Cascade Superfusion Research Articles

Inhibitory Effects of Urothelium-related Factors.

The urothelium of the bladder has long been recognized as a protective barrier between detrusor and urine. In recent years, it has become more evident that the urothelium plays a role as an active source of mediators. The urothelium can release neurotransmitters and modulators such as acetylcholine, ATP, nitric oxide, prostaglandins and neuropeptides. They exert both excitatory and inhibitory effects in modulating urinary tract motility. In addition, several studies have reported the existence of an urothelium-derived unknown inhibitory factor in the urinary bladder. By the use of a new serial cascade superfusion bioassay on guinea pig ureter, recent studies confirm that the guinea pig bladder urothelium releases a substance with inhibitory bioactivity, which was resistant to treatment with nitric oxide synthase inhibitor and cyclooxygenase inhibitor and to adenosine A1/A2 receptor blockade. Lately, a marked and quickly inactivated novel release of PGD2 from the bladder urothelium was discovered, together with localization of prostaglandin D synthase therein. PGD2 was found to have an inhibitory influence on nerve-induced contractions in guinea pig urinary bladder and on spontaneous contractions in the out-flow region. An altered release of excitatory and inhibitory factors is likely to play an important part in bladder motility disturbances, of which the prostanoids are a notable group. Due to the fact that the bladder is relaxed 99% of the time, not only excitatory mechanisms in the bladder are necessary to study, but also inhibitory mechanisms need considerable attention, which will contribute to the discovery of new targets to treat bladder motility disorders.

Cascade bioassay evidence for the existence of urothelium-derived inhibitory factor in Guinea pig urinary bladder.

Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1–5 µM in the presence of scopolamine 5–30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor.

Bioassay by cascade superfusion using a highly sensitive laminar flow technique.

Journal Article Bioassay by cascade superfusion using a highly sensitive laminar flow technique Get access H Bult, H Bult Department of Pharmacology, Erasmus University Rotterdam, Rotterdam, The Netherlands Correspondence: Department of Pharmacology, Erasmus University Rotterdam, Rotterdam, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar M J Parnham, M J Parnham Department of Pharmacology, Erasmus University Rotterdam, Rotterdam, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar I L Bonta I L Bonta Department of Pharmacology, Erasmus University Rotterdam, Rotterdam, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Journal of Pharmacy and Pharmacology, Volume 29, Issue 1, September 1977, Pages 369–370, https://doi.org/10.1111/j.2042-7158.1977.tb11338.x Published: 12 April 2011 Article history Received: 21 December 1976 Published: 12 April 2011

Parallel assay of prostaglandin-like activity in rat inflammatory exudate by means of cascade superfusion.

Parallel assay of prostaglandin-like activity in rat inflammatory exudate by means of cascade superfusion.

Effects of eicosanoids, neuromediators and bioactive peptides on murine airways

The effects of several mediators including prostanoids, neuromediators, bioactive peptides and leukotrienes were investigated on the trachea, upper bronchi, lower bronchi and lung parenchyma of selected strains of mice mounted in a cascade superfusion system. The upper airways (trachea, upper bronchi) responded with greater maxima than lower airways (lower bronchi, lung parenchyma). Acetylcholine, carbachol, serotonin and 9,11-dideoxy-9α,11α-epoxymethano-prostaglandin F 2α (U-44069) contracted the mouse airways (acetylcholine>carbachol>serotonin≥U-44069). However, the rank order of potency for these compounds was U-44069>carbachol>serotonin≥acetylcholine. Prostaglandins E 2, F 2α and D 2 relaxed the carbachol precontracted isolated mouse airways achieving >90% relaxation in some cases. The rank order of potency for the prostaglandins was E 2≥F 2α>D 2 with the exception of the lower bronchi on which prostaglandins had the following order of potency: F 2α≥E 2>D 2. The effects of prostaglandins were similar in four commonly used strains of mice (CD-1, BALB/c, C57BL/c6 and C3H) with some variations in efficacy. Iloprost was a weak mouse airway relaxant. It had the greatest effect on the trachea and bronchi of BALB/c and C57BL/c6 mice, whereas it had little or no effect on the airways of the CD-1 and C3H mouse strains. Vasoactive intestinal peptide potently relaxed the carbachol and precontracted the mouse trachea and bronchi. However, vasopressin, another bioactive peptide, potently and efficaciously contracted the mouse trachea and upper bronchi but had little effect on the lower bronchi. Vasopressin was the most potent and efficacious contractile agonist tested in this study. Contractions were observed with endothelins-1, -2 and -3 on mouse trachea and bronchi, but marked tachyphylaxis was present. Sarafotoxin s6c followed the same pattern suggesting the presence of endothelin ET B receptors on the mouse airways. Of all leukotrienes assayed (B 4, C 4, D 4 and E 4) only leukotriene C 4 weakly contracted the mouse trachea and upper bronchi, but tachyphylaxis was most evident.

The technique of superfusion

Jack Gaddum was one of the giants of pharmacology in the 1950's. Along with Harold Burn, George Brownlee, Willie Bain, A.D. MacDonald and Gladwin Buttle, he dominated meetings of the British Pharmacological Society. This paper illustrates several of his attributes including conciseness, clarity and directness of approach. These were the days when isolated organs such as the guinea-pig ileum and the rat uterus were used not only to assay solutions of known mediators such as histamine but also for the serendipitous discovery of unknown but potentially important pharmacological activity. This paper on “the technique of superfusion” is widely cited as the beginning of the method. His idea of dribbling a suitable solution over the surface of an isolated tissue derived from Finkleman (1930). Important aspects of this paper were the coining of the word “superfusion”, the front page illustration of the superfusion chamber and Gaddum's simple trick of stopping the normal flow of fluid in order to replace the drops with the test solution. This added even further to the improvement in sensitivity over and above the isolated organ bath that was commonly used. Of substantial importance in those days was to reduce the coefficient of friction between the writing lever and the smoked drum, normally achieved by regularly tapping the bench. Here, however, Gaddum deliberately used a stream of bubbles through the warming jacket in order to cause a slight vibration of the whole apparatus which could be optimised by finding the appropriate height for the apparatus on a vertical bar. Much of this paper is concerned with substance R, a slow reacting substance which he discovered was released from a superfused piece of small intestine and which contracted a rat uterus superfused below it. He then began experiments to try to identify substance R released from the perfused mesenteric bed of the rat into fluid which then superfused the rat uterus. He used various chemical manoeuvres and enzymes such as trypsin or chymotrypsin to try to identify the class of chemical to which substance R belonged. In a classical statement typical of Gaddum, he says “substance R is not histamine, because histamine does not cause contraction of the rat's uterus”. He also distinguished it from acetylcholine, 5-hydroxytryptamine, bradykinin, oxytocin and substance P by the use of various manoeuvres, including dialysis and antagonists. He concluded that the properties of substance R were similar to those of kallikrein and warned that the appearance of substance R may complicate experiments on the pharmacological activity of extracts of intestine. Interestingly, Rod Flower and his colleagues (1991) took up the challenge of substance R. Because of the preciseness of Gaddum's work and of his description of it, they were able totally to reproduce the formation of this unknown slow reacting substance in the perfused mesentery of the rat, even to reproducing the exact time course of its appearance. They concluded that it was a kininogenase enzyme, distinguishable from plasma or urinary kallikrein but did not elucidate the exact nature of the protein. The significance of Gaddum's paper was several-fold. First, it popularised the technique of superfusion, leading to a substantial increase in the sensitivity of bioassays. It also formed the basis for my own work on cascade superfusion (Vane 1964), a technique which allowed more accurate identification of substances in solution by simultaneous differential bioassay. Overall, this is a gem of a paper, especially because it describes a simple technique in such minute detail that it can be reproduced easily by others. Bioassay continued to have a major importance over the next 40 years or so, especially in identification of more unknown substances. The advent of electronic pen recorders and of powerful transducers further increased the sensitivity of bioassay, dispensing with the need for a vibrator or for tapping the bench! The whole discovery of endothelium-derived relaxing substance (EDRF) and its eventual identification as nitric oxide in the 1980's would not have been possible without the type of superfusion bioassay technique described in this paper by Gaddum and developed further by me.

Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells. 1985.

A cascade superfusion technique has been developed for the differential bioassay of prostacyclin and endothelium‐derived relaxing factor (EDRF) released from porcine aortic endothelial cells cultured on microcarriers, packed into a column and perfused. Bradykinin (Bk; 20–100 Nm) released prostacyclin (9.6 ± 1.5 Nm per 106 cells; mean ± s.e.mean, n = 9) and prostaglandin E2 (PGE2; 2.1 ± 0.6 Nm per 10° cells) from the column measured by relaxation of strips of bovine coronary artery (BCA) and rabbit mesenteric or coeliac artery, respectively. The presence of these prostanoids in the effluent was confirmed by specific radioimmunoassays. A23187 (500–2000 Nm) also released both prostacyclin and PGE2 from the cells. This release was long‐lasting and not reproducible. Bk (20–100 Nm) and A23187 (30–300 Nm) released EDRF from the column. This was detected in a cascade of four rabbit aortic strips (RbA), denuded of endothelium and contracted with U46619 or phenylephrine. The relaxation of the RbA strips caused by EDRF was progressively attenuated down the cascade (half‐life < 7 s) and was not affected by indomethacin. EDRF and prostacyclin could be differentially bioassayed in a cascade of alternating RbAs and BCAs as prostacyclin did not relax RbAs and the time delay to the BCAs destroys EDRF. EDRF could be bioassayed on its own when the endothelial cells were treated with indomethacin. 5‐Hydroxytryptamine 0.2, noradrenaline 1.0, platelet‐activating factor (Paf‐acether) 1.0, formylmethionyl‐leucyl‐phenylalanine 1.0, acetylcholine 0.5, bethanecol 0.5, adenosine diphosphate 0.25 and angiotensin II 0.1 μm did not release either prostanoids or EDRF from the column.

Endothelium-derived relaxing factor as a mediator of bradykinin-induced perinatal pulmonary vasodilatation in fetal sheep.

Studies in vivo in fetal sheep have shown that bradykinin is released following oxygenation of the lungs and is at least partly responsible for normal pulmonary vasodilatation in the transition from fetal to extrauterine life. Part of this action involves secondary release of prostaglandin I2 (PGI2). In various adult vessels, bradykinin also stimulates the release of a powerful endothelium-derived relaxing factor (EDRF). Studies in vitro were designed (using a modification of the bioassay cascade superfusion technique) to determine whether non-PGI2-related perinatal pulmonary vasodilatation is mediated by an EDRF. Superfused, precontracted, endothelium-denuded strips of fetal sheep thoracic aorta and the maternal sheep main pulmonary artery served as detectors of an EDRF released from isolated, perfused fetal sheep pulmonary arteries. Bradykinin, in the presence of indomethacin to block PGI2 synthesis, caused perfused fetal pulmonary arteries to release an EDRF, which generated a dose-dependent relaxation (24% for 1.0 microM, 16.8% for 0.1 microM, and 10% for 0.01 microM bradykinin). Thus, bradykinin can produce perinatal pulmonary vasodilatation via a mechanism involving the endothelium-dependent synthesis of an EDRF.

Characterization of the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells as an NG-hydroxyl-L-arginine-nitric oxide adduct.

The nature of an L-arginine-derived relaxing factor released from vascular smooth muscle cells cultured on microcarrier beads and stimulated for 20 h with interleukin 1 beta was investigated. Unlike the unstable relaxation elicited by authentic nitric oxide (NO) in a cascade superfusion bioassay system, the effluate from vascular smooth muscle cells induced a stable relaxation that was susceptible to inhibition by oxyhemoglobin. Three putative endogenous NO carriers mimicked this stable relaxing effect: S-nitroso-L-cysteine, low molecular weight dinitrosyl-iron complexes (DNICs), and the adduct of NG-hydroxy-L-arginine (HOArg) with NO. Inactivation of S-nitroso-L-cysteine by Hg2+ ions or trapping of DNICs with agarose-bound bovine serum albumin abolished their relaxing effects, whereas that of the vascular smooth muscle cell effluate remained unaffected. In addition, neither S-nitrosothiols nor DNICs were detectable in the effluate from these cells, as judged by UV and electron spin resonance (ESR) spectroscopy. The HOArg-NO adduct was instantaneously generated upon reaction of HOArg with authentic NO under bioassay conditions. Its pharmacological profile was indistinguishable from that of the vascular smooth muscle cell effluate, as judged by comparative bioassay with different vascular and nonvascular smooth muscle preparations. Moreover, up to 100 nM HOArg was detected in the effluate from interleukin 1 beta-stimulated vascular smooth muscle cells, suggesting that sufficient amounts of HOArg are released from these cells to spontaneously generate the HOArg-NO adduct. This intercellular NO carrier probably accounts for the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells and also from other NO-producing cells, such as macrophages and neutrophils.

Humoral factor mediates acetylcholine-induced endothelium-dependent relaxation of chicken aorta

A superfusion cascade system was used to determine whether endothelium-dependent vasorelaxation induced by acetylcholine (ACh) is mediated by a humoral factor(s) in the domestic fowl. An abdominal aorta (5 cm in length) with intact endothelium, excised from the donor bird, was mounted in an organ bath and perfused with avian Ringer solution. The vasorelaxing activity of the effluent was determined by an endothelium-denuded aortic ring (bioassay ring) equilibrated and precontracted with phenylephrine (PHE) (10 −6 M). The effluent (containing PHE) from the donor aorta with intact endothelium did not significantly change the PHE-induced tension of the bioassay rings. ACh (10 −6 M), when added to the perfusion medium that runs through the control polyethylene tubing, produced further contraction of the PHE-precontracted endothelium-denuded bioassay rings, whereas ACh caused relaxation of the rings when added to the perfusate that passes through the endothelium-intact donor aorta. The ACh-induced relaxation of the bioassay ring was sightly potentiated by superoxide dismutase (100 U/ml). Hemoglobin (10 −5 M) increased the basal tension of the bioassay rings and completely inhibited the ACh-induced relaxation of the rings. These results suggest that ACh-induced relaxation of fowl aorta is, at least partially, mediated by a humoral factor(s) that resembles endothelium-derived relaxing factor demonstrated in mammals.

Characterization and actions of human umbilical endothelium derived relaxing factor

1. A bioassay cascade superfusion technique was utilized to study the properties of endothelium derived relaxing factor (EDRF) from human umbilical vein (HUV) and compare its actions on umbilical, chorionic plate and bovine pulmonary arterial strips. 2. Histamine (1 microM), bradykinin (1 microM) and A-23187 (0.3 microM, 1 microM) but not acetylcholine (1 microM) released EDRF. 3. The non-innervated human foetoplacental vessels, i.e., umbilical and chorionic plate arteries, do relax to EDRF by a guanosine 3':5'-cyclic monophosphate (cyclic GMP)-mediated mechanism. 4. The sensitivity of the human umbilical arterial strips to EDRF was less than that of the chronic plate arterial strips. Bovine pulmonary arterial strips were the most sensitive to the relaxant actions of human umbilical EDRF.

Involvement of prostanoids in the pulmonary pressor effect of histamine

The role of prostanoids in the pulmonary actions of histamine(HA) was investigated using various antagonists and assay organs in superfusion cascade. Intraarterial injection of HA to the perfused lung caused contractions of rabbit aorta, rat stomach strips, guinea pig trachea, but contraction followed by relaxation of bovine coronary artery, superfused with lung effluent. Lung effluent produced no significant changes in resting tensions of guinea pig ileum or rat colon. The thromboxane synthetase inhibitor, UK 38485, reduced and the thromboxane/prostaglandin receptor antagonist, SQ 29548 abolished HA-induced contractions of all superfused assay tissues. Both contractile and relaxant responses of bovine coronary artery were completely inhibited by indomethacin. The pulmonary pressor effect of HA was also significantly inhibited by all of these antagonists. Our results indicate that HA releases thromboxane A 2, prostaglandin endoperoxides, and prostacyclin but not leukotriene and prostaglandin E 2- like substances from the guinea pig lung.

Endothelium-derived nitric oxide relaxes nonvascular smooth muscle

A bioassay cascade superfusion procedure was used to compare and contrast the actions of arterial and venous endothelium-derived relaxing factor (EDRF) with authentic nitric oxide (NO) on several nonvascular smooth muscle preparations. EDRF was released from human umbilical vein or bovine pulmonary artery by A23187 and allowed to superfuse two nonvascular and one vascular precontracted smooth msucle strips arranged in a cascade. NO or S-nitroso-N-acetylpenicillamine was delivered by superfusion. Both EDRF and NO relaxed bovine trachea, although artery was 10 times more sensitive than trachea to either relaxant. Similarly, rabbit taenia coli and rat fundus relaxed in response to high concentrations of NO or large amounts of EDRF released from umbilical vein. Vascular and nonvascular relaxant responses to both EDRF and NO were inhibited by oxyhemoglobin, methylene blue or superoxide, and were enhanced by superoxide dismutase. Perfusion of pulmonary artery or umbilical vein with A23187 resulted in contraction of guinea pig ileum and relaxation of pulmonary artery, whereas NO relaxed both preparations. Oxyhemoglobin enhanced the contractile and abolished the relaxant responses. Thus, ileum is more sensitive to endothelium-derived contracting factor(s) than to EDRF. No raised cyclic GMP levels in all smooth muscle preparations, but a greater fold increase was observed in artery than in nonvascular smooth muscle. EDRF released from human umbilical vein was identified chemically as NO or a nitroso compound, as was done previously for EDRF from bovine pulmonary artery and vein. These observations support the view that one EDRF from artery and vein is NO or a labile nitroso compound.

Pharmacology of Endothelin-1 in Isolated Vessels

Sensitive bioassay tissues for porcine endothelin-1 (ET-1) were developed in a cascade superfusion system and in organ baths. Venous preparations of the rabbit and rat were more sensitive than arterial preparations. ET-1 had a different pharmacological profile than Bay K 8644 on the various preparations. Nicardipine (0.1-1 microM) abolished the responses to Bay K 8644 without affecting those induced by ET-1. Thus, ET-1 contracts venous and some arterial vessels via specific receptors or channels that differ from dihydropyridine-sensitive calcium channels. Methylene blue and hemoglobin potentiated responses to ET-1 in endothelium-denuded venous vessels, whereas gossypol had no effect.

EDRF generation and release from perfused bovine pulmonary artery and vein

A bioassay cascade superfusion procedure was utilized to study the release and effects of endothelium-derived relaxing factor (EDRF) from isolated bovine pulmonary artery and vein. Three precontracted, endothelium-denuded strips of pulmonary artery or vein mounted in series served as the detector of EDRF. Perfusion of artery with acetylcholine, bradykinin or A23187, in the presence of indomethacin, caused the release of EDRF ( t 1 2 = 3−5 s ) which relaxed superfused strips of both artery and vein. Similarly, perfusion of vein with bradykinin or A23187, but not acetylcholine, caused the release of EDRF ( t 1 2 = 3−5 s ) which relaxed superfused strips of both vein and artery. EDRF-elicited vascular smooth muscle relaxation was associated with cyclic GMP accumulation in the superfused strips. EDRF activity was increased and its duration prolonged by superoxide dismutase or low oxygen tension, whereas oxyhemoglobin and methylene blue were markedly inhibitory. The similar patterns of release, instability, and properties of EDRF from intact artery and vein suggest that arterial and venous EDRF are closely similar.

Endothelium-derived relaxing factor released from cultured cells: differentiation from nitric oxide.

1. Endothelium-derived relaxing factor (EDRF) is an extremely labile mediator thought to be identical to nitric oxide (NO). 2. A cascade superfusion technique was used to bioassay EDRF released from bovine aortic endothelial cells grown to confluence on microcarrier beads. 3. Bradykinin (1-100 nmol/l), infused through a 1 cm column of endothelial cells on microcarriers, released an EDRF-like substance that caused relaxations of de-endothelialized strips of rabbit aorta (precontracted with phenylephrine). These relaxations diminished on successive tissues in the cascade, when compared with those produced by glyceryl trinitrate as a stable standard. 4. Haemoglobin (1 mumol/l), infused directly over the bioassay tissues, abolished bradykinin-induced relaxations and these were restored within 5 min after removal of haemoglobin. The infusion did not affect the relaxations produced by glyceryl trinitrate in this system. 5. Methylene blue (20 mumol/l) inhibited bradykinin-induced relaxations when infused over the rabbit aortae, and reduced those relaxations produced by glyceryl trinitrate. The effects of bradykinin, but not glyceryl trinitrate, were partially restored after removing methylene blue. 6. These data are consistent with the known effects of these compounds on the activity of NO, and on EDRF in isolated blood vessels. 7. The activity of EDRF (released by bradykinin) was compared directly with NO on strips of guinea-pig trachea (de-epithelialized) interposed in cascade between two rabbit aortae; all strips were precontracted with histamine and phenylephrine. 8. A submaximal dose of NO that matched the relaxation produced by EDRF on the uppermost aorta, caused relaxation of the trachea, but EDRF had no effect on this tissue. In addition, the NO-induced relaxation of the lower aorta was greater than that produced by EDRF. 9. These data indicate that EDRF does not have identical biological activity to NO. EDRF could contain an NO moiety attached to a carrier molecule that is bound and stabilized in tracheal tissue.

Recombinant human lipocortin 1 inhibits thromboxane release from guinea-pig isolated perfused lung.

The guinea-pig perfused isolated lung, used in conjunction with the cascade superfusion system to measure the release of thromboxane A2(TXA2), is a simple and convenient model for assessing the inhibition by glucocorticoids of eicosanoid formation. Dexamethasone inhibits the release of TXA2 from the lung when it is stimulated by agents such as RCS-RF2 of leukotrienes, but not when bradykinin or arachidonic acid are used. Using this model we have shown that the glucocorticoids suppress eicosanoid generation by cells through the induction of a family of phospholipase A2-inhibitory proteins now termed the 'lipocortins'. Recently the primary structure of one form of lipocortin has been elucidated and the human gene cloned. Lipocortin 1 is a polar monomeric protein with anti-phospholipase properties in vitro and we now report that when infused into guinea-pig lung preparations this protein has the same inhibitory profile as the glucocorticoids but with a more rapid onset of action. This is the first demonstration that eicosanoid formation can be inhibited by a recombinant phospholipase inhibitory protein applied extracellularly.

Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells.

A cascade superfusion technique has been developed for the differential bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) released from porcine aortic endothelial cells cultured on microcarriers, packed into a column and perfused. Bradykinin (Bk; 20-100 nM) released prostacyclin (9.6 +/- 1.5 nM per 10(6) cells; mean +/- s.e.mean, n = 9) and prostaglandin E2 (PGE2; 2.1 +/- 0.6 nM per 10(6) cells) from the column measured by relaxation of strips of bovine coronary artery (BCA) and rabbit mesenteric or coeliac artery, respectively. The presence of these prostanoids in the effluent was confirmed by specific radioimmunoassays. A23187 (500-2000 nM) also released both prostacyclin and PGE2 from the cells. This release was long-lasting and not reproducible. Bk (20-100 nM) and A23187 (30-300 nM) released EDRF from the column. This was detected in a cascade of four rabbit aortic strips (RbA), denuded of endothelium and contracted with U46619 or phenylephrine. The relaxation of the RbA strips caused by EDRF was progressively attenuated down the cascade (half-life less than 7s) and was not affected by indomethacin. EDRF and prostacyclin could be differentially bioassayed in a cascade of alternating RbAs and BCAs as prostacyclin did not relax RbAs and the time delay to the BCAs destroys EDRF. EDRF could be bioassayed on its own when the endothelial cells were treated with indomethacin. 5-Hydroxytryptamine 0.2, noradrenaline 1.0, platelet-activating factor (Paf-acether) 1.0, formylmethionyl-leucyl-phenylalanine 1.0, acetylcholine 0.5, bethanecol 0.5, adenosine diphosphate 0.25 and angiotensin II 0.1 microM did not release either prostanoids or EDRF from the column.

The influence of epithelium on the responsiveness of guinea-pig isolated trachea.

This study was designed to investigate the possibility that tracheal epithelium generates a relaxant factor analogous to the endothelial-derived relaxant factor (EDRF) of vascular tissue. The absence of such a factor following epithelial damage in diseases such as asthma might help to explain the airway hyperreactivity characteristic of such diseases. Removal of epithelium by rubbing enhanced the sensitivity of guinea-pig isolated tracheal chains to 5-hydroxytryptamine, histamine, acetylcholine, adenosine, isoprenaline and also minimally to KCl. Responses to LaCl3 and electrical field stimulation were not affected. Low concentrations of adenosine produced contractions only in tissues denuded of epithelium. In the presence of indomethacin 1.4 microM or dithiothreitol (DTT) 1 microM, dose-response curves to histamine were moved to the left in both control and rubbed tissues, and the maximum response was increased. The difference in sensitivity between tissues with and without epithelium was not affected by indomethacin, but was slightly reduced by DTT. Phenidone (0.1 mM) also increased the maximum responses, but increased the sensitivity only of the tissues with intact epithelium, to the same level as that seen in the tissues denuded of epithelium. Superfusion cascade studies provided no evidence for the generation of a relaxant factor from tracheal epithelium. It is suggested that the supersensitivity produced by removal of the epithelium is not due to the removal of a relaxant factor, but rather to the removal of a permeability barrier, allowing a greater concentration of agonist at the level of the underlying smooth muscle.

Correlation between the myotropic activity of leukotriene A 4 on guinea-pig lung, trachea and ileum and its biotransformation in situ

Leukotrienes A 4 and D 4 displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD 4 was much higher than that of LTA 4. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD 4 was very active whereas LTA 4 was inactive. Since the activities of both leukotrienes were blocked by FPL-55712, our results suggested that the transformation of LTA 4 by the smooth muscle preparations was a prerequisite to its biological activity. LTA 4 was then incubated for 10 min with homogenates of guinea pig lung parenchyma, trachea and longitudinal muscles of ileum, and the metabolites were analysed by bioassay using strips of guinea pig ileum and lung parenchyma in a cascade superfusion system and also by reversed phase high performance liquid chromatography (RP-HPLC). Homogenates of lung parenchyma rapidly transformed LTA 4 to LTB 4, LTC 4, LTD 4 and LTE 4. Incubation of LTA 4 with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB 4 and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA 4 undergoes distinctly different metabolic transformations in these tissues which correspond to the biological activites of the products recovered. These results strongly suggest that the myotropic activity and potency of LTA 4 is related to the tissue levels of enzymes which catalyse its biotransformation.