Michel Ansay

Modulation of acetylcholine, capsaicin and substance P effects by histamine H3 receptors in isolated perfused rabbit lungs.

The modulatory role of histamine H3 receptors in pulmonary oedema induced by acetylcholine, capsaicin and by exogenous substance P was investigated in isolated, ventilated rabbit lungs. Endothelial permeability was evaluated by measuring the capillary filtration coefficient (Kf,c). Acetylcholine (10(-8) to 10(-4) M), substance P (10(-10) to 10(-6) M), capsaicin (10(-4) M) and 5-hydroxytryptamine (5-HT) (10(-4) M) induced an increase in the Kf,c. Carboperamide, a novel histamine H3 receptor antagonist, induced a significant leftward shift of the concentration-response curve to acetylcholine and also enhanced the effect of capsaicin on the Kf,c, while it had no significant effect on the response to substance P and 5-HT. Imetit, a new histamine H3 receptor agonist, strongly inhibited the effects of acetylcholine and capsaicin. Imetit also strongly protected the lung against substance P effects but did not prevent the 5-HT-induced increase in the Kf,c. Carboperamide completely blocked the inhibitory effect of Imetit on the acetylcholine response. (R)-alpha-Methylhistamine, an other histamine H3 receptor agonist, had the same protective effect against acetylcholine response as Imetit. We conclude that histamine H3 receptors could protect the lung against acetylcholine- and capsaicin-induced oedema via a prejunctional modulatory effect on the C-fibres. However, since the response to exogenous substance P was also inhibited by histamine H3 receptor stimulation, the presence of such receptors at a postsynaptic level, probably on mast cells, was also suggested.

Altered capillary filtration coefficient in parathion- and paraoxon-induced edema in isolated and perfused rabbit lungs

Changes in pulmonary endothelium permeability and in microvascular hemodynamics induced by parathion (Pth) and paraoxon (Pox), its active metabolite, were investigated in isolated, perfused rabbit lungs. Blood-free perfusate was recirculated through isolated and ventilated lungs in an isogravimetric state and in zone III conditions. The arterial/venous/double occlusion technique was used to divide the total vascular resistance (Rt) into four components: arterial, precapillary, postcapillary, and venous. The capillary filtration coefficient (Kfc) was evaluated by measuring the amount of fluid filtering through the endothelium when the arterial and venous pressures were suddenly increased. Pth and Pox induced pulmonary edema by increasing endothelium permeability without changing the hemodynamic parameters at any level of the vascular bed. The Kfc value increased progressively, reaching a maximum (Emax) 60 min after administration of organophosphate (558 +/- 65% (n = 5) and 707 +/- 109% (n = 5) of baseline values, for Pth and Pox, respectively). During the next 60 min, it decreased. The time course of Pox-induced changes in Kfc was similar to that obtained with Pth. The concentration-response curve (Emax) expressed as a percentage of the baseline value versus the logarithm of the molar Pth concentration, ranging from 2 x 10(-5) to 4 x 10(-4) M) was linear (y = 1661.1 + 327.3x, r = 0.89, p < 0.001, n = 14). Piperonyl butoxide (4 x 10(-4) M), an inhibitor of cytochrome P450, had a strong protective effect against Pth (4 x 10(-4) M)-induced alterations of endothelium permeability (n = 5, p < 0.001). The effects of Pox (4 x 10(-4) M) on Kfc were completely abolished by pretreatment with 10(-5) M atropine, as shown by the significantly lower Emax value recorded in atropine-pretreated lungs (129 +/- 33%, n = 4) than in Pox-treated lungs (707 +/- 109%, n = 5, p < 0.001). The effects of Pth, on the other hand, were only partially inhibited, since the Emax value recorded in atropine-pretreated lungs (196 +/- 20%, n = 4) remained significantly higher than that recorded for control lungs (129 +/- 15%; n = 5; p < 0.05). These results show that isolated and perfused rabbit lungs constitute an appropriate model for studying the direct pulmonary effects of organophosphates. The edema-inducing action of Pth depends on its activation by conversion to Pox in the lung tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute Effects of Endotoxin Inhalation on the Respiratory Tract in Pigs: Interaction with Ammonia

AbstractThe acute respiratory effects of endotoxin inhalation (0–400 μ/kg) have been evaluated in pigs. Endotoxin (LPS from Escherichia coli 0127:B8 and 055:B5) nebulization had no direct effect on the nasal mucosa. An in vitro bronchial hyperreactivity to acetylcholine and histamine, related to the endotoxin dosage, was recorded. In the lung, significant endotoxin dose-dependent increases in the total cell, alveolar macrophage, and neutrophil counts in the BAL fluid were recorded. Following intrabronchial administration of endotoxin, the cellular response was significantly higher than that obtained after nebulization. Neither the dry to wet lung weight ratio (DW/WW) nor the albumin concentration in the BAL fluid was affected. When endotoxins were infused intravenously, the lung cellular response was accompanied by an increase in the albumin concentration in the BAL fluid. In pigs exposed for 6 days to 50 ppm ammonia, endotoxin nebulization induced a significant increase in the albumin concentration and t...

Permeability of the endothelium and partitioning of the pulmonary blood flow resistance in isolated perfused pig lungs: effects of breed and age.

The right and left lungs of 5 healthy Minipigs and of 13 healthy Landrace piglets were isolated, perfused at constant pressure and maintained in an isogravimetric state under zone III conditions (pulmonary venous pressure>alveolar pressure). By applying the double, arterial and venous, occlusion technique, the total blood flow resistance (Rt) was partitioned into four components: arterial (Ra), pre-(Ra′) and post-capillary (Rv′) and venous (Rv). The capillary filtration coefficient (Kf,c) was evaluated by measuring the weight gained by the lungs when the arterial and venous pressures were suddenly increased. In the youngest Landrace piglets (5 weeks old), there was an uncontrolled vasoconstriction which sometimes prevented perfusion of the lungs and induced a large increase inRt. These high values ofRt were decreased by tolazoline administration. The values ofRt recorded in older pigs (12–13 weeks old) were lower in Minipigs (33.66±3.77 cmH2O min L−1 per 100 g of lungs;n=5) than in Landrace piglets (55.20±6.18 cmH2O min L−1 per 100 g;n=5). This breed difference was due to the differences inRa′ andRv′. The mean values ofKf,c were 0.193±0.015 and 0.202±0.029 ml min (cmH2O)−1 per 100 g of the lungs in Minipigs and Landrace piglets respectively. All these parameters were stable for the 3 hours following the equilibrium period. It was concluded that: (1) There is an age-related maturation of the control of the vasomotor tone in porcine lungs. (2) Pulmonary microvascular haemodynamics are influenced by the breed of the pigs. (3) There was no difference in theKf,c values between both the breeds. (4) A comparison of the values reported for dogs and rabbits with our data shows that the pre- and post-capillary resistances and, to a lesser extent, the arterial and venous resistances are relatively high in pigs.

Interactions between acetylcholine and substance P effects on lung mechanics in the rabbit.

Summary— The pharmacological mechanisms involved in the acetylcholine (ACh)‐ and substance P (SP)‐induced changes in pulmonary mechanics were studied in isolated perfused rabbit lungs. Tracheal pressure (Ptr) and airflow were measured by a Fleisch pneumotachograph and pressure transducers. Air volume, lung resistance (RL) and dynamic compliance (Cdyn) were calculated. ACh induced a dose‐dependent increase in Ptr and RL, and a decrease in Cdyn. These effects were strongly prevented by atropine, and partly by SR140333, a neurokinin NK1 receptor antagonist; SR48968, a neurokinin NK2 receptor antagonist; indomethacin and antihistaminics. Ketanserin had no significant protective effect against ACh. SP also induced concentration‐dependent increases in RL and decreases in Cdyn. SR140333 and atropine strongly inhibited the effects of SP, while ketanserin, SR48968, antihistaminics and indomethacin did not protect the lungs against this drug. 5‐hydroxytryptamine induced no significant change in lung mechanic parameters. Cumulative concentrations of histamine increased RL and decreased Cdyn. We conclude that ACh‐induced changes in lung resistance and compliance are in part mediated by a direct effect on airway smooth muscle and in part by the stimulation of C fibers, by the release of histamine from mast cells and by the synthesis of arachidonic acid metabolites. In turn, the effects of SP on lung mechanics are partly due to cholinergic activation.

In vitro short-term study of ammonium-nitrogen production from cattle urine : influence of ampicillin, hydroquinone and animal litter materials

The production of NH + 4 -N following in vitro incubation of cattle urine was monitored for 24 h in the presence of ampicillin sodium salt (0, 32, 64, 128 mg/l), hydroquinone (0, 16·7, 33·4, 66·8 mg/l), wheat straw (0, 3·3, 6·6, 13·2 g/l) or spruce sawdust (0, 3·3, 6·6, 13·2 g/l) with (20 IU) or without urease. Each concentration of ampicillin, hydroquinone, wheat straw or spruce sawdust was tested in triplicate. The equipment consisted of Woulff flasks containing 300 ml of a buffered solution (0·02 M ; pH 7·50) with 1 ml of cattle urine. The cattle urine was characterized by measuring the main nitrogen contents, which were 6·52 mg total-N/ml, 5·96 mg urea-N/ml and 0·026 mg ammonium-N/ml. The initial pH of urine was 7·84. Ammonium and nitrate concentrations, and pH were monitored at zero- time and after 3, 6 and 24 h of incubation with the cattle urine. The addition of urease to the flasks containing urine induced a significant increase in the production of ammonium-N, from 1·83 to 6·32 mg NH + 4 -N/flask after 24 h of incubation. In the presence of urease, an inhibitory effect was recorded in NH + 4 -N production with ampicillin and spruce sawdust. In contrast, hydroquinone inhibited urease activity and wheat straw adsorbed the NH + 4 -N produced, both causing a dose-dependent relationship. In the absence of urease, ampicillin, hydroquinone, wheat straw or spruce sawdust caused a dose- related decrease in NH + 4 -N production. However, the highest amounts of wheat straw (6·6 and 13·2 g/flask) exhibited a temporary increase in NH + 4 -N production during the first 6 h. This is probably due to a generation of extra NH + 4 -N as compared to the control flasks without straw. However, at 24 h, the situation paralleled the other materials. Although pH increased approximately from 7·50 to 7·65 during the hydrolysis of urea in the presence of urease, no gaseous ammonia was volatilized into a 0·1 M HCl flask. Moreover, no increase in nitrate concentration was found during the incubation. The present results suggest that ampicillin, hydroquinone and spruce sawdust could be used in order to reduce NH + 4 -N production from cattle urine. The NH + 4 -N already produced could probably be absorbed on wheat straw.

Mechanisms of substance P‐induced pulmonary oedema in the rabbit: interactions between parasympathetic and excitatory NANC nerves

Summary— The pharmacological mechanisms involved in the substance P (SP)‐induced pulmonary oedema were studied in isolated perfused rabbit lungs. Substance P induced a dose‐dependent increase in the capillary filtration coefficient (Kf,c), responsible for oedema. Atropine, hemicholinium‐3 and ruthenium red pretreatment partly protected the lungs against SP effects, while tetrodotoxin and hexamethonium did not significantly modify them. (±)CP96,345, a NK1 receptor antagonist, completely inhibited the SP‐induced increase in the Kf,c. Like SP, acetylcholine (ACh) and capsaicin also increased the Kf,c. Atropine and (±)CP96,345 completely blocked the oedema induced by both drugs. Tetrodotoxin and ruthenium red strongly inhibited the response to capsaicin and acetylcholine. It was concluded that SP‐induced pulmonary oedema is in part mediated by a stimulating action on cholinergic efferent nerves, with subsequent release of endogenous acetylcholine. Acetylcholine can, in turn, stimulate the release of SP from excitatory non adrenergic, non cholinergic nerves. The effects induced by capsaicin and exogenous acetylcholine, thus endogenous SP, involve tetrodotoxin‐sensitive mechanisms, while those produced by exogenous SP are tetrodotoxin‐resistant.

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B1 (AFB1). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB1 inhibited RNA and protein synthesis at a concentration of 10−7 M and 10−6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10−3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB1 inhibited the accumulation of glycogen from 10−6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB1 on glycogen accumulation. If the “in vivo” protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB1-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB1. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.

ENDOTOXIN-INDUCED MICROVASCULAR INJURY IN ISOLATED AND PERFUSED PIG LUNGS

The lungs of 13 healthy Landrace piglets were isolated, perfused and maintained in an isogravimetric state under zone III conditions. By applying vascular occlusion methods, the total blood flow resistance (Rt) was partitioned into four components: arterial (Ra), pre- (Ra′) and post-capillary (Rv′), and venous (Rv). The capillary filtration coefficient (Kf,c) was evaluated using a gravimetric technique. A bolus of 55 µg ofEscherichia coli endotoxins (LPS) per 100 g of lung was injected into the arterial reservoir of eight lungs, followed by an infusion of LPS at a rate of 55 µg per 100 g of lung per hour for 180 min. A bolus of theophylline (85 mg per 100 g of lung weight) was injected into the arterial reservoir after the last determination of theKf,c value. All the parameters were evaluated again when the lungs reached a new steady state. Endotoxin induced a significant increase inRt from 54.7 ± 7.0 at zero time to 184.7±44.2 cm H2O min L−1 (100 g)−1 180 minutes later, which can be ascribed to the increase inRa′ andRv′. These haemodynamic modifications were related to the increases in the arterial pressure and in the pressure at the distal end of the arterial segment and to the decreases in the pressure at the proximal end of the venous segment and in the blood flow. The capillary pressure and the lung weight remained unchanged. Endotoxin infusion infusion induced an increase in theKf,c value from 0.208±0.011 (att=0) to 0.391±0.034 ml min−1 (cmH2O)−1 (100 g)−1 (att=180). Administration of theophylline significantly reducedRt,Ra,Ra′ andRv′ towards or under the baseline values and also induced a significant increase in the lung weight and in theKf,c value. It was concluded that the endotoxin-induced increase in the total blood flow resistance can be ascribed to a vasospasm occurring at the level of the pre- and post-capillary small vessels and that changes in the permeability of the endothelium greatly contribute to the development of the pulmonary oedema observed in endotoxaemic pigs.

Synergism between aflatoxin B1 and oxytetracycline on fatty acid esterification in isolated rat hepatocytes.

The individual and combined effects of aflatoxin B1 (AFB1) and oxytetracycline (OXT) on the synthesis and secretion of triacylglycerols in isolated rat hepatocytes maintained in suspension during 2.5 h were studied. Secretion of triacylglycerols was inhibited by both drugs when administered separately. This inhibition was accompanied by a concomitant elevation of intracellular triacylglycerols only at the highest AFB1 dose tested. Total synthesis of triacylglycerols was not inhibited by AFB1 or by OXT. When the two drugs were simultaneously added to the incubation medium, the AFB1-induced accumulation of intracellular triacylglycerols was no longer observed; the inhibition of secretion was nevertheless identical to that observed with AFB1 alone. Finally, total esterification of palmitate was inhibited by 20% compared to the AFB1-treated cells. These data suggest that OXT inhibits lipid accumulation induced by AFB1 but that this effect is due to an inhibition of total synthesis of triacylglycerols. The mechanism of AFB1s effect and of the interaction between both molecules is discussed.