Transmural Stimulation Research Articles

Targeting IL-5 pathway against airway hyperresponsiveness: A comparison between benralizumab and mepolizumab.

Airway hyperresponsiveness (AHR) is a central abnormality in asthma. IL-5 may modulate AHR in animal models of asthma, but the available data is inconsistent on the impact of targeting IL-5 pathway against AHR. The difference between targeting IL-5 or the IL-5 receptor, α subunit (IL-5Rα) in modulating AHR remains to be investigated in human airways. The aim of this study was to compare the role of the anti-IL-5Rα benralizumab and the anti-IL-5 mepolizumab against AHR and to assess whether these agents influence the levels of cAMP. Passively sensitized human airways were treated with benralizumab and mepolizumab. The primary endpoint was the inhibition of AHR to histamine. The secondary endpoints were the protective effect against AHR to parasympathetic activation and mechanical stress, and the tissue modulation of cAMP. Benralizumab and mepolizumab significantly inhibited the AHR to histamine (maximal effect -134.14 ± 14.93% and -108.29 ± 32.16%, respectively), with benralizumab being 0.73 ± 0.10 logarithm significantly more potent than mepolizumab. Benralizumab and mepolizumab significantly inhibited the AHR to transmural stimulation and mechanical stress. Benralizumab was 0.45 ± 0.16 logarithm significantly more potent than mepolizumab against AHR to parasympathetic activation. The effect of these agents was significantly correlated with increased levels of cAMP. Targeting the IL-5/IL-5Rα axis is an effective strategy to prevent the AHR. Benralizumab was more potent than the mepolizumab and the concentration-dependent beneficial effects of both these monoclonal antibodies were related to improved levels of cAMP in hyperresponsive airways.

Perivascular Adipose Tissue Modulation of Neurogenic Vasorelaxation of Rat Mesenteric Arteries.

Perivascular sympathetic-sensory interactions have been shown to regulate calcitonin gene-related peptide (CGRP)-mediated vasodilation in rats. We investigated whether perivascular adipose tissue (PVAT) modulates the neurogenic vasorelaxation of isolated rat mesenteric arteries. Mesenteric arterial rings were prepared with or without PVAT (PVAT+ or PVAT-) and with either an intact or denuded endothelium (EC+ or EC-). The results of myography analysis revealed that vasocontraction to phenylephrine was highest in EC-PVAT-, lowest in EC+PVAT+, and intermediate in EC-PVAT+ and EC+PVAT-. Transmural nerve stimulation (TNS) induced the tetrodotoxin-sensitive relaxation of the phenylephrine-precontracted mesenteric arteries. However, nicotine induced minor relaxation in EC-PVAT+, whereas vasorelaxation was significantly enhanced in EC-PVAT-. Nicotine-induced vasorelaxation was insensitive to propranolol and also significantly lower in sympathetically-denervated and guanethidine-treated EC-PVAT-, whereas TNS-induced vasorelaxation persisted. In EC-PVAT- depleted of CGRP via capsaicin, nicotine- and TNS-induced vasorelaxation was almost absent. Lowering the pH of Krebs' solution using HCl led to pH-dependent vasorelaxation that was sensitive to CGRP8-37. Furthermore, nicotine-induced relaxation of EC-PVAT-, which was not affected by leptin, was blocked by methyl palmitate. Methyl palmitate did not affect TNS- or HCl-induced vasorelaxation. These results suggest that PVAT plays a modulatory role in regulating sympathetic-sensory interaction-mediated CGRPergic vasorelaxation via the release of methyl palmitate.

Pharmacological characterization of the interaction between tiotropium bromide and olodaterol on human bronchi and small airways

Combining a long-acting β2-agonist (LABA) with a long-acting muscarinic antagonist (LAMA) is the cornerstone to treat patients with chronic obstructive pulmonary disease (COPD). In this study we have characterized the interaction between the LAMA tiotropium bromide, and the LABA olodaterol, on the contractile tone of human medium bronchi and small airways. The response to a combination of tiotropium bromide and olodaterol was assessed at sub-maximal contractile tone induced by carbachol. The duration of action was studied in tissue contracted by transmural stimulation. Relaxation of bronchial tone was expressed as % of maximal response to papaverine. Drug interactions were analyzed by the Bliss Independence method and Unified Theory. Tiotropium bromide/olodaterol combination induced a significant synergistic relaxant response (P < 0.05 vs. expected additive effect) in medium bronchi and small airways pre-contracted by carbachol, by enhancing relaxation +22.13 ± 4.42% and +26.31 ± 12.39%, respectively. The combination of tiotropium bromide and olodaterol also reduced the airway smooth muscle contractility elicited by transmural stimulation by 73.60 ± 3.10%. The extent of synergy was strong to very strong, and was supported by the release of neuronal acetylcholine, cyclic adenosine monophosphate levels, and activation of iberiotoxin-sensitive KCa++ channels. Conversely, the interaction between tiotropium bromide and olodaterl was independent of the activity at M2 muscarinic receptors. These results indicate that tiotropium bromide/olodaterol combination leads to a potent and durable synergistic relaxation of human medium bronchi and small airways. Further pharmacological studies are needed to confirm these results in clinical settings.

Glucagon-Like Peptide 1 Receptor: A Novel Pharmacological Target for Treating Human Bronchial Hyperresponsiveness.

Asthma is associated with several comorbidities, such as type 2 diabetes mellitus, which may lead to bronchial hyperresponsiveness (BHR). Because glucagon-like peptide (GLP) 1 regulates glucose homeostasis, we pharmacologically investigated the influence of the GLP1 receptor (GLP1-R) agonist, exendin-4, on BHR induced in human isolated airways. The effect of exendin-4 was assessed in human isolated airways undergoing overnight passive sensitization and high-glucose stimulation, two conditions mimicking ex vivo the BHR typical of patients with asthma and diabetes, respectively. GLP1-R activation modulated the bronchial contractile tone induced by transmural stimulation (maximum effect -56.7 ± 3.6%; onset of action, 28.2 ± 4.4 min). Exendin-4 prevented BHR induced by both high-glucose stimulation and passive sensitization (-37.8 ± 7.5% and -74.9 ± 3.9%, P < 0.05 versus control, respectively) through selective activation of GLP1-R and in an epithelium-independent manner. The cAMP-dependent protein kinase A inhibitor, KT5720, reduced the protective role of exendin-4 (P > 0.05 versus passively sensitized tissues). The GLP1-R stimulation by overnight incubation with exendin-4 induced the overexpression of adenylyl cyclase isoform V (+48.4 ± 1.3%, P < 0.05 versus passively sensitized tissues) and restored the cAMP levels depleted by this procedure (+330.8 ± 63.3%, P < 0.05 versus passively sensitized tissues). In conclusion, GLP1-R may represent a novel target for treating BHR by activating the cAMP-dependent protein kinase A pathway in human airways, and GLP1-R agonists could be used as a "new" class to treat patients with asthma and patients with type 2 diabetes mellitus with BHR.

Cardiac autonomic innervation of the western pygmy possum (Cercatetus concinnus) and golden bandicoot (Isoodon auratus)

Evidence for a functional ventricular parasympathetic innervation of the mammalian heart between and within taxa remains controversial. We have previously proposed that the presence of a functional parasympathetic innervation of the ventricle was indicative of heterothermy, and is essential for maintaining ventricular stability at low body temperature. However, it is possible that the presence of such an innervation is also representative of the primitive mammalian state. In this study, we aimed to determine whether a functional parasympathetic innervation of the ventricle, that is capable of actively reducing the force of contraction, is present across metatherian mammals. Using in vitro isolated cardiac preparations, we examined evidence for a functional ventricular parasympathetic innervation of the ventricle in two species of metatherian mammal, one heterotherm (Western pygmy possum; Cercatetus concinnus) and one homeotherm (Golden bandicoot; Isoodon auratus), from different families to complement existing data from a heterothermic dasyurid. Both C. concinnus and I. auratus had a potent biphasic response to transmural electrical stimulation in both atrial and ventricular preparations. Both the decrease and increase in the force of contraction in response to stimulation were almost entirely blocked by the cholinergic and adrenergic antagonists, atropine and propranolol, respectively. These observations provide clear evidence for a parasympathetic innervation of the ventricle that is capable of directly influencing the force of contraction across metatherian mammals with different thermoregulatory strategies. While this innervation may facilitate heterothermy, this suggests that the presence of such an innervation pattern is indicative of the primitive mammalian state.

Pharmacological assessment of the onset of action of aclidinium and glycopyrronium versus tiotropium in COPD patients and human isolated bronchi

Preclinical studies suggested that aclidinium and glycopyrronium might have a faster onset of action than tiotropium. In this study we assessed the onset of action of aclidinium and glycopyrronium versus tiotropium, all administered at the approved clinical doses, in patients with moderate-to-severe chronic obstructive pulmonary disease (COPD) and in human isolated bronchi by using different concentrations. Sixteen COPD patients inhaled single doses of aclidinium 400µg, glycopyrronium 50μg and tiotropium 18µg and FEV1 was measured to assess their onset of action. In human isolated bronchi the time to evoke half maximal relaxation of transmural stimulation was tested from 10nM to 1µM for each drug. Nine, eight and twelve patients did not achieve 15% increase of FEV1 after inhalation of aclidinium, glycopyrronium and tiotropium, respectively. Aclidinium (15.6±7.5min) and glycopyrronium (17.9±10.4min) enhanced 15% FEV1 more rapidly than tiotropium (42.5±19.4min), with no significant difference (P>0.05). In isolated airways, glycopyrronium elicited a dose-dependent onset of action (10nM: 8.2±1.3min, 100nM: 7.1±2.1min, 1μM: 3.4±0.4min) that was faster compared to that induced by aclidinium (1μM: 6.4±0.5min) and tiotropium (1μM: 8.4±1.1min) (P<0.05), that halved the contractile tone only at the highest concentration. Bronchodilation induced by aclidinium and glycopyrronium was faster than that induced by tiotropium, but since our analysis was restricted to the acute effect of these LAMAs and the inhaled doses were not isoeffective, the real differences in their impact on the onset of bronchodilation will be definitely determined after a long-term challenge of these treatments at isoeffective doses in COPD patients.

Functional properties of submucosal venules in the rat stomach.

Venules in the stomach may have intrinsic properties for maintaining active microcirculation drainage even during gastric filling. Properties of spontaneous and nerve-mediated activity of submucosal venules in the rat stomach were investigated. Changes in vasodiameter and intracellular Ca(2+) in venular smooth muscle cells (SMCs) were monitored by video tracking and Fluo-8 Ca(2+) imaging, respectively. Venular SMCs developed synchronous spontaneous Ca(2+) transients and corresponding rhythmic constrictions of the venules. Nominally Ca(2+)-free solution or an L-type Ca(2+) channel blocker (1 μM nifedipine) disrupted the Ca(2+) transient synchrony and abolished spontaneous constrictions. Spontaneous constrictions were also prevented by inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase (10 μM cyclopiazonic acid (CPA)), IP3 receptors (100 μM 2-APB) or Ca(2+)-activated Cl(-) channels (100 μM niflumic acid). Transmural nerve stimulation (TNS) induced a long-lasting venular constriction that was abolished by α-adrenoceptor antagonist (1 μM phentolamine), while TNS evoked a sympathetic transient constriction of arterioles that was abolished by a combination of phentolamine and a P2 purinoceptor antagonist (10 μM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS)). Consistently, P2X1 purinoceptor immunoreactivity was detected in arteriolar but not venular SMCs. Primary afferent nerve stimulation (300 nM capsaicin) caused a venular dilatation by releasing calcitonin gene-related peptide. Thus, Ca(2+) release from the sarcoplasmic reticulum may play a fundamental role in the generation of spontaneous Ca(2+) transients, while electrical coupling amongst venular SMCs via L-type Ca(2+) channel activation appears to be critical for Ca(2+) transient synchrony as well as spontaneous contractions. Sympathetic venular constrictions appear to be exclusively mediated by noradrenaline due to the lack of P2X1 receptor in venular SMCs.

High Glucose Enhances Responsiveness of Human Airways Smooth Muscle via the Rho/ROCK Pathway

Glucose moves into airway secretions after a glucose load. Therefore people with diabetes or hyperglycemia spend a significant proportion of each day with glucose in their airways secretions. This study investigated the effects of glucose on isolated human airways and on cultured airway smooth muscle (ASM) cells. Human isolated bronchi were stimulated with acetylcholine, histamine, and transmural stimulation and treated with the selective ROCK inhibitors Y27632 and SB772077B under high-glucose conditions. The effect of high glucose concentrations on intracellular calcium flux and the phosphorylation of MYPT1 in ASM cells was also investigated. High (44 mM for 6 h) glucose, but not mannitol, concentrations led to an enhanced responsiveness of ASM to contractile agents. Y27632 and SB772077B completely abolished (P < 0.05) the enhanced contractile effects with a high-concentration glucose solution, compared with control tissues. In cultured ASM cells, incubation with high glucose concentrations significantly (P < 0.05) enhanced bradykinin-induced intracellular calcium flux and the levels of pMYPT1, which were inhibited by Y27632 (P < 0.05). Our study has demonstrated that high glucose concentrations leads to hyperresponsiveness of human isolated bronchi and enhances intracellular calcium release in cultured ASM cells via a Rho/ROCK- and pMYPT1-dependent pathway, suggesting that this crucial pathway may contribute to the reduced lung function observed in patients with diabetes. These data propose novel targets for the treatment of patients with respiratory diseases that also suffer from diabetes mellitus.

Potentiation of the responses of the vas deferens of the guinea-pig to transmural stimulation and to noradrenaline, by triethylcholine and tetraethylammonium.

Potentiation of the responses of the vas deferens of the guinea-pig to transmural stimulation and to noradrenaline, by triethylcholine and tetraethylammonium.

Effect of cocaine on tritium overflow evoked from vasa deferentia previously loaded with [3H]noradrenaline by stimulation using different types of electrode.

In mouse and guinea-pig vasa deferentia previously incubated with [3H]noradrenaline, electrical stimulation applied through parallel electrodes (transmurally) increased overflow of tritium 2- to 5-fold above the resting value. Electrical stimulation applied using methods involving more substantial conduction of nerve impulses in neuronal elements in the tissues evoked a tritium overflow which was smaller (70%) than that evoked by transmural stimulation. Cinchocaine (25 microM), tetrodotoxin (0.5 microM) or the absence of calcium effectively abolished evoked overflow in both tissues whichever method of stimulation was used. In mouse vas deferens, cocaine (10 microM) did not alter overflow evoked by either transmural or axonal stimulation while 100 microM produced a reduction. In guinea-pig vas deferens, cocaine (10 microM) produced a statistically significant increase in evoked overflow of about 50% or more with both transmural and axonal stimulation. As in mouse vas deferens, 100 microM cocaine produced a reduction. It is concluded that the action of cocaine is independent of these methods of stimulation and that some difference in the arrangement of the noradrenergic nerves in the two species may account for the differential effect of cocaine observed.

Some effects of pilocarpine and acetylcholine on the responses of the guinea-pig vas deferens to hypogastric nerve stimulation and transmural stimulation and applied (–)-noradrenaline

Enhancement by acetylcholine and by pilocarpine of the contractile responses of the guinea-pig isolated vas deferens to hypogastric nerve and transmural stimulation and to exogenous (minus)-noradrenaline was examined. At high concentration (600 nM) acetylcholine enhanced responses in the order:hypogastric nerve greater than transmural greater than exogenous (minus)-noradrenaline. Pilocarpine produced a similar maximal degree of enchancement with all three stimuli. All these effects were reversibly inhibited by 1-4 mum atropine, a concentration which alone did not affected responses to the stimuli. Pilocarpine (50-400 muM) gave a concentration-related inhibition of the enhancement of responses to hypogastric nerve stimulation by 600 nM acetylcholine.

Acetylcholine in extracts and perfusates of urinary bladder.

Abstract Tissue extracts of the urinary bladder of the rat, cat and guinea-pig contain an acetylcholine-like substance which is considered to be stored predominantly in post-ganglionic nerve fibres. An acetylcholine-like substance was liberated into the fluid bathing the isolated bladder of the guinea-pig and its output was increased 200-fold after transmural stimulation. The acetylcholine-like substance, both from bladder extracts (rat, cat and guinea-pig) and perfusates (rat), was found at the same Rf value as acetylcholine chloride when separated by paper chromatography in three solvent systems. It is concluded that the substance was acetylcholine.

Alphaxalone reveals GABAA receptor-mediated inhibition in guinea-pig ileum

Contractions of the transmurally stimulated guinea-pig ileum were not affected by 10 microM bicuculline, a competitive GABAA receptor antagonist. The GABAA potentiator alphaxalone depressed the responses to 2 and 20 Hz stimulation by 20% and 40%, respectively, but had no effect on 0.2 Hz responses. The depressions were prevented by 10 microM bicuculline. This suggests that endogenous activation of GABAA receptors during transmural stimulation was normally subthreshold but, when pharmacologically potentiated, inhibitory effects were seen.

A NOTE ON THE ELECTRICALLY TRANSMURALLY STIMULATED ISOLATED TRACHEA OF THE GUINEA-PIG.

Abstract The isolated trachea of the guinea-pig was stimulated electrically transmurally to give a biphasic response. The initial contraction was abolished by atropine and was rapidly succeeded by relaxation which was blocked by low concentrations of dichloro-isoprenaline. The relaxation was also blocked by procaine, guanethidine, or by previous treatment of the animal with reserpine, but not with hexamethonium. It was concluded that the effects of transmural stimulation were mediated by post-ganglionic adrenergic nerves innervating beta receptors.

The actions of atropine, tropenziline and N-butyl hyoscine bromide on the isolated distal colon of the guinea-pig: a comparison of their activities and mechanisms of action.

Abstract In the guinea-pig isolated distal colon, the order of anticholinergic activity is as follows: atropine &amp;gt; tropenziline bromide &amp;gt; N-butyl hyoscine bromide. The reduction in the responses to pelvic and transmural stimulation produced by tropenziline and N-butyl hyoscine bromide is due partly to their ganglion blocking activity. This effect also explains the reduction they cause in acetylcholine output on pelvic nerve and transmural stimulation. Since atropine also reduces acetylcholine release on pelvic nerve stimulation, it is suggested that muscarinic receptors of the parasympathetic ganglia are involved in transmission of pelvic nerve impulses.

Pharmacodynamic effects of dehydroemetine.

Abstract The pharmacodynamics of dehydroemetine were studied in isolated preparations and in whole animals. Dehydroemetine had no effect on the guinea-pig isolated ileum but non-specifically antagonized the spasmogenic effect of histamine, acetylcholine and nicotine on this tissue. Dehydroemetine in small concentrations inhibited relaxations of the guinea-pig and rabbit ileum produced by stimulating the sympathetic nerve without inhibiting the effect of noradrenaline. Twitches of the guinea-pig ileum elicited by transmural stimulation were blocked by dehydroemetine while higher doses of the drug also inhibited the indirectly elicited contractions of the isolated rat diaphragm. In vivo, neuromuscular blocking action could not be demonstrated for dehydroemetine while toxic doses were required to demonstrate the presence of an adrenergic neuron blocking action. Dehydroemetine had no anaesthetic action when applied locally to the rabbit cornea. Concentrations of the drug which caused local anaesthesia when injected intradermally into guinea-pigs also caused skin necrosis. It is concluded that dehydroemetine lacks any specific adrenergic neuron blocking action or neuromuscular blocking action or local anaesthetic action. It is suggested that the effects demonstrated are due to non-specific action at the nerve endings.

The effect of morphine pretreatment on the sensitivity of mouse and guinea-pig ileum to acetylcholine and to morphine.

Abstract Guinea-pigs and mice were subjected to morphine pretreatment for at least 14 days culminating in subcutaneous injections of 1 g/kg daily in 2 divided doses. In mice, tolerance to the effects of morphine on gastrointestinal propulsion had developed by the end of the pretreatment course. The sensitivity to acetylcholine of the ilia from pretreated animals and the effects of morphine on transmural stimulation were compared with the ilia from normal animals. Ilia from pretreated guinea-pigs were marginally more sensitive to frequency of stimulation but significantly less sensitive to the depressant effect of morphine than ilia from normal animals. There was no significant difference in the sensitivity to acetylcholine. There was a highly significant decrease in sensitivity to morphine of ilia from pretreated mice, but this also was not associated with any alteration in sensitivity to acetylcholine. It is concluded that on the isolated ileum preparation the development of tolerance to morphine is not associated with an increased sensitivity to the transmitter whose release morphine inhibits.

Effects of four cholinesterase inhibitors on the response of the guinea-pig isolated ileum to transmural electrical stimulation.

Effects of four cholinesterase inhibitors on the response of the guinea-pig isolated ileum to transmural electrical stimulation.

Evidence that sensory neurons participate in the non-cholinergic, non-adrenergic contractile response of the guinea-pig ileum

The possible role of non-cholinergic, non-adrenergic (NCNA) nerves in responses of the guinea-pig terminal ileum to transmural nerve stimulation (TNS) and that of sensory nerves in NCNA responses were investigated. The action of acetylcholine was almost abolished in the presence of histamine, whereas the contractions elicited by TNS were changed to frequency-dependent contraction followed by a secondary relaxation. Guanethidine did not alter the contractions or secondary relaxations. Atropine abolished the action of acetylcholine and transiently suppressed the responses to low (up to 2 Hz) and attenuated (by about 50%) those to high (4 to 20 Hz) frequency stimulation. The remaining complex NCNA response was the sum of the excitatory and inhibitory responses. During desensitization to capsaicin, and in its presence, the NCNA contractions were reduced, whereas the relaxations were not significantly enlarged. The present results suggest that besides the cholinergic innervation, the excitatory and inhibitory NCNA innervation also participates in the responses of the guinea-pig ileum to TNS even without suppression of cholinergic and adrenergic transmission, and that the sensory nerves are, at least to some extent, involved in the NCNA excitatory response.

In-vitro study of the enkephalinergic hypothesis for non-adrenergic, non-cholinergic innervation in the cat stomach.

It has been suggested that enkephalins are involved in the gastric relaxation induced by stimulation of the non-adrenergic, non-cholinergic vagal fibres in the cat stomach. Experiments were therefore performed on strips of cat stomach. With longitudinal and circular gastric fundus and corpus strips from reserpinized cats, non-adrenergic, non-cholinergic relaxatory responses could be elicited by transmural electrical stimulation in Tyrode solution containing atropine and 5-hydroxytryptamine. Morphine, leu-enkephalin and met-enkephalin did not influence the tone of the strips or the relaxation evoked by stimulation at 8 Hz, and neither did the opioid antagonist, naloxone. These results do not support the enkephalinergic hypothesis for the non-adrenergic, non-cholinergic vagal fibres in the cat stomach.