneath the varicosity. Calcium fluxes have been observed in all individual varicosities of a nerve terminal on the arrival of an impulse even though recordings made from these varicosities of the electrical signs of transmission with loose-patch electrodes over the varicosities show that they have very different probabilities for the secretion of a quantum. The fact that some varicosities seldom release a quantum on the arrival of an impulse is supported by the observation that antibodies against the N-terminus of synaptotagmin, which uniquely label the inside of synaptic vesicles when they undergo exocytosis, fail to do so in some varicosities during nerve stimulation whereas they do in others. It is suggested that the probability for secretion from a varicosity depends on the number of secretosomes that the varicosity possesses, where a secretosome is a complex of syntaxin, synaptotagmin, an N-type calcium channel, and a synaptic vesicle.

Calcium transients evoked by action potentials in the somata of chick ciliary neurons.

The effect of action potentials on the calcium concentration in the somata of chick ciliary neurons ([Ca2+]s) was determined by loading these with the calcium indicator calcium green-1. Following trains of 1-10 impulses (30 Hz) to the postganglionic nerve, the [Ca2+]s increased rapidly and then declined along a single exponential with a time constant of 0.70 +/- 0.04 s (fast phase). After trains of 20 or 50 impulses, the elevated [Ca2+]s declined as the sum of two exponentials, with time constants of 0.78 +/- 0.12 s (fast phase) and 4.0 +/- 0.4 s (moderate phase). After a 600-impulse postganglionic train of impulses, the elevated [Ca2+]s declined quickly over about 1 s, and then as the sum of two exponentials: that of the moderate phase and a slower component with a time constant of 109 +/- 16 s (slow phase). Similar time courses were observed following stimuli to the preganglionic nerve. Caffeine (3 mM) and ryanodine (20 microM) both sped the fast phase and slowed the moderate phase of [Ca2+]s decline. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 2 microM) slowed the slow phase, without affecting the other phases of decline. These results are discussed in relation to identifying the mechanisms responsible for these different phases of Ca2+ removal.

Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition.

1. The sympathetic nerve terminals of the mouse vas deferens were loaded with the calcium indicator Oregon Green 488 BAPTA-1 by orthograde transport along the postganglionic nerves. Changes in the calcium concentration in the varicosity (delta [Ca2+]v) were determined following single impulses, and short (5-impulse) and long (200-impulse) trains at 5 Hz. 2. All varicosities showed a significant delta [Ca2+]v in response to every single impulse. The elevated delta [Ca2+]v declined in two phases with similar kinetics for all varicosities: a fast phase (time constant, 0.42 +/- 0.05 s) and a moderate phase (3.6 +/- 0.4 s). 3. Line scanning confocal microscopy revealed that the delta [Ca2+] of a single terminal following single impulses was smaller for the intervaricose regions than for the varicosities. 4. Blockade of the voltage-sensitive calcium channels with Cd2+ (in calcium-free solution) completely blocked the delta [Ca2+]v on stimulation. The addition of either nifedipine (10 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin TK (100 nM) showed that 47 +/- 6% of the evoked response was mediated by N-type calcium channels. 5. Ryanodine (10 microM) did not significantly change the amplitude of delta [Ca2+]v in response to short trains. 6. Spontaneous increases in delta [Ca2+]v were observed in individual varicosities, with coupling in the increase of delta [Ca2+]v between varicosities. 7. The presynaptic alpha 2-receptor antagonist yohimbine (10 microM) increased the amplitude of delta [Ca2+]v in response to five impulses (5 Hz) by 54 +/- 14%, while the alpha 2-receptor agonist clonidine (1 microM) decreased the delta [Ca2+]v by 55 +/- 4%. 8. These results are discussed in terms of the hypotheses that the increased probability for secretion at sympathetic nerve terminals which accompanies facilitation and augmentation is due to the residual delta [Ca2+]v remaining after the calcium influx following impulses and that noradrenaline acts presynaptically to decrease the probability of secretion by modifying calcium influx.

Varicosities of single sympathetic nerve terminals possess syntaxin zones and different synaptotagmin N-terminus labelling following stimulation.

A study has been made of the probability of exocytosis of synaptic vesicles at different varicosities in single sympathetic terminal axons in the mouse was deferens. An antibody (SV2Ab) against SV2. a proteoglycan in synaptic vesicles, labelled an area of individual sympathetic varicosities that was slightly less than that occupied by dextran-rhodamine, previously orthogradely transported into the varicosities. In contrast plasma membrane bound protein syntaxin, found at active zones of motor nerve terminals, occupied an area of the varicosity that was approximately one-third that of SV2. This suggests that sympathetic varicosities possess specialized zones for exocytosis on their plasma membranes. Antibodies against the N-terminal sequence of synaptotagmin 1 (SNAb), a sequence exposed within synaptic vesicles, were used to determine the probability of exocytosis at different varicosities of single terminal branches. The area of SNAb labelling was not significantly different from that of the SV2 labelling, which implies vesicles that have undergone exocytosis may eventually return to the main pool of vesicles. Varicosities belonging to the same terminal axon, and identified with SV2Ab, showed different extents of labelling with SNAb when secretion was evoked with high potassium concentrations (80 mM) for 30 min in the presence of SNAb. There was up to an order of magnitude difference in the average intensity of SNAb labelling between different varicosities of the same terminal axon whereas there was little difference in the average intensity of SV2Ab labelling. These observations suggest that there is considerable variability in the probability of exocytosis at the specialized zones in different varicosities.

Vesicle-associated proteins and calcium in nerve terminals of chick ciliary ganglia during development of facilitation.

1. The developmental appearance of synaptic vesicle-associated proteins and nerve terminal calcium ([Ca2+]i) sequestering processes were determined for the chick ciliary ganglia in relation to the maturation of the different phase of increased efficacy of transmitter release following nerve impulses. The maturation phases studied were from stages 34-35, at the time of synapse formation, to stage 46 at hatching. 2. Western blots and immunohistochemical localization indicated that synaptotagmin 1 and synapsin IIa were detectable at stages 34-35 and were clearly localized at the nerve terminals by stage 37. Syntaxin was clearly localized at the nerve terminals at stage 34. 3. The relative size of the postganglionic compound action potential, used to measure the transmission efficacy through the ganglion, showed that the slope of the relationship between log efficacy and log extracellular calcium concentration ([Ca2+]o) in low [Ca2+]o was about 4 by stage 46. 4. A mature facilitatory mechanism for transmission was not present at stage 34 and did not emerge until stage 38. A mature augmentation was not present at stages 34 or 38 and was not established until stages 41-42. Post-tetanic potentiation (PTP) was not present at stage 34; it was evident at stages 37-38 and only reached maturity by stages 41-42. 5. The time course of calcium changes in the nerve terminals following trains of impulses that give rise to facilitation, augmentation and PTP was determined for different stages of development using the indicator Calcium Green-1 in the nerve terminal. The mature time course of the phases of calcium decline in the nerve terminal associated with facilitation and augmentation was observed as early as stage 38, whereas that of the PTP phase did not mature until after stage 42. 6. These results are discussed in terms of the maturation of the vesicle-associated proteins and calcium influx into the terminal following trains of impulses that give rise to the different components of increased synaptic efficacy.