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Adventures and Excursions in Bioassay-The Stepping Stones to Prostacyclin J

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Adventures and Excursions in Bioassay-The Stepping Stones to Prostacyclin J Postgraduate Medical Journal (December 1983) 59, 743-758 Postgrad Med J: first published as 10.1136/pgmj.59.698.743 on 1 December 1983. Downloaded from NOBEL LECTURE Adventures and excursions in bioassay-the stepping stones to prostacyclin J. R. VANE F.R.S. Wellcome Research Laboratories, Langley Court, Beckenham, Kent Physiology has spawned many biological sciences, pation of prostaglandins in inflammatory reactions amongst them my own field ofpharmacology. No man (5, 6), the contribution of prostaglandins to the has made a more important contribution to the fields autoregulation and maintenance of blood flow to the ofphysiology and pharmacology than Sir Henry Dale kidney (7-9), the inhibitory effect of aspirin-like (1875-1968, Nobel Laureate in Physiology and Medi- drugs on the biosynthesis of prostaglandins (10-12), cine in 1936). Dale had a great influence not only on the mediation of pyrogen fever by prostaglandins British pharmacology in general but also on my own (13), and the release of rabbit aorta-contracting scientific endeavours. Indeed, I can put forward a substance (RCS; now identified as thromboxane A2, strong case for considering myself as one of Dale's TXA2) from lungs during anaphylaxis (14, 15). scientific grandchildren. My early days as a pharma- Moreover, in 1976, bioassay made possible the Protected by copyright. cologist were influenced not only by Dale himself but discovery of PGX, now renamed prostacyclin (PGI2), also by his own school of colleagues, including Burn, the latest member of the prostaglandin family Gaddum and von Euler. It was Burn who taught me (16-19). Indeed, it is doubtful whether the biological the principles and practice of bioassay. Some of significance of any of the unstable products of Gaddum's first publications were on the development arachidonic acid metabolism would have been recog- of specific and sensitive methods for biological assay nised without bioassay techniques. With extraordi- and he maintained a deep interest in this subject for nary simplicity and convenience, by its very nature, the rest ofhis life (1). In 1964 he said "the pharmacolo- bioassay distinguishes between the important bio- gist has been a 'jack of all trades' borrowing from logically active compounds and their closely related physiology, biochemistry, pathology, microbiology but biologically unimportant metabolites. and statistics-but he has developed one technique of In this review I shall discuss the development of his own, and that is the technique of bioassay" (2). the cascade superfusion bioassay technique and some Expensive, powerful and sophisticated chemical of the discoveries and concepts which arose from its methods, such as gas chromatography and mass application, leading up to the discovery and develop- http://pmj.bmj.com/ spectrometry, have been developed and perfected for ment of prostacyclin. The effects of prostacyclin in detection and quantification of prostaglandins (PGs) man and its clinical assessment (another application and related substances. One should not forget, of bioassay) will also be discussed. however, that starting with the discovery and isola- tion of prostaglandins by von Euler [3; see also Bergstrom (1983)t], biological techniques and bio- assay have contributed very substantially to the 1. Cascade superfusion bioassay development of the field. Bioassay has provided (a) Development- on September 23, 2021 by guest. crucial information on the role of the lungs in the removal of circulating prostaglandins (4), the partici- Most uses of bioassays involving smooth muscle demand high sensitivity and specificity. These aspects have been achieved, first by limiting the volume of © The Nobel Foundation 1983. fluid bathing the isolated tissue and, second, by using Dr John Vane delivered the Francis Fraser lecture at the Academic Assembly of the British Postgraduate Medical Federation on 3rd an assay organ sensitiye to, and relatively specific for, June 1983 on the subject of 'Prostacyclin-from discovery to the the test substances under study. Further specificity clinic'. can be achieved by using a combination of several tBERGSTROM, S. (1983) Lecture to be published in 'Les Prix tissues which present a characteristic pattern of Nobel 1982'. Nobel Foundation (in press). response to the test substance or substances. This 0032-5473/83/1200-0743 $02.00 © 1983 Tdc Fellowship of Postgraduate Medicine Postgrad Med J: first published as 10.1136/pgmj.59.698.743 on 1 December 1983. Downloaded from 744 J. R. Vane ......................... ;tw8.|<1:.q 8',X'tt t..-.:.,.. Xr<_*e8....':'"-. ;¢t ,l@ XOf,.. @ .,. R':rR............... A.g-. t,..-,:-...o.. ASI' .- fX e )S , P : : u FIG. 1. Diagram of the blood-bathed organ technique. Blood is continuously withdrawn from a convenient vessel by a roller pump, kept at 37TC by a waterjacket and then allowed to superfuse a series ofisolated organs, the longitudinal movements ofwhich are recorded. The blood is then collected in a reservoir and returned to the animal. In some experiments, the blood flows through a length ofsilicone tubing in a water bath (incubating circuit) before superfusing the isolated tissues. Drugs can be applied directly to the isolated tissues by infusions or injections into the bathing blood (I.B.B.) or with a time delay into the incubating circuit (from Vane 1969, reference 33, by permission ofThe Macmillan Press Ltd). Protected by copyright. takes advantage of the principle of parallel pharma- Plainly, when perfusate from an organ or blood cological assay, regarded by Gaddum (20) as strong from an animal is used for superfusion, substances evidence for the identity of a compound. can reach the assay tissues within a few seconds of Magnus (21) introduced the idea of suspending an generation or release. This element of 'instantaneity' isolated portion of smooth muscle in a chamber is an important aspect of cascade superfusion bioas- containing a nutrient fluid and measuring changes in say in that it detects the biological activity of tissue tone. The organ baths used today are modified chemically unstable compounds whose activity versions of that used by Dale (22). Gaddum (23) would otherwise be lost in an extraction process. applied the experimental design developed by Fink- Another important feature of the method is that it leman (24) to the assay of minute amounts of gives the maximum opportunity for serendipity. The biologically active substances. He called his tech- dynamic nature of the assay also allows the measure- nique 'superfusion' in contrast to perfusion. This ment of inactivation of an infused substance across a consisted of bathing an assay tissue with a stream of particular vascular bed. Further modifications of thehttp://pmj.bmj.com/ fluid which was momentarily stopped at the moment bioassay technique have been developed by Collier of addition of the test substance. Vane (25) intro- (27), Ferreira and Souza Costa (28) and Gryglewski duced the idea of superfusing several tissues in and colleagues (29). cascade (generally up to six, arranged in two banks) (Fig. 1). Besides being useful for the parallel assay of individually injected samples, this arrangement also (b) Choice of bioassay tissues allows parallel analysis of the active components It is usually possible to find a piece of smooth present in a fluid stream (most commonly Krebs' muscle which is particularly sensitive to the hormone on September 23, 2021 by guest. solution) taken from the outflow of a perfused organ. under investigation and relatively insensitive to other Another innovation introduced by Vane (25) was substances. Indeed, think of any part ofthe body and to use blood as the superfusion medium (the blood- you can be sure that the pharmacologist has cut it bathed organ technique). The anaesthetized animal is out, put it into an isolated organ bath, or perfused its heparinized and blood is continuously removed at a vessels in order to study the effects of drugs. For constant rate of 10-15 ml/min (dogs, cats and bioassay, segments of the gastrointestinal tract or rabbits). Lower rates can be used from guinea-pigs spirally-cut strips of vascular tissue have mainly been (26). The blood (either from a vein or from an artery) used. Such procedures are the backbone not only of superfuses the assay tissues and is then returned by bioassay but also of classical pharmacology. gravity to a large vein. Fig. 2 depicts the reactions of some superfused Nobel Lecture 745 Postgrad Med J: first published as 10.1136/pgmj.59.698.743 on 1 December 1983. Downloaded from I- - - t l - FIG. 2. Diagram showing the reactions of some blood-bathed organs to various endogenous substances in concentrations of 0 1-5 0 ng/ml. The actions of the catecholamines can be abolished by treatment with suitable blocking agents. isolated tissues to various endogenous substances in of the limitations of the cascade superfusion bioassay concentrations likely to be found in circulating technique, the reader is referred to Vane (33) and Protected by copyright. blood. It should be remembered that when blood is Moncada, Ferreira and Vane (34). used as the superfusing medium, some smooth muscle preparations (but not others) exhibit an (c) Measurement ofsubstances by cascade superfusion increased resting tone often lasting for the duration of bioassay the experiment. Such increased tone reduces sensitiv- ity to substances which cause contraction but in- The technique is well suited for measuring sub- creases sensitivity to those which induce relaxation. stances released into the circulation,
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