Transmural Field Stimulation Research Articles

Pentobarbitone and skeletal muscle contractions: on the interaction with the effect elicited by the beta-adrenoceptor agonist, terbutaline.

The soleus, a slow-contracting muscle, and the extensor digitorum longus (EDL), a fast-contracting muscle from guinea-pig were prepared for isometric recording in vitro. Subtetanic contractions were evoked by transmural field-stimulation. Pentobarbitone increased the force of contraction in both muscles. In the soleus it shifted the stimulation frequency-response curve to the left. Terbutaline caused a decrease in the force of subtetanic contractions of the soleus, an effect which was dependent on the stimulation frequency. In the presence of pentobarbitone, the stimulation frequency had to be lowered by about 2 HZ in order to maintain the optimum response to terbutaline. The EDL responded to terbutaline with an increased force of contraction. In this case the stimulation frequency was less critical and the effects were the same in the presence and in the absence of pentobarbitone. Experiments with alpha-chloralose yielded results similar to those obtained with pentobarbitone.

Analysis of the beta-receptor mediated effect on slow-contracting skeletal muscle in vitro.

Subtetanic contractions of the guinea-pig isolated soleus, a slow-contracting skeletal muscle, were evoked by transmural field-stimulation. Isoprenaline caused a dose-dependent depression of the contractions. This effect was inhibited by propranolol and H 35/25 (1-(p-tolyl-2-isopropylamino-1-propanol) but not by practolol. Similar results were obtained for terbutaline. Tazolol and H 80/62 (1-isopropylamino-3-(p-hydroxyphenoxy)-2-propanol (HCl), selective beta1-agonists, had no effect per se but inhibited the effect of terbutaline. Adrenaline, noradrenaline, and dopamine all caused a dose-dependent decrease in the force of the soleus contractions, their potencies being in that order. Tyramine did not appreciably affect the contractions nor did it inhibit the effect of terbutaline. Pretreatment with reserpine, if anything, increased the response to terbutaline. It is concluded, in conformity with previous in vivo studies, that the adrenergic receptor mediating the effect on the soleus muscle contractions is of the beta2-type. Indirect sympathomimetic effects do not contribute to the responses observed on the isolated soleus muscle.

Effects of ginsenosides on vasodilator nerve actions in the rat perfused mesentery are mediated by nitric oxide.

This study was designed to explore the effect of ginsenosides, saponins from Panax ginseng, on the vasodilator nerve actions in the rat perfused mesentery and the mechanism of this effect. In the rat perfusion mesentery, when adrenergic nerves were blocked by guanethidine (5 x 10(-6) M) and vascular muscle tone was increased with methoxamine (5 x 10(-6)-10(-5) M), transmural field stimulation produced a frequency-dependent vasodilator response, which is due to the release of calcitonin gene-related peptide; ginsenosides significantly suppressed this vasodilator response in a concentration-dependent manner (3-30 micrograms mL-1). After pretreatment with saponin (50 micrograms mL-1, 3 min) to damage endothelial cells, this suppressing effect of ginsenosides was unaltered. However, the effect was abolished by N omega-nitro-L-arginine methyl ester (L-NAME) (10(-4) M), an inhibitor of nitric oxide synthesis and addition of L-arginine (3 x 10(-4) M) restored this suppressing effect. Methylene blue (10(-5) M), an inhibitor of guanylate cyclase, also abolished the suppressing effect of ginsenosides. However, ginsenosides did not alter the relaxation responses caused by exogenous calcitonin gene-related peptide administration. We conclude that ginsenosides can produce an inhibitory effect on the vasodilator response prejunctionally in the rat perfused mesentery and that this effect of ginsenosides may be mediated by nitric oxide released from non-adrenergic, non-cholinergic nerves.

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

Membrane properties and the neuro-effector transmission of smooth muscle cells in the canine internal anal sphincter.

The most distal part of the circular muscle layer functions as the internal anal sphincter, which constitutes a high pressure zone at rest, but maintains a relaxed state during defecation. To elucidate such sphincter mechanisms of the smooth muscle cells, the circular muscle layer in the canine anal canal was examined within 2 cm from the anal verge. Both the mechanical and intracellular electrical activities were recorded simultaneously. The examined region could be divided into three different regions according to the pattern of spontaneous activity and innervation and consisted of an upper region (20-15 mm from the anal verge), a transitional region (15-5 mm from the anal verge) and a lower region (within 5 mm from the anal verge), respectively. The spontaneous membrane activity was characterized by ongoing slow potential changes and each potential change was associated with a phasic contraction in the three regions. The mean frequencies of spontaneous electrical activity were 6.8, 15.9, and 24.1 c/min in the upper, transitional and lower regions, respectively. In the transitional and lower region, muscle tone generation was observed. Transmural field stimulation (0.4 msec in pulse duration) evoked membrane depolarization and contractions in the lower region. The application of an alfa adrenergic blocking agent completely suppressed the generation of excitatory responses, leaving a long lasting hyperpolarization associated with relaxation. In the transitional and upper region, stimulation consistently evoked membrane hyperpolarization with relaxation. The characteristics of this hyperpolarization response varied among the three regions. The total duration of hyperpolarization increased distally, while the time to peak hyperpolarization became decreases in a reverse direction. These regional differences in the characteristics of spontaneous membrane activity and innervation indicate that the transitional and lower region might therefore function as the internal anal sphincter.

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.

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.

Transmitter interactions in rabbit internal anal sphincter.

The aim of the present study was to investigate the relative importance of the different putative nonadrenergic noncholinergic (NANC) mediators and their interplay with cholinergic nerves in the rabbit internal anal sphincter (IAS). IAS preparations were mounted in organ baths for recording of isometric tension. Transmural field stimulation (TMS; 5-s trains; supramaximal voltage, 140-160 V; 0.4-ms impulse duration) was applied every 2 min with frequencies varying from 0.2 to 32 Hz. TMS induced frequency-dependent relaxations that amounted to 89.3 +/- 2.2% (n = 7). N omega-nitro-L-arginine (L-NNA; 10(-7)-10(-4) M; 8 Hz) reduced relaxations and this effect was partially inhibited by preincubation with L-arginine (10(-4) M). The effect of L-NNA was attenuated by atropine preincubation. Apamin (10(-6) M) shifted the frequency-response curve to the right but left maximal relaxations in response to TMS unaffected. In the presence of L-NNA (10(-4) M) and atropine (10(-6) M), the action (area between the frequency-response curve with or without a substance) of apamin was more pronounced, but, despite the presence of both L-NNA and apamin, some relaxation still remained. The frequency-response curve (control) was significantly shifted to the right by carbachol (10(-6) M). Concentration-response experiments showed that the response to exogenous nitric oxide (NO; 10(-7)-10(-4) M) was unaffected by carbachol (10(-6) M) preincubation, whereas responses to vasoactive intestinal polypeptide (VIP) and ATP were significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological identification of different inhibitory mediators involved in the innervation of the internal anal sphincter.

1. Inhibitory non-adrenergic, non-cholinergic (NANC) responses were studied in isolated strips from the rabbit internal anal sphincter. 2. In the presence of atropine and guanethidine, transmural field stimulation induced frequency-dependent relaxations that reached a plateau at frequencies > or = 4 Hz. 3. These relaxations were inhibited by apamin (10(-6) M) and by N omega-nitro-L-arginine (L-NOARG, 10(-4) M). With these two substances in combination, relaxations were still seen in response to field stimulation, but only at frequencies > 2 Hz. 4. In the presence of both apamin (10(-6) M) and L-NOARG (10(-4) M), responses at high frequencies consisted of a fast relaxation followed by a slow return to prestimulus tension level. alpha-Chrymotrypsin hastens the return of tension to prestimulus level after high frequency stimulation. 5. Zinc-protoporphyrin IX, an inhibitor of haeme oxygenase, had a significant inhibitory effect on relaxations induced by transmural field stimulation. It was found, however, that responses to sodium nitroprusside and to isoprenaline (both 10(-9)-10(-4) M) were reduced comparably, indicating that the effect of zinc-protoporphyrin IX was unspecific. 6. It is concluded that pharmacological analysis allows identification of at least three distinguishable components of the inhibitory NANC innervation of the rabbit internal anal sphincter. The study does not allow conclusions about the role of carbon monoxide, a recently proposed mediator of NANC responses in opossum internal anal sphincter.

Mechanical basis of ANP secretion in beating atria: atrial stroke volume and ECF translocation.

To investigate the mechanism by which an increase in pacing frequency or distension increases the secretion of atrial natriuretic peptide (ANP), the changes in atrial volume during contraction (atrial stroke volume), transmural transport of the extracellular fluid (ECF), and ANP secretion were quantified in the beating perfused rabbit atria. The atrium was stimulated by transmural field stimulation or by atrial distension induced by an increase in intraatrial pressure. Atrial stretch and incremental increases in pacing frequency up to 2 Hz activated the secretion of ANP coincident with an increase in atrial stroke volume and the transendocardial translocation of the ECF. These results showed positive relationships between changes in the secretion of ANP and the atrial stroke volume or the translocation of the ECF. The translocation of the ECF was also positively correlated with the change in atrial stroke volume. The accentuated secretion of ANP and translocation of the ECF waned at higher stimulating rates to show a peak value. Even under this condition, the secretion of ANP was a function of the translocation of the ECF. These data suggest that the increases in atrial stroke volume and translocation of ECF are fundamental factors in the ANP stimulation in response to atrial stretch and increases in atrial rate.

Effect of magnesium ions on rabbit detrusor contractility and intracellular free calcium.

Magnesium (Mg2+) is one of the most abundant ions in the body. In the human body, Mg2+ plays important roles including cofactors in many crucial enzyme systems, especially those involving energy transfer, storage and utilization. Alteration of the concentration of Mg2+ may cause neuromuscular hyperactivity, psychiatric disturbances, calcium/potassium abnormalities, and overactivity of cardiac muscle. Most information on the effect of Mg2+ on muscle contraction has been obtained from studies on cardiac, skeletal, and vascular muscle; much less is known about the effect of Mg2+ in other smooth muscle systems. In the current study, we investigated the effect of Mg2+ on the contraction and intracellular free calcium of rabbit urinary bladder detrusor muscle in response to carbachol and transmural field stimulation (FS). The results can be summarized as follows: (1) Reduction of the concentration of magnesium [Mg2+] from normal Tyrode's solution enhanced the spontaneous basal activity, whereas addition of Mg2+ gradually abolished this spontaneous activity. (2) Muscle contraction induced by FS or carbachol was enhanced in Mg(2+)-free Tyrode's solution. Addition of Mg2+ inhibited the response to both forms of stimulation in a dose-dependent manner. (3) Inhibitory effects of Mg2+ were potentiated when the Ca2+ concentration in the Tyrode's solution was reduced to 0.6 mM, whereas increasing the extracellular concentration of Ca2+ (5.4 mM) reduced the inhibitory effects of Mg2+. (4) Using FURA-2 to monitor intracellular free calcium simultaneous with contractile tension, we demonstrated that the alterations in the contractile responses observed at different concentrations of extracellular Mg2+ correlated with similar changes in intracellular free calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Effects of transmural field stimulation in isolated muscle strips from rabbit sphincter of Oddi and duodenum

The purpose of the study was to compare the effect of transmural field stimulation (TMS) on isolated smooth muscle strips from rabbit sphincter of Oddi (SO), duodenal circular layer (Dc) and duodenal longitudinal layer (D1). The strips were suspended in thermostatically controlled 5-ml organ baths containing Krebs solution constantly bubbled with 5% CO2 in O2. TMS was delivered through platinum electrodes (140 V, 0.4 ms, 5 s trains, 40 Hz). The TMS responses could be divided in two main responses: (1) contraction initiated after cessation of the stimulus train, preceded by an inhibitory phase during TMS ('off'); and (2) contraction initiated during TMS ('duration'). The 'duration' response was observed in one out of 20 strips in the SO and Dc compartments, whereas 11 D1 strips (55%) showed 'duration' responses (P < 0.001). Atropine (10(-6)) converted all 'duration' responses to an 'off' response preceded by an inhibitory phase during TMS and reduced the contractile amplitudes with 40-65%. L-NNA significantly increased the number of 'duration' responses in all types of muscle, and caused a 40% increase in D1 contractile amplitude. Inhibitory responses could not be removed by atropine, propranolol and phentolamine. The results suggest that the intrinsic innervation of SO and duodenal muscle consists of a mixture of excitatory, cholinergic and inhibitory NANC pathways. The latter may utilize, wholly or partly, NO or a related compound as transmitter. A relative dominance of excitatory, cholinergic responses was present in the D1 strips, whereas inhibitory responses were dominating in the SO and Dc strips.

Functional relation between nitric oxide and noradrenaline for the modulation of vascular tone in rat mesenteric vasculature

As previously reported, N omega-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide (NO) synthesis, decreased transmural field stimulation (TFS)-induced noradrenaline overflow from the isolated perfused rat mesenteric vasculature attached to the intestine. The decrease was attenuated by L-arginine. This suggests that NO may increase noradrenaline release (Yamamoto et al. 1993). The present experiments with this preparation were done in order to monitor changes in vascular perfusion pressure caused by TFS or by noradrenaline infusion in parallel with those in the noradrenaline outflow caused by TFS in the presence of atropine (0.1 mumol/l) (to block acetylcholine-induced release of endothelial NO) and of indomethacin (3 mumol/l) (to inhibit L-NNA-induced production of vasoconstrictor prostanoids). (1) TFS (2-10 Hz) caused a frequency-dependent increase in noradrenaline overflow and perfusion pressure. (2) L-NNA (10 and 30 mumol/l) caused a concentration-dependent inhibition of TFS-induced noradrenaline overflow, whereas the TFS-induced pressure increase was augmented by L-NNA in a concentration-dependent manner. At any given concentration of L-NNA, the potentiation of vasoconstriction by L-NNA became greater in magnitude as the frequency of the TFS was raised. (3) Infusion of noradrenaline (0.38-6 nmol) caused a dose-dependent increase in perfusion pressure up to a value comparable with that caused by TFS. The pressure increase in response to noradrenaline infusion was also enhanced by L-NNA, relatively, to a greater extent than the enhancement, by L-NNA, of the pressure response to TFS.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide is involved in muscle relaxation but not in changes in short-circuit current in rat ileum.

L-Arginine (L-Arg)-nitric oxide (NO) pathways in rat ileum were studied in an Ussing chamber modified so that a strain gauge transducer could be attached longitudinally on the serosal side of the intestine. Ileal segments from 22 rats were mounted as flat sheets and voltage clamped at zero transmural potential (PD). Changes in short-circuit current (delta ISC) in the absence of carbachol and longitudinal muscle relaxations in the presence of carbachol in response to transmural field stimulation (TMS; 5-s trains of impulses, 0.4-ms impulse duration, 1-10 Hz) were recorded during a control period, in the presence of N omega-nitro-L-arginine (L-NNA; 10(-4) M), and in the presence of L-Arg after treatment with L-NNA. In the control period, the delta ISC and muscle relaxation were frequency dependent with maximal responses generated at a frequency of 10 Hz. Tetrodotoxin (5 x 10(-6) M) blocked muscle relaxation and decreased delta ISC by 94% during TMS at 10 Hz. L-NNA blocked the muscle relaxation induced by TMS but failed to alter delta ISC. Muscle relaxation to TMS was restored dose dependently by L-Arg. In segments from another group of eight rats, saturated NO solutions relaxed the muscle but failed to change ISC either in the presence or absence of carbachol. These results support a role for NO as a neurotransmitter mediating relaxation of ileal smooth muscle but not mediating changes in epithelial ISC.

P-543 - Involvement of nitric oxide (NO) in cooling-induced decrease in transmural field stimulation (TFS)-evoked noradrenaline (NA) overflow in isolated rat vasculature.

P-543 - Involvement of nitric oxide (NO) in cooling-induced decrease in transmural field stimulation (TFS)-evoked noradrenaline (NA) overflow in isolated rat vasculature.

Effects of dicentrine, a novel α1-adrenoceptor antagonist, on human hyperplastic prostates

The effects of dicentrine, an α 1-adrenoceptor antagonist, on human hyperplastic prostates were investigated by radioligand binding and in vitro isometric tension experiments. In human hyperplastic prostates, α 1-adrenoceptors were characterized by a binding assay using [ 3H]prazosin as a radioligand. Specific [ 3H]prazosin binding was saturable and of high affinity ( K d = 0.2 ± 0.02nM) with a maximal number of binding sites ( B max = 55.2 ± 3.2 fmol/mg protein). α-Adrenoceptor antagonists competed with [ 3H]prazosin for binding in the order: dicentrine > phentolamine > rauwolscine. Norepinephrine (0.3–100 μM) or phenylphrine (1–300 μM) produced gradual contractions of human hyperplastic prostates. The concentration-response curve of norepinephrine or phenylephrine was shifted in parallel to the right by dicentrine, consistent with a competitive blockade. The pA 2 values of dicentrine against norepinephrine and phenylephrine were 8.04 ± 0.09 and 8.33 ± 0.11, respectively. These experiments were conducted to confirm that there was no interaction between α 1- and α 2-adrenoceptors in the tissue. Rauwolscine (1 μM) caused 1.7-fold, while dicentrine (0.1 μM) caused 15.8-fold shift of norepinephrine-induced contraction of human hyperplastic prostates. Combination of rauwolscine with dicentrine caused 17.8-fold shift of norepinephrine-induced prostatic tissue contraction. The contractile response to transmural field stimulation was abolished by pretreatment with tetrodotoxin, and suppressed concentration dependently by dicentrine or prazosin, whereas rauwolscine had little effect. It is concluded that dicentrine inhibits human hyperplastic prostate contractions in response to exogenous and endogenous adrenergic stimulation. Dicentrine may thus hold potential to relieve bladder outlet obstruction caused by benign prostatic hyperplasia via α 1-adrenoceptor blockade.

Evidence for functional dissociation of dependence and tolerance in guinea‐pig isolated ileal segments following 20 hour exposure to morphine in vitro

1. In the present study we have examined the relationship between tolerance and dependence in isolated ileal segments from the guinea-pig under three different conditions: fresh preparations not previously exposed to morphine (fresh/morphine naive); preparations stored overnight at 4 degrees C in modified Krebs-Henseleit saline (overnight-stored/morphine-naive); preparations stored overnight at 4 degrees C in Krebs-Henseleit saline containing 10 microM morphine and extensively washed with modified Krebs-Henseleit saline to remove residual morphine (overnight-stored/morphine-exposed). 2. Morphine produced a concentration-dependent inhibition of the response of ileal segment to 0.1 Hz, 1 ms and 10 V transmural field stimulation in fresh/morphine-naive, overnight-stored/morphine-naive and overnight-stored/morphine-exposed preparations. The maximum effect observed was similar in all three preparations-approximately 80% inhibition. Although, morphine was significantly more potent in the fresh/morphine-naive preparations (pD2 6.72 +/- 0.05, n = 8) than either the overnight-stored/morphine-native (pD2 6.42 +/- 0.11, n = 8) or the overnight-stored/morphine-exposed (pD2 6.44 +/- 0.14, n = 8), there was no significant difference between the overnight exposure to ileal segments to 10 microM morphine at 4 degrees C failed to induce tolerance to morphine. 3. The mu opiate receptor antagonist, naloxone (10 microM), produced contractions in both fresh/morphine-naive and overnight-stored/morphine-naive ileal segments following acute exposure to 10 microM morphine. Naloxone (10 microM) also produced contractions in 2/9 fresh/morphine-naive, 1/9 overnight-stored/morphine-naive and 7/9 overnight-stored/morphine-exposed preparations in the absence of morphine. The greater incidence of naloxone-induced contractions in overnight-stored/morphine-exposed preparations,suggests that dependence in this model is the product of adaptive changes that outlive the presence of morphine.4. The selective alpha2-adrenoceptor agonists, clonidine (0.3 microM) and 5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate (UK-14304, 1 microM), inhibited naloxone-induced contractions in overnight-stored/morphine-exposed preparations of ileal segments (n = 4 preparations for each agonist), suggesting that the response is due to transmitter release from the myenteric plexus.5. The findings in the present study indicate that tolerance and dependence to morphine in ileal segments of the guinea-pig can be functionally dissociated by overnight exposure to morphine at 4 degrees C.The development of tolerance to morphine, unlike dependence, appears to be a temperature-dependent process. This also raises the possibility that naloxone possesses intrinsic negative agonism at morphine sensitive receptors, which is manifested as a functional response only after adaptive changes in the myenteric plexus following exposure to morphine.

The role of the L‐arginine/nitric oxide pathway for relaxation of the human lower oesophageal sphincter

Smooth muscle specimens were taken from the oesophagogastric junction (OGJ) in patients operated on for gastrointestinal malignancies not involving the OGJ. The smooth muscle bundles of the inner, circular layer of the OGJ were richly innervated by fine nerve fibres staining positively for NADPH diaphorase. The outer longitudinal layer had a markedly lower number of NADPH-diaphorase positive nerve fibres. When the preparations were suspended in organ baths for recording of isometric tension, they developed active tension. Transmural field stimulation (TMS) induced frequency-dependent relaxations, which were abolished by NG-nitro-L-arginine (L-NNA; 10(-4) M), and were often converted to atropine-sensitive contractions. The effect of L-NNA was concentration-dependent, and the concentration-response curve for L-NNA was shifted to the right by L-arginine pre-incubation. The enantiomer NG-nitro-D-arginine (10(-4) M) also showed inhibitory actions on the responses to TMS, but significantly less than L-NNA. Relaxant responses to vasoactive intestinal polypeptide (VIP), forskolin, and sodium nitroprusside were unaffected by L-NNA pre-incubation. Exposure to a 124 mM K+ solution resulted in a biphasic relaxation of the preparations. This relaxation was not seen in preparations treated with scorpion venom (20 micrograms ml-1) or L-NNA (10(-4) M). Instead, a contractile response to 124 mM K+ solution was found. The results suggest that NANC responses to electrical stimulation of nerves in the human OGJ are mediated by a product generated from L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.