Modification of spontaneous contractility of smooth muscle preparations from the bovine abomasal antrum by serotonin receptor agonists

The effects of serotonin (5-hydroxytryptamine, 5-HT), a 5-HT2-receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-M-5-HT) and RS-67506, a 5-HT4 receptor partial agonist, on spontaneous contractility of bovine abomasal smooth muscle preparations were investigated in vitro. Preparations from the abomasal antrum of freshly slaughtered healthy dairy cows were cut parallel to the longitudinal fibres, suspended in isolated organ baths, and concentration-response curves were performed by cumulative application of the 5-HT receptor agonists. Blockade of 5-HT2-induced response was tested with atropine and hexamethonium. Serotonin evoked a significant increase in the area under curve (AUC), whilst the 5-HT2 receptor agonist alpha-M-5-HT significantly increased the AUC and resting tone (RT). RS-67506 induced a significant increase in AUC and RT and a significant decrease in the maximum force. The effect of alpha-M-5-HT was mediated by a muscarinic cholinergic pathway, as the effect of alpha-M-5-HT was inhibited in the presence of atropine but not hexamethonium. It is concluded that 5-HT2 and 5-HT4 receptors are present in the bovine abomasal antrum. Muscarinic receptors are involved in the increase in RT seen after 5-HT2 receptor stimulation.     

Central and peripheral serotonergic control of forestomach motility in sheep

Participation of tryptaminergic receptors in the control of forestomach motility was investigated in conscious sheep using strain-gauges and chronically implanted electrodes. Two hours after feeding the sheep, serotonin (5-HT) was infused into the jugular vein (i.v.), or the carotid artery (i.c.), or into the lateral cerebral ventricles (i.c.v.), over a 10-min period. An i.v. dose of 16 micrograms/kg/min abolished the cyclic propagated contractions throughout the forestomach, increased ruminoreticular tone, and induced simultaneous contractions of all the parts of the rumen. A dose of 1.6 micrograms/kg/min i.c. or i.v. 5-HT inhibited phasic contractions. The effects of 5-HT were blocked completely by i.c.v. administration of methysergide (20 micrograms/kg) and imipramine (200 micrograms/kg), and blocked partially by naloxone (25 micrograms/kg), but unaffected by atropine (50 micrograms/kg). The inhibitory effects of i.v. 5-HT were antagonized by methysergide (200 micrograms/kg, i.v.) but unaffected by imipramine (2 mg/kg, i.v.) and atropine (250 micrograms/kg, i.v.). Only the i.v. administration of methysergide blocked the inhibition induced by i.c. infusion (1.6 micrograms/kg/min) of 5-HT. It is suggested that 5-HT exhibits an inhibitory control on forestomach phasic contractions through hypothalamic and bulbar 5-HT receptors, and exerts peripheral excitatory effects on the tone of the rumen wall.     

Effects of tryptamine antagonists on the anaphylactic contractions of the bovine pulmonary smooth muscles

Calves were sensitized with horse plasma (H.P.), 0.2 ml/kg, i.v., and H.P. (0.2 ml/ kg) in Freund's complete adjuvant, s.c. The latter injection was repeated 1 week later and the animals were killed 10 days after the second injection. Spirally cut strips of pulmonary artery and vein and the trachealis muscle from the sensitized calves contracted to 5-hydroxytryptamine (5-HT) and specific antigen (horse plasma). Antigen-induced contractions of the pulmonary smooth muscles were significantly blocked (P<0.05) by the 5-HT antagonists, methysergide and ketanserin. The trachea, however, appeared less sensitive to the antagonists than the pulmonary vessels.
The results suggest that 5-HT participates in the pulmonary anaphylactic reactions of cattle and that ketanserin may be useful in suppressing bovine pulmonary hypersensitivities.     

Pharmacological evaluation of sheep lung strip

Isolated sheep lung parenchymal strips responded to histamine > carbachol > PGF_2a > 5-HT with contractions, and to isoproterenol (Isop), and to large doses of epinephrine (E), norepinephrine (NE) and phenylephrine (PE) with relaxations. PGF_2a-contracted lung strip responded to PGE₁ and PGE₂ with relaxation. The strips which were partially contracted to histamine, PGF_2a, 5-HT and carbachol also responded to isop, E and NE with relaxations. Histamine responses were not modified by metiamide (an H₂-receptor antagonist). Mepyramine and atropine selectively antagonized contractions to histamine and carbachol, respectively. After β-blockade with propranolol, lung strips responded to NE > E > PE > isop with contractions, which were inhibited or reversed by phentolamine and dibenzyline. It is concluded that H₁ receptors are present in sheep peripheral airway smooth muscles, and that a- and β-adrenoceptors mediate contraction and relaxation, respectively, in sheep lung strips.     

Equine coronary artery responds to 5-hydroxytryptamine with relaxation in vitro

Isolated equine coronary arteries responded to 5-hydroxytryptamine (5-HT) with relaxations in both endothelium-dependent and endothelium-independent mechanisms. Experiments were designed to characterize the 5-HT receptor sub type mediating these relaxations. Both 5-HT and alpha-methyl-5-HT (α-Me-5- HT; 5-HT₂ agonist) produced concentration-dependent relaxations in equine coronary arteries precontracted with a thromboxane A₂ derivative (0N011113). The degree of the maximal relaxation induced by α t-Me-5-HT was about one-half of that induced by 5-HT. In the coronary arteries without endothelium, α -Me-5-HT produced no relaxation, but 5-HT caused relaxation, which was inhibited by a 5-HT₁ antagonist (methysergide, mianserin and methiothepin), but was inhibited neither by ketanserin (5-HT₂ antagonist) nor by MDL72222 (5-HT₃ antagonist). In the coronary arteries with endothelium, however, the relaxation induced by α -Me-5-HT was inhibited by ketanserin, L-nitro-arginine (NO synthase inhibitor) and methylene blue (soluble guanylate cyclase inhibitor). These results suggest that the relaxation induced by 5-HT in equine coronary arteries depends mainly on the stimulation of both 5-HTi receptor subtype on smooth muscle cells directly, and 5-HT₂ receptor subtype on endothelial cells indirectly by liberating endothe-lium-derived NO.     

Responses of pulmonary vasculature of cattle to dopamine and apomorphine

Both dopamine and apomorphine caused concentration-dependent contractions of the bovine pulmonary artery from rest. Both of these compounds caused relaxation of histamine-precontracted arterial and venous strips after α-adrenoceptor blockade. Arterial contraction elicited by dopamine was inhibited either by phentolamine (α-blocker) or by the dopamine-selective antagonists, spiperone and butaclamol. Apomorphine in the highest concentration (<10^-5 M) inhibited dopamine-induced contractions. Dopamine- and apomorphine-induced vascular relaxations were attenuated by propranolol but not by spiperone or butaclamol. These data suggest that dopamine- and apomorphine-induced relaxation in these preparations is most likely mediated through β-adrenergic mechanisms, whereas dopamine-induced contractions seem to involve both α-adrenergic and dopa-minergic receptors.     

The vascular responses of the isolated perfused bovine external ear to exogenous histamine and 5-HT (serotonin)

The responses of the vasculature of isolated, non-sensitized, bovine external ears to histamine and serotonin (5-HT) were evaluated while they were being perfused with Krebs-Henseleit solution, Histamine (10^–5 mol/L to 5×10^–3 mol/L) and 5-hydroxytryptamine (5-HT) (10^–9 mol/L to 10^–2 mol/L) caused increased vascular resistance. Mepyramine (10^–7 mol/L), cimetidine (10^–5 mol/L) and atropine (10^–6 mol/L) inhibited the responses to histamine. The responses to 5-HT were inhibited by methysergide (10^–9 mol/L) and potentiated by morphine (10^–5 mol/L). These results suggest the presence of H₁ and H₂ histamine, and 5-HT receptors in bovine auricular vessels, all of which cause net vasoconstriction.     

Effect of bombesin and substance P on the smooth muscle of the chicken crop

The effect of bombesin and of substance P was investigated in smooth muscle strips of the chicken crop. Bombesin in picomolar concentration (0.1×10^-12–5×10^-12mol/l) caused a concentration-related contraction of the muscle strips. Substance P in nanomolar concentration (0.1×10^-9–10×10^-9mol/l) was effective in the same manner. Tetrodotoxin (2×10^-7mol/l) did not influence the contraction responses to either bombesin or substance P. The excitatory effect of bombesin and of substance P did not follow activation of cholinergic receptors since their effect on the crop smooth muscle was not antagonized by atropine (10^-4mol/l) or by hexamethonium (10^-4mol/l). Strips stored for 24 hours in the Tyrode's solution at 4°C without a supply of oxygen maintained their full sensitivity to bombesin and to substance P. These results are consistent with a direct action of bombesin and substance P on the crop smooth muscle.     

Distribution of methionine-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves in the forestomach of cattle

The distribution of methionine-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves in the forestomach of calves and cows was studied, using immunohistochemical methods. Methionine-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerves were found in all regions of the bovine forestomach. Cell bodies were found mainly in myenteric ganglia, and nerve fibers were found principally in muscular layers. Nerve fibers and ganglia were concentrated in the lips of the reticular groove, but were least numerous in ruminal and omasal walls. The reduced number of immunoreactive nerves in cows, compared with the number of those in calves, was striking, especially in the lips of the reticular groove and the omasal leaves. Results may indicate that the importance of methionine-enkephalin-Arg6-Gly7-Leu8-containing nerves in the physiologic regulation of the forestomach of the calf is different from that of the cow.     

Food intake and rumen motility in dwarf goats. Effects of some serotonin receptor agonists and antagonists

The serotonergic regulation of feeding behaviour has not so far been studied in ruminants. Therefore, the effects of some serotonin (5-HT) receptor agonists and antagonists on food intake and forestomach motility were studied in dwarf goats.Goats ate less food when treated intravenously (IV) with the 5-HT precursor 5-HTP (25 µg, 50 µg or 100 µg kg^–1 min^–1 over 15 min) than when they were treated with 5-HT (which does not pass the blood-brain barrier) or with saline. Accordingly, IV dexfenfluramine infusions (50 µg or 100 µg kg^–1 min^–1 over 15 min), which induces release of brain 5-HT, also led to dose-related reductions in food intake. In contrast, no anorectic effects were observed after IV infusions with the selective 5-HT reuptake inhibitor fluoxetine (100 µg kg^–1 min^–1 over 15 min), the selective 5-HT1A agonist 8-OH-DPAT (0.5 µg kg^–1 min^–1 over 15 min), or eltoprazine (4 or 8 µg kg^–1 min^–1 over 15 min), a mixed 5-HT1A/5HT1B receptor agonist. None of the 5-HT antagonists tested gave any increase in food consumption in this model. Interestingly, the non-selective 5-HT receptor antagonist methysergide (360 µg/kg IV) reduced food intake. This effect was most noticeable at 3 h after injection. The 5-HT3 receptor antagonist ondansetron (IV 10 µg kg^–1 min^–1 over 15 min) and the peripheral 5-HT2 receptor antagonist xylamidine (IV 100 µg kg^–1 min^–1 over 10 min) failed to modify food intake. These results provide evidence for central serotonergic involvement in the control of feeding. However, this control system differs markedly in goats and rodents.Dexfenfluramine, 5-HTP and eltoprazine administered at similar dose rates to those used in the food intake experiments induced some clinical signs including inhibition of forestomach contractions. These results, together with our earlierin vivo andin vitro observations, suggest that the inhibitory effects of serotonin receptor agonists on forestomach contractions are due to interactions with both peripheral and central serotonergic receptors. The change in smooth muscle tension, which leads to a change in the signals transmitted via vagal afferents to the central nervous system, appears not to modify feeding behaviour in dwarf goats.     

Selective blockade of the responses of reticuloruminal muscle to 5-HT in sheep

The effects of intravenous administration of 5-HT on the patterns of reticulo-rumen contractions were studied in conscious sheep. Contractions were measured by strain gauges applied to the external wall of the reticulum and the ventral and/or dorsal sac of the rumen. The responses to 5-HT comprised (i) a short-lived contraction followed by (ii) a sustained increase in muscle tone and (iii) a concomitant inhibition of the extrinsic reticulo-rumen contractions. The corresponding blockades produced by (i) atropine, (ii) 5-HT antagonists and (iii) chemical sympathectomy suggest involvement of a peripheral cholinergic mechanism in the initial contractile response and a central adrenergic mechanism in the reflex inhibition of extrinsic reticulo-rumen contractions.     

Effect of hypoxia/reoxygenation on the contractility of the isolated bovine digital vein

The bovine digital vasculature contractility has been implicated in the development of laminitis. To investigate the effect of hypoxia/reoxygenation on the contractility of isolated peripheral bovine digital veins (BDVs), vessel rings were studied under isometric conditions and submitted to 30 min of hypoxia (95%N(2)-5%CO(2)) and reoxygenation (95%O(2)-5%CO(2)) conditions, respectively. The BDVs contracted with a high K(+) depolarizing solution, developed hypoxia-induced relaxation, followed by an increase in tension upon reoxygenation. In contrast, phenylephrine-contracted BDVs displayed a rapid, sustained and reversible hypoxia-induced contraction. Reoxygenation caused a rapid relaxation in phenylephrine-contracted BDVs. The presence of the endothelium did not modify the hypoxia/reoxygenation effects and hypoxia-induced contraction was still observed in a nominal Ca(2+)-free Krebs, however, the last effect was not maintained over time. The hypoxia-induced contraction in an isolated peripheral vein may contribute to the understanding of the physiology and pathophysiology of superficial venous smooth muscle contractility, particularly in the alteration of bovine digital haemodynamics under hypoxia/reoxygenation conditions.     

Effect of intravenous and aerosol administration of 5-hydroxytryptamine on pulmonary function values in healthy calves.

Effects of IV and aerosol administration of 5-hydroxytryptamine (5-HT) on ventilation, pulmonary mechanics values, pulmonary arterial pressure, and heart rate were investigated in healthy unsedated Friesian calves. Minute volume increased significantly, mainly because of an increase in respiratory rate. Except for total pulmonary resistance after bolus injection, continuous administration of 5-HT given by either route caused significant alterations of lung dynamic compliance and total pulmonary resistance, the former decreasing to one-fifth of its baseline value and the latter increasing twofold. Pulmonary arterial pressure increased significantly, whatever the speed or route of administration. Administration of a bolus did not affect heart rate, whereas continuous IV administration of 5-HT as well by perfusion or by aerosol resulted in sustained tachycardia. It was concluded that 5-HT induces reversible bronchoconstriction and pulmonary vasoconstriction in healthy unsedated calves, 5-HT-induced functional alterations depend on the speed of administration, and excess of 5-HT production or depression in uptake by the lungs during bovine respiratory tract diseases could contribute to pulmonary dysfunction.     

PDE4 and PDE5 regulate cyclic nucleotide contents and relaxing effects on carbachol-induced contraction in the bovine abomasum

The effects of various selective phosphodiesterase (PDE) inhibitors on carbachol (CCh)-induced contraction in the bovine abomasum were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), vardenafil (type 5), BRL-50481 (type 7) and BAY73-6691 (type 9), inhibited CCh-induced contractions in a concentration-dependent manner. Among the PDE inhibitors, Ro20-1724 and vardenafil induced more relaxation than the other inhibitors based on the data for the IC₅₀ or maximum relaxation. In smooth muscle of the bovine abomasum, we showed the expression of PDE4B, 4C, 4D and 5 by RT-PCR analysis. In the presence of CCh, Ro20-1724 increased the cAMP content, but not the cGMP content. By contrast, vardenafil increased the cGMP content, but not the cAMP content. These results suggest that Ro20-1724-induced relaxation was correlated with cAMP and that vardenafil-induced relaxation was correlated with cGMP in the bovine abomasum. In conclusion, PDE4 and PDE5 are the enzymes involved in regulation of the relaxation associated with cAMP and cGMP, respectively, in the bovine abomasum.     

In vitro effects of 5-hydroxytryptamine and cisapride on the circular smooth muscle of the jejunum of horses

OBJECTIVE: To determine effects of cisapride and 5-hydroxytryptamine (5-HT) on the jejunum of horses. SAMPLE POPULATION: Jejunal muscle strips from 8 horses. PROCEDURE: Muscle strips were suspended in isolated muscle baths. Isometric stress responses to 5-HT and cisapride, with and without specific antagonists, were determined. RESULTS: Muscle strips incubated with atropine and tetrodotoxin responded to 5-HT and cisapride with an increase in contractile force. The 5-HT caused a concentration-dependent increase in contractile amplitude, with a maximum response (Emax) of 1,151+/-214 g/cm2 and a molar concentration that induces contractile force equal to 50% of maximum response (EC50) of 0.028+/-0.002 microM. Prior incubation with the 5-HT2 antagonist ketanserin decreased the Emax (626 +/-147 g/cm2) and potency (EC50, 0.307+/-0.105 microM) of 5-HT Prior incubation with the 5-HT3 antagonist tropisetron decreased the efficacy (Emax, 894+/-184 g/cm2) to 5-HT Cisapride also caused a concentration-dependent increase in contractile amplitude, with an Emax of 331+/-82 g/cm2 and an EC50 of 0.302+/-0.122 microM. Prior incubation with ketanserin decreased the Emax (55+/-17 g/cm2) and potency (EC50, 0.520+/-0.274 microM) of cisapride. CONCLUSION AND CLINICAL RELEVANCE: Stimulatory effects of 5-HT and cisapride on circular smooth muscle of equine jejunum are mediated primarily through a noncholinergic effect. The effects of 5-HT are mediated, at least partially, by 5-HT2 and 5-HT3 receptors, whereas the effects of cisapride are mediated primarily by 5-HT2 receptors. This may impact treatment of horses with postoperative ileus.     

Adrenergic, cholinergic, and nonadrenergic-noncholinergic intrinsic innervation of the jejunum in horses.

OBJECTIVE: To determine the major neurotransmitters that regulate contractile activity in the jejunum of horses. SAMPLE POPULATION: Jejunal specimens from 65 horses without gastrointestinal tract lesions. PROCEDURE: Jejunal smooth muscle strips, oriented in the plane of the circular or longitudinal muscular layer, were suspended isometrically in muscle baths. Neurotransmitter release was induced by electrical field stimulation (EFS) delivered at 30 and 70 V intensities and at various frequencies on muscle strips maintained at low or high muscle tone. To detect residual nonadrenergic-noncholinergic neurotransmission, the response of muscle to EFS in the presence of adrenergic and cholinergic blockade was compared with the response in the presence of tetrodotoxin. RESULTS: Atropine (ATR) decreased the contractile response of muscle strips to EFS under most conditions. However, ATR increased the contractile response of high-tone circular muscle. Adrenergic blockade generally increased the muscle responses to 30 V EFS and in high-tone longitudinal muscle but decreased contractile responses in high-tone circular muscle. Tetrodotoxin significantly altered the responses to EFS, compared with adrenergic and cholinergic receptor blockade. CONCLUSIONS: Acetylcholine and norepinephrine appear to be important neurotransmitters regulating smooth muscle contractility in the equine jejunum. They induce contraction and relaxation, respectively, in most muscle preparations, although they may cause opposite effects under certain conditions. In addition, nonadrenergic-noncholinergic excitatory and inhibitory influences were detected. CLINICAL RELEVANCE: Acetylcholine or norepinephrine release within the myenteric plexus of horses may alter gastrointestinal motility.     

Endothelium‐dependent relaxation mechanisms involve nitric oxide and prostanoids in the isolated bovine digital vein

Endothelial dysfunction contributes to the development of ungulate's laminitis. Although extensively studied in equines, the endothelial function is not fully examined in bovine digital veins (BDVs). BDVs were studied under isometric conditions to describe the acetylcholine (ACh) endothelium‐dependent relaxation. Concentration–response curves were constructed to phenylephrine, ACh, and sodium nitroprusside (SNP). Relaxation responses were evaluated using either phenylephrine or depolarizing high‐potassium Krebs solution (DKS) as precontraction agents. Endothelium denudation and incubation with L‐NAME (300 μM), indomethacin (10 μM) or both were used to explore endothelial‐mediated mechanisms. Endothelium denudation did not modify phenylephrine and SNP responses, however, significantly (p < 0.05) converted a relaxation (63.2 ± 5%) response to ACh into a contraction (30.3±9%). The ACh‐evoked relaxation was significantly (p < 0.05) reduced in the presence of indomethacin (37.5 ± 6%) and L‐NAME (6.40 ± 2%). The presence of both inhibitors abolished the ACh‐evoked relaxation. Although DKS caused a higher precontraction than phenylephrine, ACh‐evoked relaxation (22.4 ± 3.4%) was still observed and was reduced by the combination of inhibitors (7.0 ± 1.0%). The ACh endothelium‐dependent relaxation in BDVs is essentially mediated by nitric oxide and endothelium‐derived prostanoids. The BDV endothelium function is a dynamic component in the control of the bovine digital blood flow, particularly under endothelial dysfunction conditions when venoconstriction might lead to postcapillary resistance increase.     

Responses of goat ruminal musculature to substance P in vitro and in vivo

A concentration-response curve obtained for contraction of goat rumen strips indicated that substance P (SP) is a very potent spasmogenic agent. In vivo, SP caused a dose-dependent inhibition of the extrinsic rumen contractions possibly due to an increase in ruminal smooth muscle tone, resulting in reflex inhibition. SP did not induce significant changes in heart rate. Further studies are necessary to elucidate a possible physiological role of SP in the modulation of forestomach motility.     

Responses of goat ruminal musculature to substance P in vitro and in vivo

A concentration-response curve obtained for contraction of goat rumen strips indicated that substance P (SP) is a very potent spasmogenic agent. In vivo, SP caused a dose-dependent inhibition of the extrinsic rumen contractions possibly due to an increase in ruminal smooth muscle tone, resulting in reflex inhibition. SP did not induce significant changes in heart rate. Further studies are necessary to elucidate a possible physiological role of SP in the modulation of forestomach motility.     

Central and local actions of opioids upon reticulo-ruminal motility in sheep

The effects of opioids and naloxone on cyclical forestomach motility were determined in anaesthetized and conscious sheep. To assess central or peripheral opioid actions, differential routes of administration were used. Possible dynamic effects along the innervating vagovagal reflex arc were investigated electrophysiologically at the cervical level of the vagus nerve. Further, direct influences on the smooth muscle were evaluated in vitro on isolated longitudinal reticular strips. Additionally, the effects of some spasmogenic agents were studied for comparative purposes. In anaesthetized sheep, opioids depressed in an identical manner both the amplitude of spontaneous cyclical contractions and contractions evoked by electrical stimulation of the distal end of the cut cervical vagus. In conscious sheep, low doses of normorphine and loperamide inhibited frequency and amplitude centrally (20 micrograms/kg and 4 micrograms/kg via carotid artery respectively), whereas locally higher dose levels (200 micrograms/kg and 10 micrograms/kg via coeliac artery respectively) affected only the amplitude of cyclical contractions. Furthermore the opioid peptides Leu-, Met-enkephalin and [D-Ala2-Met5]-enkephalinamide preferentially depressed the amplitude of cyclical motility most efficiently if administrated via the coeliac artery. These results indicate the presence both of a central opioid action depressing frequency and amplitude and of a local opioid action depressing only the amplitude of cyclical reticulo-ruminal motility. Opioids did not alter the resting discharge of afferent tension units and similarly failed to modulate tone of reticular strips in vitro, suggesting that the opioids act locally on the intramural neuronal plexus, possibly by diminishing the output of excitatory transmitter. Whether substance P could play a role as a vagal excitatory transmitter besides the classically implicated acetylcholine has been discussed. The central opioid mechanism is probably not situated within the gastric centres but elsewhere in the brain. Naloxone (greater than or equal to 100 micrograms/kg, jugular vein) stimulated the frequency of cyclical ruminal motility only in well-defined experimental conditions, probably via a central mechanism.