Antioxidant treatment restores prejunctional regulation of purinergic transmission in mesenteric arteries of deoxycorticosterone acetate-salt hypertensive rats.

Norepinephrine (NE) and ATP are co-released by periarterial sympathetic nerves. In mesenteric arteries (MA) from deoxycorticosterone-acetate (DOCA)-salt hypertensive rats, ATP, but not norepinephrine, release is impaired suggesting that their release may be regulated differently. We tested the hypothesis that different calcium channels contribute to ATP and norepinephrine release from sympathetic nerves in vitro in MA from normotensive and DOCA-salt hypertensive rats and that oxidative stress disrupts prejunctional regulation of co-transmission. Excitatory junction potentials (EJPs) were used to measure ATP release. Norepinephrine release was measured amperometrically with carbon-fiber microelectrodes. CdCl2 (30 microM) inhibited norepinephrine release in sham and DOCA-salt arteries by 78% and 85%, respectively. The N-type calcium channel antagonist, omega-conotoxin GVIA (CTX, 0.1 microM) inhibited norepinephrine release by 50% and 67% in normotensive and DOCA-salt arteries, respectively while CTX blocked EJPs. The P/Q-type calcium channel antagonist omega-agatoxin IVA (ATX; 0.03 microM) reduced norepinephrine release in sham but not DOCA-salt arteries and increased EJPs in sham but not DOCA-salt arteries. ATX did not increase EJPs in sham arteries in the presence of the alpha(2)-adrenergic receptor antagonist, yohimbine (1 microM). alpha(2)-Autoreceptor-sensitive EJP facilitation is impaired in DOCA-salt hypertension but this response is restored in DOCA-salt rats treated chronically with the antioxidant, apocynin. Apocynin restored alpha(2)-autoreceptor regulation of norepinephrine release. We conclude that ATP released from periarterial sympathetic nerves is controlled directly by N-type calcium channels. Norepinephrine release is controlled by N and P/Q type calcium channels. Norepinephrine release controlled by P/Q channels acts at alpha(2)-adrenergic receptors to inhibit norepinephrine release suggesting that there may be multiple pools of norepinephrine in periarterial sympathetic nerves. Regulation of norepinephrine release by alpha(2)-autoreceptors and P/Q-type channels is impaired in DOCA-salt hypertension and alpha(2)-autoreceptor function is disrupted by oxidative stress.

N-hydroxyl derivatives of guanidine based drugs as enzymatic NO donors.

Recent research suggests that NO may play a role in the physiological effects of some guanidine-containing drugs. In this report, three guanidine-containing drugs (guanadrel, guanoxan, and guanethidine) together with their N-hydroxyl derivatives were synthesized and their NO-releasing abilities catalyzed by nitric oxide synthases (NOSs) and horseradish peroxidase were evaluated. The guanidine containing compounds could not release NO in the presence of NOS or peroxidase. The corresponding N-hydroxyl compounds exhibited weak NO-releasing ability under the catalyzed of NOS and good NO-releasing ability under the oxidation by horseradish peroxidase in the presence of H(2)O(2). These compounds also displayed vasodilatory activity.

Increased blood pressure responses in neuropeptide Y transgenic rats.

Considering the coexistence of neuropeptide Y (NPY) and norepinephrine in perivascular sympathetic nerves and the known vasoconstrictor cooperation of NPY with norepinephrine, we investigated the involvement of NPY in long-term control of cardiovascular functions using NPY transgenic (NPY-tg) rats. These rats were developed by injection of the rat (Sprague-Dawley) pronuclei with a 14.5-kb clone of the rat structural NPY gene. When compared with nontransgenic littermates, NPY concentrations were significantly increased in a number of cardiovascular tissues of NPY-tg hemizygotes. Direct basal mean arterial pressure and heart rate were not changed, but calculated total vascular resistance was significantly increased in NPY-tg subjects. Arterial pressure increases, in response to norepinephrine injection, were greater in the NPY-tg rats. Also, the hypotension and bradycardia in response to hemorrhage were significantly reduced in NPY-tg subjects. These results indicate that NPY, when expressed in increased amounts, potentiates the pressor effects of norepinephrine and contributes to maintaining blood pressure during hemorrhage, but it does not alter resting blood pressure. These transgenic rats will facilitate studies of the role of NPY signaling in cardiovascular regulation, particularly regarding its functional cooperation with norepinephrine.

Guanethidine evokes vasodilatation in guinea pig mesenteric artery by acting on sensory nerves.

In precontracted, endothelium-free guinea pig mesenteric artery rings, in which adrenergic vasoconstrictor responses had been eliminated, guanethidine (1-30 microM) produced a vasodilatation of 69.3+/-4.4%. The vasodilatation was reduced 89% by capsaicin (10 microM) or 55% by tetrodotoxin (10 microM), indicating mediation of this effect by primary sensory nerves. The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (100 microM, 30 min) but not its stereoisomer reduced the guanethidine vasodilatation by 70%. Blockade of monoamine uptake with ouabain (25 microM, 15 min) or cocaine (5 microM, 5 min) reduced the guanethidine-induced vasodilatation by 85 and 67%, respectively. These results suggest that guanethidine produced vasodilatation by being transported into capsaicin-sensitive primary sensory nerves where it functioned as a substrate for nitric oxide synthase to generate a vasodilatory substance.

Patterns of innervation of sympathetic vascular neurons by peptide-containing primary sensory fibers.

The purpose of this study was to determine whether there is a specific organization of the primary sensory innervation on to identified vascular neurons in the inferior mesenteric ganglion (IMG) in guinea-pig. Retrograde tracers were placed intraluminally in inferior mesenteric artery (IMA) or inferior mesenteric vein (IMV) in vitro to identify ganglionic neurons as arterial, venous or unlabeled neurons. The distribution of primary sensory nerve fibers containing calcitonin gene-related peptide (CGRP), neuronal nitric oxide synthase (NOS) and substance P immunoreactivity (SP-IR) was compared before and after treatment with capsaicin. In control animals the density of immunoreactivity varied both with the transmitter and the type of neuron innervated. The density of immunoreactivity for all the three substances was reduced by capsaicin treatment. The degree of reduction of immunoreactivity in the fibers varied with the transmitter and the type of neuron. The density of CGRP and SP immunoreactive fibers was greatest around unlabeled neurons; 78% of the CGRP fibers were of primary sensory origin and all of the SP fibers were primary sensory. Around arterial neurons 44% of the CGRP fibers were of primary sensory origin and around venous 68% were primary sensory. NOS positive innervation around venous neurons was denser than around arterial neurons and all of it was completely (97%) eliminated by capsaicin, indicating that it was solely of primary sensory origin. We conclude that the primary sensory fibers innervating the IMG are differentially distributed to arterial and venous neurons and that the pattern of distribution is characteristic for each sensory neurotransmitter.

Two populations of sympathetic neurons project selectively to mesenteric artery or vein.

The objective of this study was to determine whether sympathetic neurons of the inferior mesenteric ganglion (IMG) projecting to mesenteric arteries could be distinguished by their localization, neurochemical phenotype, and electrophysiological properties from neurons projecting to mesenteric veins. In an in vitro intact vasculature-IMG preparation, neurons were labeled following intraluminal injection of Fluoro-Gold or rhodamine beads into the inferior mesenteric artery (IMA) or vein (IMV). The somata of neurons projecting to IMA were localized in the central part of the IMG, whereas those projecting to IMV were localized more peripherally. None of the labeled neurons was doubly labeled. Neuropeptide Y immunoreactivity was found in 18.9% of neurons innervating the IMA, but not in neurons innervating the IMV. Identified neurons were dissociated and characterized using whole cell patch-clamp recording. After direct soma depolarization, all of the labeled arterial and venous neurons were classified as tonic firing, compared with only 40% of unlabeled neurons; the remaining 60% of unlabeled neurons were phasic firing. The results indicate that IMG neurons projecting to mesenteric arteries are distinct from neurons projecting to mesenteric veins.

Effects of nitric oxide in cultured prevertebral sympathetic ganglion neurons.

The effects of the nitric oxide donor, S-nitrosoacetylpenicillamine (SNAP), were tested on cultured dissociated guinea pig celiac ganglion neurons using whole cell patch-clamp recordings. S-nitrosoacetylpenicillamine induced a concentration- and voltage-dependent inwardly directed shift in holding current (inward current shift) in 89% of neurons. The inward current shift was prevented by pre-treatment with the nitric oxide scavenger reduced hemoglobin and was abolished by intra- or extracellular cesium. The amplitude of the inward current shift was also sensitive to the extracellular potassium concentration. The S-nitrosoacetylpenicillamine-induced inward current shift was mediated by a decrease in calcium-dependent potassium currents (IAHPs); apamin (100 nM), charybdotoxin (10 nM) or tetraethylammonium (5 mM) reduced but did not abolish the amplitude of its inward current shift and a combination of apamin and tetraethylammonium abolished the S-nitrosoacetylpenicillamine-induced inward current response. In the presence of extracellular cobalt, SNAP produced an outward current that was concentration- and voltage-dependent, abolished by reduced hemoglobin and extracellular cesium and reduced by 4-AP (1 mM); in the absence of cobalt, 4-AP increased the SNAP-induced inward current shift. These data indicate that NO exerts dual opposing effects on neuronal potassium conductances, namely an inward current shift mediated through an inhibition of IAHP and induction of an outward current mediated by activation of the potassium delayed rectifier.

Nitric oxide is a sensory nerve neurotransmitter in the mesenteric artery of guinea pig.

Previous studies have shown that the guinea pig inferior mesenteric artery receives spinal sensory vasodilatory innervation, which can be activated by colon distention and electrical nerve stimulation. In the present study, we investigated the hypotheses that nitric oxide synthase (NOS) is present in guinea pig primary sensory neurons in the dorsal root ganglion (DRG) and in nerve fibers surrounding the mesenteric arteries, and that nitric oxide (NO) is a sensory neurotransmitter in the inferior mesenteric artery in vitro. Double-labeling immunohistochemistry showed that neuronal NOS-IR was found in 12% of cells of guinea pig thoracic and lumbar DRGs; in 95.1% of these cells it was colocalized with substance P (SP), and SP immunoreactivity (SP-IR) was present in 23% of cells of the same DRGs. Neuronal NOS-like immunoreactivity was localized in nerve fibers surrounding guinea pig mesenteric artery and 25% of them were double stained with SP-IR. Endothelium-denuded inferior mesenteric artery preparations in vitro were incubated with guanethidine (30 microns, 30 min) and pre-contracted with methoxamine (30 microns). The NO donors, sodium nitroprusside (1 micron) and L-nitrosocysteine (300 microns), produced 91.0 +/- 5.5 and 90.4 +/- 9.6% vasodilatation of total vasodilatation in the vessel segments, respectively, which was capsaicin- or tetrodotoxin-insensitive. Repetitive electrical field stimulation of the preparations produced a frequency-dependent vasodilatation which was reduced by pretreatment with capsaicin or by tetrodotoxin (10 microns). The NOS inhibitor N omega-nitro-L-arginine (L-NNA) (100 microns, 30 min) diminished the nerve-evoked vasodilatation from 41.8 +/- 8.4 to 21.4 +/- 9.7% at 2 Hz and from 50.8 +/- 5.6 to 19.0 +/- 7.3% at 15 Hz (P < 0.05), whereas NG-nitro-L-arginine methyl ester (L-NAME, 100 microns-1 mM) did not significantly inhibit the relaxation. The stereo isomer nitro-D-arginine (D-NNA, 100 microns, 30 min) was ineffective. These findings suggest that NO is a neurotransmitter released from primary sensory nerves which mediates vasodilation in vitro.

Roles of peptides and other substances in cotransmission from vascular autonomic and sensory neurons.

Blood vessels may be innervated by up to three major classes of neurons: sympathetic vasoconstrictor neurons; sympathetic or parasympathetic vasodilator neurons; and peripheral fibres of small diameter sensory neurons, which can mediate vasodilation. Most vascular neurons utilise multiple transmitters, including neuropeptides and small nonpeptides such as ATP or nitric oxide, often in addition to noradrenaline or acetylcholine. Subpopulations of each major class of vascular neurons innervating different vascular segments may contain different combinations of neurotransmitters. Furthermore, the same population of neurons can release different cotransmitters in response to different patterns of stimulation. In general, peptides mediate slower and more long lasting changes in vascular resistance than do nonpeptides. Thus, autonomic and sensory neurons are well adapted to produce qualitatively different vascular effects in response to different types of afferent input. The major challenge for the future is to develop new antagonists for many of the substances colocalised in vascular neurons, particularly neuropeptides. These agents will allow us to precisely determine the relative roles of multiple cotransmitters, and are likely to provide therapeutic agents that can be targeted to specific regions of the vasculature.

The muscarinic receptor agonist oxotremorine methiodide evokes a nicotinic response in mammalian sympathetic neurons.

Oxotremorine methiodide, a congener of oxotremorine, is used as a muscarinic receptor agonist. Responses to oxotremorine methiodide and nicotinic receptor agonists were examined in cultured guinea-pig celiac ganglion neurons using whole-cell voltage clamp techniques. At holding potentials between -30 and -60 mV, a brief application of oxotremorine methiodide produced fast and slow inward current transients, depending upon the concentration applied. Slowly developing inward current transients, characteristic of muscarinic responses, were produced by lower concentrations (EC50: 0.3 microM) and were blocked by atropine. Rapid inward current transients, characteristic of nicotinic responses, were produced by higher concentrations of oxotremorine methiodide (EC50: 168 microM) and were blocked by d-tubocurarine. Thus oxotremorine methiodide, at concentrations of 10 microM and greater, produced an initial nicotinic fast inward current transient followed by a slow muscarinic inward transient. The fast inward transients were similar to responses evoked by the nicotinic receptor agonists acetylcholine, nicotine and 1,1-dimethyl-4-phenyl-piperazinium iodide and were not antagonized by atropine. We conclude that oxotremorine methiodide acts as a nicotinic and muscarinic receptor agonist in celiac sympathetic ganglion neurons.

Muscarinic inhibition of two potassium currents in guinea-pig prevertebral neurons: differentiation by extracellular cesium.

Muscarinic responses were studied in dissociated guinea-pig celiac ganglion neurons using the whole-cell voltage-clamp technique. Muscarine (0.025-1 mM; EC50 = 95 microM) administered to cells for 1.5 s evoked inward shifts in holding current in 53 of 74 cells. The amplitude of the inward current transients decreased with hyperpolarization and the null potential averaged -71 +/- 3.4 mV (n = 11). The currents that underlie the responses to muscarine were examined with hyperpolarizing voltage stepping protocols to -100 mV from a holding potential of -30 mV. Eighty-one per cent of cells displayed voltage-dependent current relaxations characteristic of the M-potassium current. Twenty per cent of responding cells displayed no M-current but only a voltage-independent current consistent with a leak current. In the latter type of cells, the muscarine-evoked inward currents reversed near EK and became outward at more hyperpolarized potentials. Analysis of steady state I-V relationships before and after bath application of muscarine showed that the two muscarine-sensitive potassium currents were distributed differently among three types of cells: (i) with M-current (18%); (ii) with leak current (18%); and (iii) with M-current and with leak current (64%). Cesium and barium were used to differentiate the M-current and the muscarine-sensitive leak current. Barium (2 mM) reduced the M-current and the leak potassium current, whereas cesium (2 mM) reduced the M-current but did not affect leak current. Thus, barium reduced the amplitude of muscarinic responses by 79% but cesium reduced them by only 14%. We conclude that muscarinic responses in guinea-pig celiac neurons are produced by suppression of two K+ currents: the M-current and a muscarine-sensitive leak current. These two currents are differentially susceptible to the potassium channel blockers barium and cesium.

A physiologically-evoked M1-muscarinic depolarization in guinea-pig inferior mesenteric ganglion neurons.

The possibility of physiologically-evoked muscarinic excitatory synaptic potentials was examined in the inferior mesenteric ganglion (IMG) with intracellular microelectrodes in vitro. Three types of depolarizing responses were evoked concurrently by colonic distension: (1) fast nicotinic excitatory postsynaptic potentials (EPSPs); (2) an 'intermediate' time course depolarization and (3) a long time course potential that persisted throughout the period of distension. After hexamethonium was superfused over the ganglia the long time course potential was observed in 91% of IMG neurons. Intermediate time course of depolarizations were observed in 100% of IMG neurons and correlated with each propulsive contraction of the distal colon. The intermediate depolarizations had an average amplitude of 1.8 +/- 0.1 mV (n = 175 individual events; 27 preparations) with an average duration of 11.9 +/- 0.8 sec (n = 28 individual events). The intermediate time course synaptic potentials were accompanied by an increase in input resistance of 15% (n = 6). Superfusion of atropine (1 microM; n = 6) on the IMG or the M1-selective antagonist pirenzepine (1 microM; n = 5) abolished the intermediate time course synaptic potentials during distension. Superfusion of the M2-selective antagonist AF-DX 116 (1 microM; n = 4) had no effect. In all preparations examined, distension-induced intermediate time course depolarizations were blocked by tetrodotoxin (TTX) (3 microM). Pressure ejection of carbachol (1 mM, 60-100 ms pulses) evoked depolarizations of similar amplitude (6.5 +/- 0.7 mV; n = 18) and duration to the intermediate depolarizations observed during propulsive contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotransmitter properties of guinea-pig sympathetic neurons grown in dissociated cell culture--I. Adult neurons.

The autonomic nervous system of mammals displays extensive neurotransmitter diversity. The guinea-pig sympathetic nervous system has served as a model for in vivo studies of neurotransmitter co-expression. We have developed methods for the dissociation and long-term culture of adult guinea-pig prevertebral sympathetic ganglia. The neurotransmitter properties of cultured adult guinea-pig sympathetic neurons from the celiac and superior mesenteric ganglia were examined. Cultured principal neurons were found to display many of their in vivo neurotransmitter characteristics, including catecholamine-specific histofluorescence and immunoreactivity for tyrosine hydroxylase and the neuropeptides, neuropeptide Y, somatostatin and vasoactive intestinal polypeptide. In addition, the cultures of both ganglia displayed the various neurotransmitter characteristics in approximately the same percentage of the cultured neurons as reported in in vivo studies. A small percentage of principal neurons and many small, intensely fluorescent-like cells labeled with antibodies against 5-hydroxytryptamine. Many of the principal neurons were found to bear 5-hydroxytryptamine3 receptors, suggesting a possible role for this neurotransmitter in neuron-neuron and small, intensely fluorescent cell-neuron transmission. We conclude that adult guinea-pig sympathetic neurons retain their neurotransmitter phenotypes when grown in dissociated cell culture. These properties include the co-expression of the classical transmitters, norepinephrine, 5-hydroxytryptamine and neuropeptides. This culture preparation will prove to be valuable in future studies on the functional properties of these neurons and their development.

Neurotransmitter properties of guinea-pig sympathetic neurons grown in dissociated cell culture--II. Fetal and embryonic neurons: regulation of neuropeptide Y expression.

We report here the neurotransmitter characteristics of neurons cultured from the same ganglia of fetal and embryonic guinea-pigs. Both the celiac ganglion and the superior mesenteric ganglion were examined. In a previous paper we described the neurotransmitter properties of adult guinea-pig prevertebral sympathetic neurons grown in dissociated cell culture, including the expression by these cells of immunoreactivity for tyrosine hydroxylase, neuropeptide Y and somatostatin. Tyrosine hydroxylase immunoreactivity was ubiquitously expressed in all fetal embryonic cultures, as was the case for adult neurons. Fetal-derived celiac and superior mesenteric gangli neurons displayed neuropeptide Y and somatostatin immunoreactivity in the same percentage of neurons as in adult cultures but at markedly lower levels. Embryonic neurons also expressed somatostatin immunoreactivity in roughly the same proportion of neurons as in adult and fetal cultures; however, the expression of neuropeptide Y immunoreactivity in both celiac and superior mesenteric gangli cultures was significantly different. Specifically, neuropeptide Y immunoreactivity in embryonic celiac cultures was greatly reduced in both the number of positive-labeled neurons and the amount of immunoreactive product, while neuropeptide Y immunoreactivity in embryonic superior mesenteric gangli cultures was markedly increased compared to their adult and fetal counterparts. The expression of neuropeptide Y immunoreactivity in celiac neurons was found to be specifically elevated by culturing the neurons in medium conditioned by disassociated vascular cells, this treatment having no effect on tyrosine hydroxylase or somatostatin immunoreactivity. Heart cell-conditioned medium did not effect neuropeptide Y or somatostatin immunoreactivity, although it did result in a significant reduction of tyrosine hydroxylase immunoreactivity and an increase in 5-hydroxytryptamine immunoreactivity. We conclude that the expression of neuropeptide Y immunoreactivity develops independently in cultures of adult and near-term fetuses but that embryonic neurons require interactions with target cells to express this phenotype. Neuropeptide Y immunoreactivity can be induced in embryonic sympathetic neurons by a target-derived factor(s).

CCKA receptors mediate slow depolarizations in cultured mammalian sympathetic neurons.

The effect of cholecystokinin octapeptide (CCK-8) was examined in guinea-pig celiac ganglion (CG) neurons in primary culture using standard intracellular recording techniques. Sulfated CCK-8 (CCK-8S; 1 microM) evoked slow depolarizing responses in 94% of CG neurons tested. In contrast, membrane potential was not affected by nonsulfated CCK-8 (CCK-8NS; 1 microM), CCK tetrapeptide (CCK-4; 1 microM), or gastrin (1 microM). The selective CCKA receptor antagonist L 364,718 potently inhibited CCK-8S-induced slow depolarizations (IC50 2.9 pM). In contrast, the selective CCKB receptor antagonist L 365,260 was a weak inhibitor of CCK-8S-induced slow depolarizations (IC50 1.3 microM). The depolarizing responses to CCK-8S were associated with an average increase in cell input resistance of 61%. Single electrode voltage clamp experiments indicated that CCK-8S-induced depolarizations were associated with a slow inward shift in holding current. Thus, the present findings indicate that guinea-pig cultured CG neurons are endowed with excitatory CCKA receptors the activation of which elicits a decrease in membrane conductance, thereby resulting in slow depolarizations.

A capsaicin-sensitive inhibitory reflex from the colon to mesenteric arteries in the guinea-pig.

1. The present in vitro study examined the effect of distension of the distal colon on membrane potential in the inferior mesenteric artery of the guinea-pig. 2. Distension of the distal colon up to an intraluminal pressure of 25 cmH2O induced a hyperpolarization in the inferior mesenteric artery. The average amplitude of hyperpolarizations induced by 2 min distensions of the colon was 3 mV and their average duration was 268 s. 3. Distension-induced hyperpolarizations (DIHs) were abolished in the presence of tetrodotoxin or a low-Ca2+ (0.5 mM) superfusion solution. 4. Superfusion of capsaicin (10 microM) induced slow hyperpolarizing responses in mesenteric arteries. Following application of capsaicin (10 microM), DIHs were abolished. 5. These findings provide strong evidence that mesenteric arteries receive an inhibitory, capsaicin-sensitive sensory innervation from the distal colon which is activated during periods of colon distension to induce hyperpolarization of the arterial smooth muscle. This extramural inhibitory reflex pathway may play a physiological role in co-ordinating mesenteric blood flow with changes in gut motility.

Response to 5-hydroxytryptamine on neurons of guinea pig celiac and inferior mesenteric ganglia in primary culture.

The electrophysiological effects of serotonin, a putative neurotransmitter in prevertebral sympathetic ganglia, were evaluated in cultured celiac and inferior mesenteric ganglia (IMG) neurons. Intracellular microelectrode recordings were performed in neurons that were maintained in culture an average of 26 days. Seventy-eight of 85 neurons responded when serotonin (10 microM) was applied by pressure ejection from a micropipette to the surface of the isolated cells. The majority of the neurons (n = 48) generated fast depolarizations, although slow depolarizations (n = 17), bipolar responses (n = 5), hyperpolarizations (n = 7), and a biphasic response (n = 1), were also seen. Hyperpolarizing responses were evoked in celiac neurons only. All responses were inhibited by the 5-HT3 antagonist MDL 72,222 (5 microM). Fast responses were not inhibited by tetrodotoxin (n = 3). These results demonstrate that serotonin evokes a variety of membrane potential changes in cultured prevertebral sympathetic neurons by activating 5-HT3 receptors.

Capsaicin-sensitive nerves mediate inhibitory junction potentials and dilatation in guinea-pig mesenteric artery.

1. The present study examined the effects of repetitive nerve stimulation on membrane potential and on contractile responses to noradrenaline in the guinea-pig inferior mesenteric artery and its distal branches. 2. Repetitive stimulation of perivascular nerves evoked slow inhibitory junction potentials (IJPs) and dilator responses. Individual nerve shocks elicited excitatory junction potentials (EJP)s. 3. Stimulation-evoked IJPs were abolished in the presence of tetrodotoxin (0.3 microM) or a low-Ca2+ (0.5 mM) superfusion solution. 4. The amplitudes and durations of IJPs were dependent on the frequency and duration of repetitive nerve stimulation. Nerve stimulation delivered at 5 Hz for 5 s induced IJPs which had an average amplitude of 2 mV and an average duration of 130 s. When the time interval between successive stimulation periods was less than 4 min, the amplitudes of IJPs were reduced in a time-dependent manner. 5. Stimulation-evoked IJPs were unaffected following endothelium removal. Furthermore, stimulation-evoked IJPs were not affected by atropine (1 microM), indomethacin (20 microM), prazosin (0.5 microM), phentolamine (10 microM), propranolol (0.5 microM) or alpha,beta-methylene ATP (0.2 microM). 6. Pre-treatment of arteries with guanethidine (30 microM) or 6-hydroxydopamine (0.4 mM) abolished stimulation-evoked EJPs but had no effect on stimulation-evoked IJPs. 7. In a similar manner to repetitive nerve stimulation, capsaicin (10 microM) itself induced membrane hyperpolarization and dilatation in mesenteric arteries. Moreover, following application of capsaicin (10 microM), stimulation-evoked IJPs and dilator responses were abolished. 8. EJPs evoked during stimulation-induced IJPs were reduced in amplitude, compared to EJPs evoked under resting conditions. 9. These findings suggest that, in addition to an excitatory sympathetic innervation, mesenteric arteries receive an inhibitory, capsaicin-sensitive innervation which is activated by low-frequency repetitive stimulation.

Comparison of neurotransmission with nerve trunk and transmural field stimulation in guinea-pig mesenteric artery.

1. Intracellular electrical and contractile responses to sympathetic nerve trunk stimulation (NTS) and transmural electrical field stimulation (TMS) were compared in the guinea-pig mesenteric artery in vitro. 2. Step increases in voltage with NTS gave rise to excitatory junctional potentials (EJPs) which reached a plateau amplitude of 5-10 mV, whereas with TMS larger amplitude EJPs and sometimes action potentials were obtained. 3. EJPs of equal amplitude (1-7 mV) elicited with TMS and NTS had the same rise time, duration and decay half-time. 4. Slow depolarization obtained with repetitive stimulation was significantly greater in amplitude with TMS than with NTS. 5. Equal amplitude EJPs were obtained throughout the preparation with NTS. With TMS, the amplitude of responses declined substantially with distance from the stimulating electrodes. 6. Tetrodotoxin (TTX) completely blocked EJPs, slow depolarization and contraction with NTS; however, with TMS a TTX-resistant component was observed. The TTX-resistant response to TMS was abolished in the presence of a low-Ca2+ superfusion solution but was not affected by endothelium removal. 7. Phentolamine or prazosin abolished slow depolarization but not EJPs with NTS or TMS. Prazosin abolished contraction with NTS and reduced but did not abolish contraction with TMS. 8. alpha, beta-Methylene ATP abolished EJPs with NTS, whereas with TMS only EJPs obtained with low stimulus intensities were abolished. alpha, beta-Methylene ATP did not block contraction with either NTS or TMS. 9. Combined TTX, prazosin and alpha, beta-methylene ATP abolished EJPs initiated with TMS at all but the highest stimulus intensities (12-20 V, 0.3 ms duration). 10. It is concluded that responses obtained with NTS can be reliably attributed to the release of transmitter by the conduction of action potentials in paravascular nerves, whereas activation by TMS is a more complex phenomenon dependent upon stimulus strength and probably involving multiple forms of activation.