Effects Of Sympathetic Nerve Stimulation Research Articles

Stimulation of extrinsic sympathetic nerves differentially affects neurogenic motor activity in guinea pig distal colon.

The speed of pellet propulsion through the isolated guinea pig distal colon in vitro significantly exceeds in vivo measurements, suggesting a role for inhibitory mechanisms from sources outside the gut. The aim of this study was to investigate the effects of sympathetic nerve stimulation on three different neurogenic motor behaviors of the distal colon: transient neural events (TNEs), colonic motor complexes (CMCs), and pellet propulsion. To do this, segments of guinea pig distal colon with intact connections to the inferior mesenteric ganglion (IMG) were set up in organ baths allowing for simultaneous extracellular suction electrode recordings from smooth muscle, video recordings for diameter mapping, and intraluminal manometry. Electrical stimulation (1-20 Hz) of colonic nerves surrounding the inferior mesenteric artery caused a statistically significant, frequency-dependent inhibition of TNEs, as well as single pellet propulsion, from frequencies of 5Hz and greater. Significant inhibition of CMCs required stimulation frequencies of 10Hz and greater. Phentolamine (3.6μM) abolished effects of colonic nerve stimulation, consistent with a sympathetic noradrenergic mechanism. Sympathetic inhibition was constrained to regions with intact extrinsic nerve pathways, allowing normal motor behaviors to continue without modulation in adjacent extrinsically denervated regions of the same colonic segments. The results demonstrate differential sensitivities to sympathetic input among distinct neurogenic motor behaviors of the colon. Together with findings indicating CMCs activate colo-colonic sympathetic reflexes through the IMG, these results raise the possibility that CMCs may paradoxically facilitate suppression of pellet movement in vivo, through peripheral sympathetic reflex circuits.

B-AB01-04 INTERACTION BETWEEN ISCHEMIA AND SYMPATHETIC ACTIVITY ON CARDIAC ELECTROPHYSIOLOGY AND ARRHYTHMIA IN THE INNERVATED LANGENDORFF-PERFUSED MOUSE HEART

Sympathetic activation of the heart plays a key role in the modulation of cardiac electrophysiology and arrhythmogenesis. It is generally believed that sympathetic activation is proarrhythmic in the setting of cardiac ischemia. However, few studies have looked at the dynamic effects of physiological sympathetic nerve activation on cardiac electrophysiological activities during ischemia. To investigate the effects of sympathetic activation in the innervated Langendorff-perfused mouse heart subjected to acute ischemia. The heart and posterior thoracic cavity from mice (C57Bl6, N=4) were dissected and perfused through the descending aorta for dual optical mapping of transmembrane potential (Vm) and intracellular Ca2+ to study the effects of sympathetic nerve stimulation (SNS). SNS, achieved through spinal cord stimulation at T1-T3, was performed before, during and immediately after 5 min of low-flow ischemia (10% of pre-ischemic flow rate). Early afterdepolarizations (EADs) and ventricular tachycardia (VT) occurred in all 4 animals within 5 min of ischemia and immediately upon reperfusion. Both SNS and ventricular pacing, during either ischemia or reperfusion, completely abolished EADs and VT induced by global low-flow ischemia and reperfusion (Fig). In the isolated innervated mouse heart, we showed that sympathetic stimulation protects against ischemia-induced cardiac arrhythmias potentially by over-driving ventricular ectopic activity.

Sympathetic Nervous Regulation of Calcium and Action Potential Alternans in the Intact Heart.

Rationale: Arrhythmogenic cardiac alternans are thought to be an important determinant for the initiation of ventricular fibrillation. There is limited information on the effects of sympathetic nerve stimulation (SNS) on alternans in the intact heart and the conclusions of existing studies, focused on investigating electrical alternans, are conflicted. Meanwhile, several lines of evidence implicate instabilities in Ca handling, not electrical restitution, as the primary mechanism underpinning alternans. Despite this, there have been no studies on Ca alternans and SNS in the intact heart. The present study sought to address this, by application of voltage and Ca optical mapping for the simultaneous study of APD and Ca alternans in the intact guinea pig heart during direct SNS.Objective: To determine the effects of SNS on APD and Ca alternans in the intact guinea pig heart and to examine the mechanism(s) by which the effects of SNS are mediated.Methods and Results: Studies utilized simultaneous voltage and Ca optical mapping in isolated guinea pig hearts with intact innervation. Alternans were induced using a rapid dynamic pacing protocol. SNS was associated with rate-independent shortening of action potential duration (APD) and the suppression of APD and Ca alternans, as indicated by a shift in the alternans threshold to faster pacing rates. Qualitatively similar results were observed with exogenous noradrenaline perfusion. In contrast with previous reports, both SNS and noradrenaline acted to flatten the slope of the electrical restitution curve. Pharmacological block of the slow delayed rectifying potassium current (IKs), sufficient to abolish IKs-mediated APD-adaptation, partially reversed the effects of SNS on pacing-induced alternans. Treatment with cyclopiazonic acid, an inhibitor of the sarco(endo)plasmic reticulum ATPase, had opposite effects to that of SNS, acting to increase susceptibility to alternans, and suggesting that accelerated Ca reuptake into the sarcoplasmic reticulum is a major mechanism by which SNS suppresses alternans in the guinea pig heart.Conclusions: SNS suppresses calcium and action potential alternans in the intact guinea pig heart by an action mediated through accelerated Ca handling and via increased IKs.

219 Stimulation of Sympathetic Nerves within Perivascular Adipose Tissue Causes the Release of Vasoactive Relaxing Factors

<h3>Introduction</h3> There is considerable evidence to implicate over-activity of the sympathetic nervous system during obesity and the subsequent development of metabolic syndrome and type 2 diabetes. It has been suggested that these conditions are dependent upon changes in small artery function. Healthy perivascular fat exerts an anti-contractile effect on circulation which is lost in obesity. Therefore it was decided to examine the effects of sympathetic nerve stimulation on perivascular adipose tissue (PVAT) function. The hypothesis to be tested was that an anti-contractile factor is released from PVAT as a result of sympathetic nerve stimulation. <h3>Methods</h3> The frequency-stimulation profiles (0.1–30 Hz) of murine mesenteric arteries (&lt;200 µM), +/-PVAT, were characterised using wire myography at various voltages (5–30 V). The accepted test for neural stimulation using 1 µM tetrodotoxin (TTX) was performed, and 1.46 µM 6-hydroxydopamine (6-OHDA) used to sympathetically denervate vessels +/-PVAT. Exogenous PVAT was incubated with TTX and 6-OHDA for 30 min before re-suspending in the bath. Specific adrenoceptor stimulation of PVAT function was investigated using noradrenaline, phenylephrine (both 1 × 10^–9–1 × 10^–5M), and CL-316,243 (10 µm). <h3>Results</h3> At 20&amp;30V PVAT elicited an anti-contractile effect (P &lt; 0.01); reproducible at 20V after a 15 min rest period. This effect is due to factors released from the PVAT and not due to a “barrier” effect, as application of exogenous PVAT elicits the same anti-contractile response (P &lt; 0.05). Sympathetic denervation of whole vessels with 6-OHDA abolished all significant contractile activity, confirming that EFS at 20V innervates sympathetic nerves (P &lt; 0.0001). PVAT incubated with TTX, demonstrated a reduced anti-contractile effect, confirming this effect to be neural and therefore physiologically relevant (P &lt; 0.05). Exogenous PVAT pre-incubated with 6-OHDA also induced a diminished PVAT anti-contractile effect, implicating the role of sympathetic nerves in PVAT (P &lt; 0.0001). In the noradrenaline but not phenylephrine concentration-response curve, PVAT elicits an anti-contractile response. The addition of β₃-adrenoceptor agonist CL-316,243 to phenylephrine-constricted +PVAT vessels induced a relaxation which replicated the PVAT anti-contractile effect seen in vessels stimulated with NA (P &lt; 0.05), indicating the role of β₃-adrenoceptors in PVAT. <h3>Conclusions</h3> These results clearly demonstrate sympathetic nerve stimulation can provoke the release of vasodilators from PVAT. The mechanism appears to be via activation of the adipocyte membrane located β3-adrenoreceptor. Further studies will be carried out in an obese model to understand how over-stimulation leads to a down-regulation and a loss of anti-contractility possibly via β3 receptor desensitisation.

The Effects of Sympathetic Nerve Stimulation on Ventricular Electrophysiology in Long QT Syndrome‐1

Congenital long QT syndrome 1 (LQTS1) results from reduced slow activating delayed rectifier potassium channel (IKs) activity. Ventricular arrhythmias occur with increases in sympathetic tone which can cause sudden cardiac death (SCD). Underlying mechanisms are not understood but may include changes in electrical restitution (RT) – the relationship between action potential duration and diastolic interval. We aimed to examine the effects of sympathetic nerve stimulation (SNS) on effective refractory period (ERP), RT slope and inducibility of ventricular fibrillation (VF) in a pharmacological model of LQTS1. The innervated heart preparation from adult male guinea pigs (n=6, 450–550g) were used. ERP and LV apical and basal RT were measured using an extrastimulus protocol. Inducibility of VF was assessed with ventricular fibrillation threshold (VFT). Parameters were measured during baseline (BL) and SNS (3Hz, 1V) in control (Ctrl) and in the presence of the IKs blocker HMR1556 to mimic LQTS1 (HMR). Data are mean±SEM, analysed using Student's T‐Test (*vs BL, **vs Ctrl). During control, SNS significantly reduced ERP and VFT and increased RT slope but only at LV base, (Table). In the presence of HMR1556, the effects of SNS on ERP and VFT were augmented whilst the effect on RT was abolished. In conclusion, SNS increases susceptibility to VF which appears to be augmented in this pharmacological LQTS1 model and related to changes in ERP but not RT.Research support from the University of Leicester

Effects of sympathetic nerve stimulation on electrical activity of Auerbach's plexus and intestinal smooth muscle tone.

Effects of sympathetic nerve stimulation on electrical activity of Auerbach's plexus and intestinal smooth muscle tone.

The effects of sympathetic nerve stimulation and guanethidine on parasympathetic neuroeffector transmission; the inhibition of acetylcholine release.

Abstract The effect of noradrenaline released either by sympathetic nerve stimulation or guanethidine added to the organ bath has been studied on acetylcholine release from parasympathetic nerve terminals and compared with the effect of exogenous noradrenaline. Sympathetic nerve stimulation, guanethidine and noradrenaline reduced the release of acetylcholine from resting rabbit intestine by up to 70%. Sympathetic stimulation and guanethidine failed to reduce acetylcholine release in preparations previously depleted of noradrenaline. Noradrenaline added to the bath still remained effective. The fact that noradrenaline released is capable of inhibiting acetylcholine release supports the concept that noradrenaline physiologically controls the release of acetylcholine.

On the Mechanism Involved in the Ability of Meptazinol to Potentiate the Effects of Sympathetic Nerve Stimulation

Mouse isolated vas deferens responded to field stimulation (0.1 Hz) with twitch responses which were abolished by alpha beta-methyleneadenosine 5'-triphosphate (0.5 microM) and were potentiated 2 to 3 fold by meptazinol (5-300 microM). Exogenous adenosine 5'-triphosphate (4-30 microM) also caused a twitch response but was unaffected by meptazinol (30 microM) as was the response to phenylephrine. The effect of meptazinol on the electrically-induced twitch was reproducible, fast in onset, easily reversed by washing and was still seen in the presence of prazosin (1 microM), yohimbine (1 microM), propranolol (0.1 microM), atropine (0.1 microM), physostigmine (1 microM), cocaine (1 microM) or desmethylimipramine (0.3 microM) indicating that the mechanism involved does not depend on adrenoceptors, cholinergic mechanisms or blockade of uptake. Mouse isolated atria responded to stimulation (1, 2 or 5 Hz) of their sympathetic nerves via transmural electrodes with chronotropic responses which were abolished by atenolol (5 and 50 microM) but were unaffected by alpha beta-methyleneadenosine 5'-triphosphate (0.5 microM). Meptazinol (100 microM) failed to potentiate the responses. It is suggested that meptazinol potentiates the effects of the non-adrenergic non-cholinergic transmitter thought to be involved in the response of the mouse vas deferens to electrical stimulation.

Interaction between α 1- and α 2-adrenoreceptors contributes to enhanced constrictor effects of norepinephrine in mesenteric veins compared to arteries

Mesenteric veins are more sensitive than arteries to the constrictor effects of sympathetic nerve stimulation and α-adrenoceptor agonists. We tested the hypothesis that α 1- and α 2-adrenoceptors interact to enhance adrenergic reactivity of mesenteric veins. We studied neurogenic and agonist-induced constrictions of mesenteric veins and arteries in vitro. Norepinephrine concentration–response curves were left-shifted in veins compared to arteries. UK 14,304 (0.01–1 μM, α 2-adrenoceptor receptor agonist) did not constrict arteries or veins but enhanced constrictions and Ca 2+ signals mediated by α 1-adrenoceptor stimulation in veins. Yohimbine (α 2-adrenoceptor receptor antagonist) and MK912 (α 2C-adrenoceptor receptor antagonist), but not α 2A- or α 2B-adrenoceptor antagonists, produced rightward shifts in norepinephrine concentration–response curves in veins. Pharmacological studies revealed that α 1D-adrenoceptors mediate venous constrictions. Norepinephrine responses in veins from α 2C-adrenoceptor knock-out (KO) mice were not different from wild type veins. Yohimbine inhibited norepinephrine constrictions in α 2C-adrenoceptor KO veins suggesting that there is upregulation of other α 2-adrenoceptors in α 2C-KO mice. These data indicate that α 1D- and α 2C-adrenoceptors interact in veins but not in arteries. This interaction enhances venous adrenergic reactivity. Mesenteric vein-specific α 2-adrenoceptor linked Ca 2+ and perhaps other signaling pathways account for enhanced venous adrenergic reactivity.

Effects of Sympathetic Nerve Stimulation on Ischemia-induced Ventricular Arrhythmias by Modulating Connexin43 in Rats

Increased cardiac sympathetic nerve activity is thought to contribute to ventricular tachyarrhythmias during acute myocardial ischemia (MI). However, the mechanism is not completely understood. This study investigated the effects of sympathetic nerve stimulation (SNS) on ventricular tachyarrhythmias and connexin43 (Cx43) during acute MI in rats. Ninety five male Wistar rats were randomly assigned into four groups receiving the following treatments: myocardial ischemia with sympathetic nerve stimulation (MI-SNS, n=25), sham-operation treated with sham stimulation (SO, n=20), myocardial ischemia with sham stimulation (MI, n=25), myocardial ischemia pretreated with sympathetic nerve stimulation (pSNS-MI, n=25). During the 30-min ischemia, the incidence of ventricular tachyarrhythmias, i.e., ventricular tachycardia or ventricular fibrillation (VT/VF) was increased in the MI-SNS group and decreased in the pSNS-MI group compared to that in the MI group (p<0.05 for both). The total amount of Cx43 protein was significantly decreased in the MI-SNS group but not in the MI group and the pSNS-MI group. The amount of phosphorylated Cx43 in the MI-SNS group was significantly lower compared to that in the SO group and the MI group (p<0.05). However, the amount of phosphorylated Cx43 was significantly increased in the pSNS-MI group compared to that in the MI group and the MI-SNS group (p<0.05). SNS promoted the degradation of Cx43 protein, especially the phosphorylated Cx43 protein, whereas pSNS inhibited the ischemia-induced loss of phosphorylated Cx43 during acute MI. These changes may be related to the pro- or anti-arrhythmic effect of SNS or pSNS during acute MI.

Effects of sympathetic nerve stimulation on connexin43 and ventricular arrhythmias during acute myocardial ischemia: experiment with rats

To investigate the effects of sympathetic nerve stimulation (SNS) on connexin43 (Cx43) and ventricular arrhythmias during acute myocardial ischemia (MI). Ninety five Wistar rats were randomly divided into four groups: MI group (n=25), undergoing: ligation of the anterior descending coronary; MII-SNS group (n=25); undergoing electric stimulation of sympathetic nerve since the beginning of ligation of the anterior descending coronary and lasting till 30 min after the ligation, sympathetic nerve stimulation preconditioning + myocardial ischemia (pSNS-MI) group (n=25), undergoing electric stimulation of sympathetic nerve since the beginning of ligation of the anterior descending coronary that ended just after the ligation; and sham operation (SO) group (n=20), without coronary ligation. Ventricular arrhythmias were monitored by electrocardiography. Western blotting and RT-PCR were used to detect the protein and mRNA expression of Cx43 respectively. Immunofluorescence analysis was used to observe the changes of Cx43 protein distribution. One and 3 rate died due to ventricular fibrillation in the MI group and MI-SS group respectively. The incidence of ventricular tachycardia (VT)/VF within 30-minute after ligation in the MI-SNS group was 80.0%, significant higher than that of the MI group (52.0%, P < 0.05). The incidence of VT/VF within 30-minute after ligation of the pSNS-MI group was 20.0%, significantly lower than that of the MI-SNS group (P < 0.05). 30 minutes after the ligation, the percentage of phosphorylated Cx43 of the pSNS-MI and MI-SNS groups were 71.2% +/- 7.0% and 73.4% +/- 6.7% respectively, both significantly higher than that of the MI group (46.7% +/- 6.3%) (both P < 0.05). The total contents of Cx43 of the MI and pSNS-MI groups were 1.29 +/- 0.14 and 1.25 +/- 0.13 respectively, both similar to that of the SO group [(1.30 +/- 0.10), both P > 0.05], while the total Cr43 content of the MI-SNS group was 0.73 +/- 0. 12, significantly lower than that of the SO group [(1.30 +/- 0.10), P < 0.05]. The Cx43 mRNA levels of the 3 experimental groups were all significantly lower than that of the SO group (all P < 0.05). Immunofluorescence analysis confirmed that ischemia and sympathetic nerve stimulation induced the changes of connexin43 distribution and sympathetic nerve stimulation preconditioning inhibited the changes of connexin43 distribution induced by ischemia. SNS promotes ventricular arrhythmias by promoting Cx43 degradation, and sympathetic nerve stimulation preconditioning inhibits ventricular arrhythmias by preventing Cx43 dephosphorylation.

Differential contributions of alpha-1 and alpha-2 adrenoceptors to vasoconstriction in mesenteric arteries and veins of normal and hypertensive mice

Mesenteric veins are more sensitive than arteries to the constrictor effects of sympathetic nerve stimulation and α-adrenergic receptor agonists. In the present study, we tested the hypothesis that α 2-adrenergic receptors (α 2-ARs) contribute to in vitro agonist-induced constriction in veins but not arteries and that α 2-AR function is down-regulated in mesenteric arteries and veins in deoxycorticosterone acetate–salt (DOCA–salt) hypertension. Norepinephrine (NE) concentration–response curves were similar in SHAM and DOCA–salt arteries and veins indicating that adrenergic reactivity of mesenteric blood vessels is not altered in murine DOCA–salt hypertension in vitro. Veins were 30-fold more sensitive to NE than arteries. The α 1-AR antagonist, prazosin (0.003–0.3 μM), produced concentration-dependent rightward shifts of the NE concentration–response curves in arteries but not veins. The α 2-AR agonists, clonidine and UK-14,304, did not constrict arteries or veins in the absence or presence of indomethacin (10 μM) and nitro- l-arginine (NLA; 100 μM). The α 2-AR antagonists, yohimbine (0.003–0.3 μM) and rauwolscine (0.1 μM) did not affect NE responses in SHAM or DOCA–salt arteries but antagonized NE responses in veins. These data indicate that there are different α-AR contractile mechanisms in murine mesenteric arteries and veins. α 1-ARs, but not α 2-ARs, mediate direct contractile responses in arteries and veins while α 2-ARs contribute indirectly to NE-induced constrictions in veins but not arteries in vitro. There may be direct protein–protein interactions between α 1- and α 2-ARs or between their signaling pathways in veins. This contribution of α 2-ARs may account for the greater sensitivity of veins compared to arteries to the contractile effects of NE.

Effects of sympathetic nerve stimulation on long posterior ciliary artery blood flow in cats.

A new technique using ultrasonic flowmetry was developed in order to directly measure blood flow in the long posterior ciliary artery (LPCA) of anesthetized cats. Basal LPCA blood flow averaged about 0.6 ml/min and was stable over the experimental period. Electrical stimulation of the cervical preganglionic cervical sympathetic nerve produced frequency-dependent anterior segment ocular vasoconstrictor responses. Ipsilateral nictitating membrane contractions were simultaneously measured as a well-established index of neural sympathetic activation. LPCA frequency-response relationships were shifted to the right in comparison with those for the nictitating membrane. When elicited at two min intervals, submaximal evoked responses of both systems were stable for more than 90 min. Ocular vasoconstrictor and nictitating membrane responses were blocked in a dose-dependent fashion by intravenous treatment with the non-selective a-adrenoceptor antagonist, phentolamine (0.3-3.0 mg/kg), as well as with the selective alpha1-adrenoceptor antagonist, prazosin (3-30 microg/kg). In contrast, neither evoked response was further antagonized by subsequent administration of the alpha2-adrenoceptor antagonist, yohimbine (500 microg/kg). These results demonstrate the usefulness of ultrasonic flowmetry to study mechanisms controlling ocular anterior segment circulation and suggest that, as previously established for the nictitating membrane and anterior choroid, adrenergic neurogenic vasoconstriction in tissues perfused by the LPCA is mediated predominantly by alpha1-adrenoceptors.

Sympathetic inhibition of ascending and descending interneurones during the peristaltic reflex in the isolated guinea-pig distal colon.

1. We investigated the effects of sympathetic nerve stimulation within ascending and descending reflex pathways underlying the peristaltic reflex in the guinea-pig distal colon. 2. A three-chambered partitioned bath was used to divide a segment of distal colon into stimulation, recording and intermediate regions. The effects of lumbar colonic nerves (LCN) could be localized to the intermediate region by surgical lesions of the mesentery and by application of guanethidine (3 microM) to the stimulation and recording chambers. 3. Brush stroking the mucosa in the anal and oral stimulation chambers elicited a synchronous contraction of the longitudinal muscle (LM) and circular muscle (CM) oral to, and transient relaxation of the LM and CM anal to, the stimulus, respectively. 4. After N omega-nitro-L-arginine (L-NA; 100 microM) in the oral and intermediate chambers, mucosal stimulation in the oral chamber elicited a prolonged descending inhibitory and excitatory complex in both the LM and CM in the anal recording chamber. This was blocked by hexamethonium (300 microM), which did not affect the transient relaxation response recorded in control conditions. 5. Stimulation of the LCN (1200 pulses, 20 Hz), delivered to the intermediate region, abolished the oral contraction and the L-NA-induced anal complex in both the LM and CM, but was without effect on the transient hexamethonium-resistant anal relaxation. These effects of LCN stimulation were reversed by phentolamine (3 microM) or yohimbine (100 nM), but not propranolol (10 microM), when added to the intermediate chamber. 6. LCN stimuli (2-20 Hz, 600 micros pulses) directed to the recording chamber elicited synchronous relaxations in the LM and CM that were unaffected by hexamethonium (300 microM), but were reduced by yohimbine and usually blocked by the further addition of propranolol (10 microM). 7. In conclusion, sympathetic nerve stimulation inhibits orally and anally projecting cholinergic interneurones underlying the peristaltic reflex in the distal colon. In addition, the LM and CM relax synchronously following release of sympathetic neurotransmitter, over a range of stimulus frequencies.

Effects of sympathetic nerve stimulation on membrane potential, [Ca2+]i, and force in the toad sinus venosus.

The effects of sympathetic nerve stimulation on beat rate, force, intracellular Ca2+ concentration ([Ca2+]i) measured using fura 2, and membrane potential were recorded from the spontaneously beating toad sinus venosus. Short trains of stimuli evoked an increase in the beat rate and force. During this tachycardia the amplitude of pacemaker action potentials was not changed, but there was an increase in the basal level of [Ca2+]i with little change in peak [Ca2+]i measured during each action potential. Depletion of intracellular Ca2+ stores with caffeine (3 mM) abolished all responses to sympathetic nerve stimulation. The effects of caffeine were fully reversible. Caffeine (3 mM), in the presence of the Ca2+-ATPase inhibitor thapsigargin (30 microM), abolished irreversibly the chronotropic and inotropic responses evoked by sympathetic nerve stimulation. Ryanodine (10 microM) attenuated, but did not abolish, these responses. These results suggest that, in the toad sinus venosus, increases in force and beat rate evoked by sympathetic nerve stimulation result from the release of Ca2+ from intracellular Ca2+ stores.

Effects of sympathetic nerve stimulation on membrane potential in the circular muscle layer of mouse distal colon.

Little is known about the effects of sympathetic nerve stimulation on the membrane potential of colonic smooth muscle. In the distal colon of the mouse, intracellular microelectrodes were used to record the effects of lumbar colonic (LCN) and intermesenteric nerve (IMN) stimulation on circular muscle membrane potential in vitro. A two-compartment organ bath was used to selectively perfuse the colon and inferior mesenteric ganglion (IMG). In the presence of nifedipine (1-2 microM) (to the colonic compartment only), spontaneous depolarizations (myoelectric complexes, MCs) were recorded about every 4 min. MCs consisted of a prolonged burst of rapid oscillations in membrane potential (approximately 2 Hz) that were superimposed on a slow depolarization (mean amplitude 12 mV). Single electrical stimuli (600 microseconds duration) delivered to the LCN or IMN did not elicit a detectable change in the membrane potential. However, trains of stimuli (e.g., 60 pulses at 10-20 Hz) to the LCN or IMN during the intervals between MCs evoked a depolarization (with superimposed action potentials in the absence of nifedipine). Trains of stimuli delivered during the plateau phase of the MC reduced or abolished the rapid oscillations, without a further membrane depolarization. The MC period was unaffected by stimulation of the IMN or LCN. Responses were abolished by the selective perfusion of guanethidine (1 microM) to the colon, or by severing the LCN. Hexamethonium (500 microM) (to the colon) abolished MCs, induced sustained depolarization and attenuated the amplitude of the sympathetic depolarizations by 74%. In hexamethonium, sympathetic responses remained attenuated when the membrane of the circular muscle was repolarised by sodium nitroprusside (1 microM). Immunohistochemical studies of the colon revealed intense immunoreactivity for tyrosine hydroxylase in the myenteric plexus but not in the circular muscle layer. It is suggested that responses to sympathetic nerve stimulation in the circular muscle layer of the mouse colon are secondary to actions on the enteric nervous system.

Regional differences in sympathetic neurotransmission to cutaneous arteries in the guinea-pig isolated ear

The effects of sympathetic nerve stimulation on different cutaneous arteries were examined in arteries isolated from guinea-pig ears, by measuring membrane potential changes in smooth muscle cells in response to electrical field stimulation. Resting membrane potential (RMP) was similar in proximal (main ear artery) and distal (3rd or 4th branch order) cutaneous arteries (mean −71 mV). Single stimuli evoked excitatory junction potentials (EJPs) in all arteries. The EJPs in proximal arteries were twice the amplitude, and the time constant of EJP decay was almost half the value, compared with distal cutaneous arteries. EJP amplitude was reduced by >90% by suramin (30 μM) or α,β,methylene-ATP (α,β,m-ATP) (1 μM) in all proximal, and most distal arteries. Residual responses in distal arteries were resistant to tetrodotoxin. The N-type calcium channel blocker, ω-conotoxin GVIA (30 nM), reduced EJP amplitude by 70–100% in both proximal and distal arteries. Successive EJPs evoked by trains of stimuli at 1 to 5 Hz were depressed in amplitude in proximal arteries, but showed facilitation in distal arteries. EJP depression in proximal arteries was reversed to facilitation by the α 2-adrenoceptor antagonist, yohimbine (30 nM). Trains of stimuli delivered at 10–20 Hz produced summation of EJPs and active membrane responses in 30% of proximal arteries. Active responses were never detected in distal arteries. Slow depolarizations following the EJPs were detected in most arteries after trains of stimuli, and were abolished by prazosin (0.3 μM) or ω-conotoxin GVIA (30 nM). The density of the perivascular plexus of axons innervating proximal arteries, demonstrated with catecholamine fluorescence histochemistry, was twice that in distal cutaneous arteries. These regional differences in sympathetic neurotransmission suggest that cutaneous vasoconstriction in response to thermoregulatory stimuli, which occurs predominantly in distal cutaneous segments, is likely to be qualitatively different from cutaneous vasoconstriction of proximal arteries in response to other physiological stimuli.

Activation of alpha- and beta-adrenoceptors by sympathetic nerve stimulation in the large intestine of the rat.

1. The effects of sympathetic nerve stimulation on the motility of the circular and longitudinal muscle of the large intestine were investigated in vitro, and the involvement of various adrenoceptor subtypes determined. A comparison between the sympathetic supply arising from the prevertebral and pelvic ganglia was also made. 2. In the longitudinal muscle of the distal colon, sympathetic nerve stimulation caused responses which were contractile (0.1-2 Hz), biphasic (5-10 Hz) or purely inhibitory (20-30 Hz). All contractile responses were removed with phentolamine (3 microM), whereas the inhibitory responses were significantly diminished by propranolol (0.1 microM) and completely abolished by alprenolol (3 microM) or nadolol (300 microM). 3. In the longitudinal muscle of the proximal colon, the effects of sympathetic nerve stimulation were predominantly inhibitory. Some of this inhibition was removed by propranolol (0.1 microM), but was largely unaffected by alprenolol (3 microM). The remainder of the inhibitory response was probably non-noradrenergic as it was not removed by a combination of phentolamine (3 microM) and alprenolol (3 microM). 4. In the circular muscle of both the proximal and distal colon, sympathetic stimulation caused a strong contractile response which was completely removed by phentolamine (3 microM) to reveal an inhibitory response. This inhibitory response was unchanged by propranolol (0.1 microM) but was removed by alprenolol (3 microM), following which a weak non-noradrenergic contractile response was unmasked. 5. Stimulation of the hypogastric nerve to activate pelvic sympathetic pathways had no effect on the motility of the longitudinal muscle, but caused a contractile response in the circular muscle which was completely removed by phentolamine (3 microM). 6. We conclude that sympathetic nerves innervate adrenoceptors of different types in the various muscle layers and regions of the colon. They innervate a mixture of alpha-, and beta(3)-adrenoceptors in the longitudinal muscle of the proximal colon, alpha-, classical beta- and beta(3)-adrenoceptors in the distal colon, and primarily alpha-adrenoceptors with a few beta(3)-adrenoceptors in the circular muscle. In addition, the pelvic sympathetic innervation of the rectum differs from the prevertebral supply by innervating only excitatory alpha-adrenoceptors.

Postjunctional alpha 2-adrenoceptors are not present in proximal arterioles of rat intestine.

The purpose of this study was to evaluate two potential stimuli for nitric oxide (NO) release in rat intestinal arterioles during sympathetic nerve activation. To determine whether these vessels contain endothelial alpha 2-adrenoceptors linked to the L-arginine-NO pathway, intravital microscopy was used to study the response of first-order arterioles (1As, 20-40 microns ID) to direct application of 1) the selective alpha 2-agonist BHT-933 and 2) norepinephrine (NE) or sympathetic nerve stimulation before and after alpha 1- or alpha 2-receptor blockade. The effect of sympathetic nerve stimulation on 1A wall shear rate (WSR) was also determined to evaluate the possibility of hemodynamic shear stress as a stimulus for NO release. BHT-933 had no effect on 1A diameter, whereas NE produced dose-dependent constrictions of 5 +/- 3 to 15 +/- 3 microns, which were usually abolished by the alpha 1-antagonist prazosin but unaffected by the alpha 2-antagonist idazoxan. Sympathetic nerve stimulation at 3, 8, and 16 Hz induced constrictions of 4 +/- 1, 8 +/- 2, and 17 +/- 4 microns, respectively, and these constrictions were also usually abolished by prazosin but unaffected by idazoxan. Resting WSR averaged 1,997 +/- 163 s-1 and decreased to 1,587 +/- 209, 1,087 +/- 195, and 537 +/- 99 s-1 during 3-, 8-, and 16-Hz nerve stimulation. These results suggest that alpha 2-adrenoceptor-dependent pathways do not influence either resting tone or sympathetic constriction of proximal arterioles in the intestinal submucosa and that luminal shear stress in these vessels significantly decreases with sympathetic constriction. It therefore appears unlikely that either alpha 2-receptor activation or changes in hemodynamic shear serve as stimuli for arteriolar NO release during periods of increased sympathetic nerve activity.

Effects of sympathetic nerve stimulation on membrane potential, [Ca2+]i and force in the arrested sinus venosus of the toad, Bufo marinus.

1. The effects of sympathetic nerve stimulation on membrane potential and on the intracellular concentration of calcium ions, [Ca2+]i, were recorded concurrently from the sinus venosus of the toad, Bufo marinus, in preparations where beating had been abolished by adding an organic calcium antagonist to the physiological saline. In a separate set of experiments the effects of sympathetic nerve stimulation on force production were examined. 2. Stimulation of the sympathetic nerves caused a membrane depolarization and a simultaneous increase in [Ca2+]i. Both responses were reduced by dihydroergotamine (20 microM). 3. The membrane depolarization and increase in [Ca2+]i evoked by sympathetic nerve stimulation were abolished by ryanodine (10 microM), or caffeine (3 mM). The effects of caffeine, but not those of ryanodine, were fully reversible. 4. Although the Ca(2+)-ATPase inhibitor thapsigargin (30 microM) itself had little effect on the responses to sympathetic nerve stimulation, in its presence caffeine (3 mM) irreversibly abolished the responses. 5. In the presence of nifedipine (10 microM), sympathetic nerve stimulation caused contractions of the sinus venosus. These responses were abolished by either ryanodine (10 microM) or caffeine (3 mM). 6. The results suggest that neuronally released transmitter activates a complex biochemical pathway which triggers the release of Ca2+ from internal stores.