Transmural Electrical Field Stimulation Research Articles

Cortistatin Inhibits Intestinal Ion Secretion via Activating the Somatostatin‐2 Receptor in the Submucosal Neurons of the Mouse Colon

Background and AimsCortistatin (CST) is a somatostatin‐like neuropeptide that is found in both the central and peripheral nervous system. CST binds with high affinity to all somatostatin receptors and also has actions not shared by somatostatin. We previously found that CST is expressed by enteric neurons in the guinea pig ileum and acts as an inhibitory neurotransmitter in the submucosal plexus. The aim of the present study was to evaluate the effect of CST on neurogenic intestinal secretion.MethodsMucosal/submucosal flat‐sheet preparations from mouse proximal colon were mounted in Ussing flux chambers for measurement of short‐circuit current (Isc) as an indicator of mucosal secretion. Transmural electrical field stimulation (EFS) evoked characteristic biphasic increases in Isc, with an early rapidly rising phase followed by a sustained phase.ResultsApplication of CST‐14, CST‐17, or CST‐29 to the basolateral side of the mucosal/submucosal preparation decreased baseline Isc in a concentration‐dependent manner with an IC50 of 90.5 nM, 52.1 nM, or 102.9 nM, respectively. The somatostatin‐2 receptor (SSTR‐2) antagonist CYN 154806 (1 mM) significantly suppressed CST‐14 (100 nM), CST‐17 (100 nM), or CST‐29 (100 nM)‐evoked ΔIsc by 36.0 ± 8.2% (n=5; P < 0.05), 47.2 ± 11.2% (n=5; P < 0.01), or 51.7.0 ± 8.4% (n=5; P < 0.01), respectively. The neuronal blocker tetrodotoxin (300 nM) significantly reduced CST‐14 (100 nM), CST‐17 (100 nM), or CST‐29 (100 nM)‐evoked ΔIsc by 36.5 ± 2.7% (n=5; P < 0.01), 48.9 ± 11.2% (n=5; P < 0.01), or 49.5 ± 3.6% (n=5; P < 0.01), respectively. All of the CSTs (1 nM – 1 mM) also produced a concentration‐dependent reduction of the EFS‐evoked biphasic secretory responses. The effect of CSTs on the EFS‐evoked responses was antagonized by CYN 154806 (1 mM). Immunohistochemical staining with a CST‐17 polyclonal antibody revealed expression of CST‐17 immunoreactivity (IR) in mouse colonic submucosal neurons. CST‐17‐IR was localized in cholinergic secretomotor neurons but was absent from non‐cholinergic secretomotor neurons and calbindin‐IR neurons.ConclusionsThe results suggest that CST acts at SST‐2 receptors in the submucosal plexus to suppress secretomotor neuronal excitability and neurogenic intestinal secretion in the mouse colon.

Calcium Imaging of Nerve-Mast Cell Signaling in the Human Intestine.

Introduction: It is suggested that an altered microenvironment in the gut wall alters communication along a mast cell nerve axis. We aimed to record for the first time signaling between mast cells and neurons in intact human submucous preparations.Methods: We used the Ca2+ sensitive dye Fluo-4 AM to simultaneously image changes in intracellular calcium [Ca+2]i (%ΔF/F) in neurons and mast cells. Data are presented as median with interquartile ranges (25/75%).Results: We recorded nerve responses in 29 samples upon selective activation of 223 mast cells by IgE receptor cross linking with the antibody mAb22E7. Mast cells responded to mAb22E7 with a median [Ca+2]i increase of 20% (11/39) peaking 90 s (64/144) after the application. Only very few neurons responded and the median percentage of responding neuronal area was 0% (0/5.9). Mast cell activation remained in the presence of the fast sodium channel blocker tetrodotoxin. Specific neuronal activation by transmural electrical field stimulation (EFS) in 34 samples evoked instantaneously [Ca+2]i signals in submucous neurons. This was followed by a [Ca+2]i peak response of 8%ΔF/F (4/15) in 33% of 168 mast cells in the field of view. The mast cell response was abolished by the nerve blocker tetrododoxin, reduced by the Calcitonin Gene-Related Peptide receptor 1 antagonist BIBN-4096 and the Vasoactive Intestinal Peptide receptor antagonist PG97-269, but not by blockade of the neurokinin receptors 1–3.Conclusion: The findings revealed bidirectional signaling between mast cells and submucous neurons in human gut. In our macroscopically normal preparations a nerve to mast cell signaling was very prominent whereas a mast cell to nerve signaling was rather rare.

Glucagon-like peptide-1 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro

Glucagon-like peptide-1 (GLP-1) acts at the G protein-coupled receptor, GLP-1R, to stimulate secretion of insulin and to inhibit secretion of glucagon and gastric acid. Involvement in mucosal secretory physiology has received negligible attention. We aimed to study involvement of GLP-1 in mucosal chloride secretion in the small intestine. Ussing chamber methods, in concert with transmural electrical field stimulation (EFS), were used to study actions on neurogenic chloride secretion. ELISA was used to study GLP-1R effects on neural release of acetylcholine (ACh). Intramural localization of GLP-1R was assessed with immunohistochemistry. Application of GLP-1 to serosal or mucosal sides of flat-sheet preparations in Ussing chambers did not change baseline short-circuit current (I(sc)), which served as a marker for chloride secretion. Transmural EFS evoked neurally mediated biphasic increases in I(sc) that had an initial spike-like rising phase followed by a sustained plateau-like phase. Blockade of the EFS-evoked responses by tetrodotoxin indicated that the responses were neurally mediated. Application of GLP-1 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-1 receptor antagonist exendin-(9-39) suppressed this action of GLP-1. The GLP-1 inhibitory action on EFS-evoked responses persisted in the presence of nicotinic or vasoactive intestinal peptide receptor antagonists but not in the presence of a muscarinic receptor antagonist. GLP-1 significantly reduced EFS-evoked ACh release. In the submucosal plexus, GLP-1R immunoreactivity (IR) was expressed by choline acetyltransferase-IR neurons, neuropeptide Y-IR neurons, somatostatin-IR neurons, and vasoactive intestinal peptide-IR neurons. Our results suggest that GLP-1R is expressed in guinea pig submucosal neurons and that its activation leads to a decrease in neurally evoked chloride secretion by suppressing release of ACh at neuroepithelial junctions in the enteric neural networks that control secretomotor functions.

Effects of Resveratrol on Ileal Smooth Muscle Reactivity in Polymicrobial Sepsis Model

To determine the effects of resveratrol on the ileal smooth muscle reactivity in polymicrobial sepsis. Polimicrobial sepsis was induced by the cecal ligation and perforation (CLP) procedure. Sprague Dawley rats were divided into three groups. Rats in resveratrol group received resveratrol after CLP (100 mg/kg, i.p.). Rats received saline immediately after CLP in the sepsis group. Control group rats underwent sham operation. The rats were sacrificed and the ileum was excised 24 h after the operation. Contractile and relaxant responses in isolated smooth muscle strips (SMS) were determined using an in vitro muscle technique. TNFα and IL-6 levels were measured in blood samples. Contractile responses to carbachol and KCl and relaxant responses to transmural electrical field stimulation (EFS) were significantly decreased in the sepsis group compared with control and resveratrol groups. No significant changes were observed for smooth muscle reactivity in the resveratrol and control groups. Sodium nitroprusside (SNP) or papaverine-induced relaxations were similar in the all groups. Resveratrol treatment supressed increased TNFα and IL-6 levels in blood seen in sepsis group. Ileal smooth muscle reactivity was improved after resveratrol treatment in rats with sepsis. The results of the present study indicate that the beneficial effects of resveratrol might be, at least in part, attributed to its effects on non-adrenergic non-cholinergic pathway and/or anti-inflammatory and antioxidant activity.

Cortistatin Inhibits Intestinal Secretion in the Guinea‐pig Ileum In Vitro

Background and AimsCortistatin (CST) is a novel neuropeptide with high structural homology with somatostatin. We previously found that CST is expressed by enteric neurons and acts as an inhibitory neurotransmitter in the submucosal plexus. The aim of the present study was to evaluate the effect of CST on neurogenic intestinal secretion.MethodsSubmucosal/mucosal preparations from guinea pig ileum were mounted in Ussing flux chambers for measurement of short‐circuit current (Isc) as an indicator of mucosal secretion. Transmural electrical field stimulation (EFS) was used to evoke neural‐mediated intestinal secretion.ResultsApplication of CST‐14 (rat), CST‐17 (human), CST‐29 (rat), or CST‐29 (human) to the submucosal side of the preparation decreased baseline Isc in a concentration‐dependent manner with an IC50 (in nM) of 90.5, 52.1, 102.9 or 71.6, respectively. The somatostatin‐2 receptor (SSTR‐2) antagonist CYN 154806 significantly suppressed CST‐14r, CST‐17h, CST‐29r, or CST‐29h‐evoked ΔIsc. The neuronal blocker tetrodotoxin significantly reduced CST‐14r, CST‐17h, CST‐29r, or CST‐29h‐evoked ΔIsc. CSTs produced a concentration‐dependent reduction of the EFS‐evoked secretory responses, which was antagonized by CYN 154806. Immunoreactivity (IR) of CST‐17 was found in submucosal neurons. CST‐17‐IR was localized in cholinergic secretomotor neurons but was absent from non‐cholinergic secretomotor neurons.ConclusionsThe results suggest that CSTs act at SST‐2 receptors in the submucosal plexus to suppress secretomotor neuronal excitability and neurogenic intestinal secretion.

Glucagon-like peptide-2 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro.

Glucagon-like peptide-2 (GLP-2) is an important neuroendocrine peptide in intestinal physiology. It influences digestion, absorption, epithelial growth, motility, and blood flow. We studied involvement of GLP-2 in intestinal mucosal secretory behavior. Submucosal-mucosal preparations from guinea pig ileum were mounted in Ussing chambers for measurement of short-circuit current (I(sc)) as a surrogate for chloride secretion. GLP-2 action on neuronal release of acetylcholine was determined with ELISA. Enteric neuronal expression of the GLP-2 receptor (GLP-2R) was studied with immunohistochemical methods. Application of GLP-2 (0.1-100 nM) to the serosal or mucosal side of the preparations evoked no change in baseline I(sc) and did not alter transepithelial ionic conductance. Transmural electrical field stimulation (EFS) evoked characteristic biphasic increases in I(sc), with an initially rapid rising phase followed by a sustained phase. Application of GLP-2 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-2R antagonist GLP-2-(3-33) significantly reversed suppression of the EFS-evoked responses by GLP-2. Tetrodotoxin, scopolamine, and hexamethonium, but not vasoactive intestinal peptide type 1 receptor (VPAC1) antagonist abolished or reduced to near zero the EFS-evoked responses. GLP-2 suppressed EFS-evoked acetylcholine release as measured by ELISA. Pretreatment with GLP-2-(3-33) offset this action of GLP-2. In the submucosal plexus, GLP-2R immunoreactivity (-IR) was expressed in choline acetyltransferase-IR neurons, somatostatin-IR neurons, neuropeptide Y-IR neurons, and vasoactive intestinal peptide-IR neurons. We conclude that submucosal neurons in the guinea pig ileum express GLP-2R. Activation of GLP-2R decreases neuronally evoked epithelial chloride secretion by suppressing acetylcholine release from secretomotor neurons.

W1374 Excitation of Neurons in the Enteric Nervous System (ENS), Degranulation of Enteric Mast Cells and Direct Action On Enterocytes Underlie Stimulatory Action of Bradykinin (Bk) On Mucosal Secretion in Guinea-Pig Small Intestine

Neurons in the g-pig small intestinal ENS express the BK-B2 receptor. Stimulation of the BK-B2 receptor elevates neuronal excitability (J Pharmacol Exp Ther 2004; 309:310-9; & Br J Pharmacol 2003; 138:1221-32). BK-2 stimulation releases prostaglandins, which feedback in autocoid manner to excite the same neurons that release the prostaglandins. Full thickness and muscle-stripped segments of g-pig ileum were mounted in Ussing flux chambers for recording short-circuit current (Isc), an indicator of Clsecretion. Application of BK in the serosal compartment of the chambers evoked increases in Isc in a concentration-dependent manner with an EC50 8.0±0.1nM. Higher concentrations of 1-5μMwere required for stimulation of Isc with application in the mucosal compartment. Pretreatment with 0.5-μM tetrodotoxin (TTX) partially suppressed the BK-evoked increases in Isc. Transmural electrical field stimulation evoked neurally-mediated increases in Isc. These responses were abolished by TTX in each of 18 preps. Application of the mast cell degranulating agent, compound 48/ 80 (60μg/ml), increased Isc (P 0.05). The BK-B2 antagonist, WIN64388 (1-10μM), partially suppressed BK-evoked increases in Isc. WIN64388 at 10μM suppressed BK-evoked Isc by 62.8% in 12 preps. Pre-incubation for 30 min with the selective cyclooxygenase-1 (COX-1) inhibitor, SC560 (10μM), abolished increases in Isc evoked by 10-nM BK in each of 10 preps. The COX-2 inhibitor, NS398 (2μM) reduced BK-evoked increases in Isc by 28.9 percent in 6 preps. Pre-treatment with a prostanoid receptor antagonist, AH6809 (5μM), significantly suppressed BK-evoked stimulation of Isc (P<0.05) for all of 6 preps. BK-evoked increases in Isc were suppressed by pre-treatment with the mast cell stabilizing agents: cromolyn (10-30μM; P<0.05); doxantrazole (30-50μM; P<0.05) or ketotifen (10μM; P<0.05) with each agent tested on 10 preps. Carbachol acted directly on the epithelium to increase Isc. This well-known action of the muscarinic agonist was suppressed by ketotifen, but not by cromolyn or doxantrazole. The results suggest a role for enteric mast cells in BK-stimulation of mucosal secretion, with a prostaglandin involved as a mediator. Once released from mast cells prostaglandins stimulate secretomotor neuronal activity; as well as, act in direct concert with BK on the enterocytes. All of which might underlie the involvement of BK in inflammation-related secretory diarrhea. Supported by: NIH RO1DK57075 & KO8 DK060468

Corpus cavernosum dysfunction in diabetic rats: effects of combined alpha-lipoic acid and gamma-linolenic acid treatment.

The effects of streptozotocin-induced diabetes on nitric oxide (NO)-mediated relaxation of rat corpus cavernosum smooth muscle to neurogenic and endothelial stimulation was examined. The aim was to assess the effects of treatment with low doses of the antioxidant, alpha-lipoic acid, and the omega-6 essential fatty acid, gamma-linolenic acid, either separately or in combination. Treatment was preventive from diabetes induction or corrective over 4 weeks after 4 weeks of untreated diabetes. Corpus cavernosum responses were examined in vitro. Neither diabetes nor treatment affected contractile responses to transmural electrical field stimulation of noradrenergic nerves. Stimulation of phenylephrine precontracted cavernosa in the presence of guanethidine and atropine caused relaxation via the nitrergic innervation. Maximum relaxation responses were 40% and 46% decreased after 4 and 8 weeks of diabetes, respectively. alpha-Lipoic acid, gamma-linolenic acid combination treatment fully prevented this deficit, and partially (52%) corrected the effect of 4 weeks of untreated diabetes. Neither alpha-lipoic acid nor gamma-linolenic components alone had significant effects, which suggests that there were synergistic interactions between the drugs. Both 4 and 8 weeks of untreated diabetes reduced maximum endothelium-dependent relaxation of phenylephrine precontracted cavernosa to acetylcholine by approximately 40%. While alpha-lipoic acid or gamma-linolenic acid were ineffective, joint treatment fully prevented and corrected this diabetic endothelial deficit. Neither diabetes nor treatment affected endothelium-independent relaxation to the NO donor, sodium nitroprusside. The data show that alpha-lipoic acid and gamma-linolenic acid interact synergistically to improve NO-mediated neurogenic and endothelium-dependent relaxation of corpus cavernosum in experimental diabetes.

Purinergic and noradrenergic cotransmission in the rat pineal gland

ATP is coreleased with noradrenaline in several noradrenergic synapses, and P2-like receptors were shown to be present in rat pineal glands. A new method of functional investigation was developed to assess the importance of both transmitters (noradrenaline and ATP) in eliciting the synthesis of melatonin and its precursor N′-acetyl-5-hydroxytryptamine ( N′-acetyl-5-HT) through transmural electrical field stimulation of cultured pineal glands. Incubation with the β-adrenoceptor antagonist propranolol (>10 −7 M) blocked almost completely the production of N′-acetyl-5-HT, whilst the P2 receptor antagonists pyridoxalphosphate-6 azophenyl-2′,4′-disulfonic acid (PPADS, >3×10 −6 M) and suramin (>10 −6 M) blocked it partially. These findings indicate a physiologically relevant role for the purinergic cotransmission in this system.

Transmural field stimulation-induced relaxation in the rat common hepatic artery.

Hepatic arteries are reportedly innervated by vasoconstrictor and vasodilator nerves. Experiments were carried out to investigate the possible involvement of calcitonin gene related peptide (CGRP) and nitric oxide as neurotransmitters during the relaxation of the rat common hepatic artery produced by transmural electrical field stimulation (ES). Common hepatic arteries were excised under ether-anesthesia from 6 weeks-old female rats, and isometric tensions recorded from endothelium-damaged ring preparations. In the presence of atropine and guanethidine, ES relaxed arteries which had been previously contracted with vasopressin. The relaxation response to ES was attenuated by either tetrodotoxin or capsaicin-pretreatment. CGRP induced a concentration-dependent relaxation, which was inhibited by the CGRP antagonist CGRP(8-37). The ES-induced relaxation was attenuated either slightly by the nitric oxide synthesis inhibitor L-nitroarginine (L-NNA) or markedly by CGRP(8-37). The relaxation response was nearly abolished in the presence of both CGRP(8-37) and L-NNA. These results may indicate that the nerve stimulation-induced vasodilatation of the rat common hepatic artery is mediated mainly by CGRP and partly by nitric oxide.

Neurogenic Control of the Ovine Gallbladder: Ultrastructural and Functional Study

The purpose of this study was to investigate the ultrastructure of the local nerve supply of ovine gallbladders as well as the functional characteristics of inhibitory nerves. We used electron microscopy of thin sections of ovine gallbladders and in vitro isometric tension recording using gallbladder strips. Specifically, we measured contractile and inhibitory responses induced by transmural electrical field stimulation (EFS). We found a ganglionated plexus with intramural nerve cells and interconnecting axons. Clear and large dense-core vesicles colocalized in axons close to smooth muscle cells. EFS elicited gallbladder contractions which were converted to relaxation after atropine. EFS-induced relaxation was reduced by the nitric oxide (NO) synthase inhibitor, L-NOARG and blocked by propranolol and/or tetrodotoxin. In conclusion, enteric ganglia and neurones with synaptic vesicles (clear and dense core) were detected close to smooth muscle bundles. Neural inhibition of gallbladder contraction was mediated by β-adrenoceptors coupled to NO generation.

Effects of calcium channel blockade on intestinal fluid secretion: sites of action.

Most intestinal secretagogues, including cholera toxin, evoke fluid secretion in part by activating the enteric nervous system (ENS). The enterotoxins that, due to size, cannot pass the intestinal epithelial lining have been proposed to activate the ENS via the release of amines/peptides from the intestinal endocrine cells. It has been shown that calcium channel blockers of the L-type attenuate intestinal fluid secretion. This study was performed on rat jejunal segments to elucidate where calcium channel antagonists interact with the secretory nervous reflex(es) of the ENS. In vivo, net fluid transport, transmural potential difference (PD) and luminal release of serotonin from the enterochromaffin cells were monitored before and after exposing the intestinal mucosa to cholera toxin (20 microg/mL) or the calcium ionophore A23187 (0.5 mM). In vitro, the effects of transmural electrical field stimulation (EFS) on short circuit current (SCC) was investigated using the Ussing chamber method. Cholera toxin and A23187 evoked a net fluid secretion, an increased PD and an augmented luminal release of 5-HT. These effects were markedly attenuated by giving the calcium channel blocker nifedipine i.v. (5.75 micromol kg(-1) body wt). On the other hand, nifedipine (0.02 mM) had no significant effect on the increased SCC caused by EFS in vitro. The results obtained in the in vivo experiments suggest that the nifedipine markedly attenuates the initial event in cholera toxin- and A23187-induced secretion, the release of amines and probably also of peptides from the intestinal endocrine cells. The in vitro experiments seem to exclude an effect of the calcium channel blockade on the efferent part of the secretory nervous reflex.

Effects of transmural field stimulation in isolated smooth muscle of human rectum and internal anal sphincter.

Smooth muscle preparations from the circular muscle layer of the most distal rectum and the proximal and distal human internal anal sphincter (IAS) mounted in organ baths to record isometric tension developed spontaneous tension. Transmural electrical field stimulation (TMS) induced frequency- and impulse duration-dependent relaxations sensitive to tetrodotoxin in the stimulation range of 0.5-40 Hz and 0.04-0.6 ms. Poststimulus contractions were most frequent and prominent in rectal preparations. Maximal relaxations were comparable in the three locations and were achieved at 10 Hz and 0.4 ms. The frequency inducing half-maximal response was lower in rectal strips compared with IAS. Phentolamine (10-(6) M) enhanced relaxations and diminished off-contractions at 40 Hz in distal IAS. N omega-nitro-L-arginine (L-NNA) concentration dependently inhibited both relaxations and off-contractions (10 Hz, 0.4 ms). The pD2 values (-log EC50) of L-NNA were lower in rectal muscle compared with those in IAS. L-Arginine (10-(4) M) inhibited the blocking effect of L-NNA. In one-half of the preparations, L-NNA reversed the relaxations to duration contractions (15-40 Hz), which were inhibited by atropine in rectal preparations and by phentolamine in IAS. In conclusion, excitatory innervation of the IAS is alpha-adrenergic and cholinergic in the rectum. A product of the L-arginine-nitric oxide pathway mediates the TMS-induced inhibition of the muscle and is also involved in poststimulus contractions.

Functional innervation of the aganglionic segment in Hirschsprung's disease—A comparison of the short- and long-segment type

The authors examined the neuro-effector transmission in the aganglionic segment from 13 patients with Hirschsprung's disease, 10 patients had short-segment type and three patients had long-segment type. Circular muscle strips were prepared and the responses to transmural electrical field stimulation were examined using the isometric tension recording technique. In the ganglionic preparations from the short- and long-segment cases, the stimulation evoked a biphasic response consisting of a relaxation and an atropine-sensitive contraction. The relaxation was partly inhibited by N-ω-nitro- l-arginine (NNLA, a nitric oxide [NO] synthase inhibitor), and the effect of NNLA was abolished completely in the presence of l-arginine, which suggested the presence of NO-mediated inhibitory innervation. In the aganglionic preparations from the short-segment-type cases, stimulation evoked only an atropine-sensitive contraction, while in the aganglionic preparations from the long-segment-type cases, a weak inhibitory response persisted after the contractile response was abolished by atropine. This NO-mediated inhibitory response was frequently detected as the examined region approached the ganglionic segment. These results suggest that the aganglionic segment in the long-segment-type cases might therefore receive NO-mediated inhibitory input from the proximal ganglionic segment.

Nonadrenergic contractile response of guinea pig portal vein to electrical field stimulation mimics response to UTP but not to ATP.

Transmural electrical field stimulation (EFS, 4-32 Hz) produced a biphasic contractile response consisting of a rapid and transient contraction (first phase) followed by a slow contraction (second phase) in ring preparations of guinea pig portal veins. Both contractions were enhanced by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 30 microM). In the presence of L-NAME, tetrodotoxin (1 microM) and guanethidine (3 microM) inhibited both contractions and phentolamine (10 microM), and reserpine treatment abolished the first-phase contraction without affecting the second-phase contraction. These results suggest that the first-phase contraction is caused by norepinephrine released from the perivascular nerves. In the presence of phentolamine and L-NAME, the second-phase contraction was inhibited by the nonselective P2-purinoceptor antagonist suramin (30-300 microM) and the P(2Y)-purinoceptor antagonist reactive blue 2 (RB2; 10-100 microM). alpha,beta-Methylene-adenosine triphosphate (alpha,beta-mATP; 3-30 microM), which desensitizes P(2X)-purinoceptors, and the P(2X)-purinoceptor antagonist 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS; 1-10 microM) had a little effect. Exogenous ATP (0.1-3 mM) and UTP (0.1-3 mM) in the presence of L-NAME produced contractions in a concentration-dependent manner. The ATP-induced contraction was enhanced by suramin, RB2, and DIDS but unaltered by alpha,beta-mATP. The UTP-induced contraction was inhibited by suramin and RB2 but unaltered by alpha,beta-mATP and DIDS. These results indicate that in the guinea pig portal vein, the classic P(2X)-purinoceptors do not contribute to the nonadrenergic component of sympathetic neurotransmission. Furthermore, the pharmacology of the nonadrenergic component of neurotransmission resembles that of vasoconstrictor responses to exogenous UTP rather than to ATP.

Prominent sympathetic purinergic vasoconstriction in the rabbit splenic artery: potentiation by 2,2'-pyridylisatogen tosylate.

1. Vasoconstrictions induced by transmural electrical field stimulation were frequency-dependent from 2 to 32 Hz in the rabbit isolated splenic artery. All contractions were abolished in the presence of tetrodotoxin 1 microM or guanethidine 100 microM. Stimulation at a frequency of more than 32 Hz induced both neurogenic and myogenic responses. 2. Prazosin (1 microM) did not significantly affect vascular contractions to electrical stimulation. Desensitization of P2X-purinoceptors with alpha, beta-methylene ATP (alpha, beta-meATP, 3 microM) abolished the contractions to stimulation at 2-8 Hz and inhibited more than 80% of the vascular response at 16 Hz, but it did not significantly change the responses at 32 Hz. Contractile responses at 32 Hz were inhibited by a combination of prazosin and alpha, beta-meATP. Effects of pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid tetrasodium salt (a selective P2X-purinoceptor antagonist) and suramin (a competitive P2-purinoceptor antagonist) on the neurogenic responses were investigated in this study. 3. 2,2'-Pyridylisatogen tosylate (PIT, 0.3-3 microM) significantly potentiated the vasoconstrictions to electrical stimulation at 2-32 Hz in a concentration-dependent manner. Potentiated responses were restored to the control level 30 min after washing. Concentration-dependent response curves for noradrenaline (NA) or alpha, beta-meATP were not significantly changed by 3 microM PIT, and vasoconstriction by adenosine 5'-triphosphate (ATP, 300 microM) was unaffected by PIT. Coomassie brilliant blue-G (1 microM), which shares the potentiating effect on a recombinant P2Y-purinoceptor with PIT (King et al., 1996), did not inhibit or potentiate the purinergically-mediated component of the response to sympathetic nerve stimulation. The selective alpha 2-adrenoceptor antagonist yohimbine (1 microM) also potentiated the vascular responses to electrical stimulation. 4. The present results indicate that ATP evokes postjunctional contractile responses at low and high frequency electrical stimulation of sympathetic nerves supplying the rabbit splenic artery. PIT potentiates the responses to sympathetic (purinergic) nerve stimulation; this appears to be mainly via prejunctional rather than postjunctional actions.

Nitrergic inhibitory innervation of porcine rectal circular smooth muscle.

This study was performed to determine whether nitric oxide contributes to inhibitory non-adrenergic non-cholinergic (NANC) neurotransmission in the circular smooth muscle layer of pig rectum. Smooth muscle strips were mounted for isometric tension recording in superfusion organ baths. In the presence of atropine sulphate and guanethidine, transmural electrical field stimulation (EFS) produced frequency-dependent relaxation of precontracted muscle strips. N omega-nitro-L-arginine (L-NOArg), a powerful competitive antagonist of nitric oxide synthase, reduced the relaxant response in dose-dependent fashion; this response was maximally reduced by mean(s.e.m.) 86.5(2.6) per cent (P = 0.0003) at a concentration of 3 x 10(-5) mol/l L-NOArg. The response was restored by the addition of L-arginine. Sodium nitroprusside, an exogenous donor of nitric oxide, mimicked the relaxant response. Responses to EFS were abolished by tetrodotoxin. These results suggest that inhibitory NANC neurotransmission in this tissue is mediated, at least in part, by nitric oxide. The failure of L-NOArg to completely abolish the relaxant response suggests that additional neurotransmitters may be involved.

Electrical field stimulation of rat mesenteric small arteries: force and free cytosolic calcium during neurogenic contractions and mechanisms of non‐neurogenic relaxations

The effect of transmural electrical field stimulation (TEFS) of rat mesenteric small arteries was studied. Stimulation parameters were selected to cause tetrodotoxin (TTX) sensitive contractions. In arteries precontracted with PGF2 alpha in the presence of phentolamine, TTX insensitive relaxation could be induced by TEFS. The relaxing effect of TEFS required higher stimulation amplitude and duration than the contractions. Thus, by appropriately choosing stimulation parameters, contractile responses could be elicited which were little affected by any relaxing effect, while contractions were abolished by TTX at any stimulation conditions in the present study. The contractions were abolished by cold storage and almost completely inhibited by phentolamine. Thus, contractions were neurogenic and primarily caused by noradrenaline. At low frequencies, TEFS caused phentolamine sensitive increases in free cytosolic calcium with no contractions. At higher frequencies, there was a further increase in free cytosolic calcium, associated with contraction. Only at high frequencies, noradrenaline from nerves caused sensitization of the contractile filaments to free cytosolic calcium as during stimulation with exogenous noradrenaline. The relaxations were associated with decreases in free cytosolic calcium and were probably non-neurogenic since they were resistant to TTX, cold storage, capsaicin, and repeated stimulation. Furthermore, relaxations were almost completely abolished by increasing extracellular potassium to 40 mM or by adding tetraethylammonium chloride or 4-aminopyridine. Relaxations were also reduced by ouabain and potassium free conditions.

Muscle responses to transmural electrical field stimulation (TMS) and nitric oxide synthase (NOS) activity in the ileum of endotoxic rat

Muscle responses to transmural electrical field stimulation (TMS) and nitric oxide synthase (NOS) activity in the ileum of endotoxic rat

Neuronal release of adrenaline by transmural field stimulation of the Atlantic salmon (Salmo salar) cardiac atrium

Abstract 1. The atrial cardiac tissue of the Atlantic salmon was pre-incubated with 3 H-adrenaline before transmural electrical field stimulation at 8°C in vitro . 2. The total contractile tension response and the total 3 H-release, but not the net 3 H-release per pulse, increased in proportion to the stimulus frequency from 1 to 4 Hz. These responses were nearly abolished by guanethidine and counteracted by propranolol. 3. The results indicate that in vitro accumulated adrenaline was released from a neuronal pool activating atrial contractility via β-adrenoceptors, and are consistent with our hypothesis of a neuronal uptake of plasma adrenaline for re-use as neurotransmitter during and following periods of stress.