Toxicon, 1976, Vol. 1~, pp . 171-t78 . Petaaman Prcu . Printed In Great Hrftain .

AN IN VITRO STUDY ON EFFECT OF PIMELEA PLANTEXTRACT ON PULMONARY VEIN SMOOTH MUSCLE

KENNETH MASONDepartment of Veterinary Clinical Studies, University of Queensland, St, Lucia,

Brisbane, Queensland, 4067 Australia

(Acceptedjor publication 30 October 1975)

K. MASON. An in vitro study on effect of Pimelea plant extract on pulmonary vein smoothmuscle. Toxicon i4,175-178, 1976.-A direct contraction of pulmonary vein smooth muscleto Plme%a plant extract was demonstrated in vitro. This effect is discussed with regard totheories on the pathogenesis of St . George disease, and it is concluded that these resultssupport Clark's hypothesis which is based on a contraction of smooth muscle . The potentialof this preparation as a bioassay of the toxins) is discussed.

INTRODUCTION5T . GBORGE disease has been recognised as a syndrome ofcattle in specific areas ofAustralia(MAiJNDER, 1947 ; CANTELLO, 1969 ; DonsoN, 1965) . The clinical picture is of cattle inpoor condition with variable diarrhoea and is characterized by jugular distension (CLARK,1971b), and oedema of the head, neck and brisket. Pathological findings include hydro-thorax and enlarged swollen bluish-purple liver and anasarca involving the subcutaneoustissues of the head, neck and brisket (MAUNDER, 1947 ; SEAWRIGHT and FRANCIS, 1971).Dilatation of the right ventricle of the heart was a consistent finding by CLARK (1971b).Clinical pathology by SEAWRIGHT and FRANCIS (1971) in the progressive stage of thedisease showed a fall in haemoglobin concentration, red cell count, packed cell volumeand plasma protein accompanied by a leucopaenia involving mainly lymphocytes.

HURST (1942) reported the toxicity of Pirnelea simplex and CANTELLO (1969) providedfield evidence that P. simplex was associated with a condition resembling St . George disease.Experimental evidence implicating P. simplex was provided by RosERTS and HEALY (1971)and McCLURE and FARROW (1971) . Another Pimelea species was implicated when CLARK(1971a) induced a syndrome identical with St . George disease by forced inhalation andingestion of dried P. trichostachya.

While McCLURE and FARROW (1971) referred to the possible role of hypoproteinaemiain the pathogeneses of St . George disease, SEAWRIGHT and FRANCIS (1971) postulated anexcess sodium retention by the kidney to be the key primary disturbance. CLARK (1971b)suggested that the primary disturbance was an increase in the pulmonary vascular resistancecaused by pulmonary venule constriction .

CLARK (1973) reports that, after intravenous administration of plant extract, the leftatrial and the systemic arterial pressure fall, as does the cardiac output, while the rightventricular pressure and pulmonary arterial pressure rise . Although Clark only used twocalves, the pulmonary vascular resistance in each of these rose markedly while the systemicvascular resistance was much less aífected . CLARK (1973) reasons thus : "The increase inpulmonary vascular resistance that intravenous injection of this plant extract causes in

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calves could result from constriction of the pulmonary arterioles were it not for the pul-monary congestion and oedema, which, if hydrostatic in origin, must be due to events onthe venous side of the capillary bed. The fall in left atrial pressure eliminates stenosis orinsufficiency of the left atrio-ventricular valve or left ventricular failure as causes of in-creased pulmonary capillary hydrostatic pressure . Constriction of part of the pulmonaryvenous system is the other possibility ."

Lungs were obtained at the abattoir within 20 min of exsanguination of the animal . The pulmonaryvein and some of its major branches were quickly dissected out, placed in warm Krebs-Henseleit solution(KREB and HENSELEIT, 1932) containing 0~4~ glucose, and immediately transported to the laboratory .During the experiment spare tissue was kept in this solution throughwhich was bubbled S~carbon dioxidein oxygen . A tension of S00 mg was applied and responses were measured isotonically on smoked drumswithamagnification of4-S times . The strips were allowed to settle until a steady base line wasachieved.A pilot study on nine tissue established an approximate dose response curve to S-hydrozytryptamine

(SHT) for this preparation (Fig . 1) . All subsequent preparations were tested with 2 ng of SHT prior toexperimentation, those not conforming with the established dose-response curves were discarded .

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MATERIALS AND METHODS

pose ,

ngFIG. 1. A DOSE-RESPONSE CURVE FOR SHT ESTABLISHED IN THE PREPARATION OF PULMONARY

VEIN, AND USED TO STANDARDIZE THE PREPARATION.Asolid line is drawn through the means. Standard error ofthe means is0322and the differences

between dosages are significant (P < 001) .

The SHT was stored at a concentration of 1 mg per ml in Krebs-Henseleit solution at 8°C. Each daydilutions to 10 ng per ml in Krebs-Henseleit were made for the purpose of testing the sensitivity of thepreparation . A crude alcoholic extract ofPimelea trichostachya was supplied by W. R. Kelly of the Depart-ment of Veterinary Preventive Medicine, University of Queensland . This was prepared by soaking milledplants in two changes of absolute ethyl alcohol for 24 hr each, the extract was then filtered and evaporateduntil a tarry residue remained . The extract was dissolved in ethyl alcohol for use in the organ bath .

After demonstrating normal function and sensitivity of the preparation (by response to 2 ng SHT) thepreparation was washed and allowed to settle to an even base line (approximately 3-S min) . Two prepara-tions were given 004ml ethyl alcohol, the solvent used and six were dosed with 10 mg of crude extract in004 ml ethyl alcohol but in three of these the dose was washed out after 6 min (Fig. 2) .

RESULTS

Figure 1 is the dose-response curve for SHTestablished in this preparation . This prepara-tion was sensitive to levels of SHT as low as 0~1 ng per ml in the organ bath . The processcould be reversed by washing; the preparation returning to the base line .

Figure 2 represents examples of results obtained with Pimelea extract . Part A shows

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A

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I0 2 4 6 8 10 12 14 16

Time, minFIG. 2. TRACING OF SPIRAL STRIP OF HOVINB PULMONARY VEIN IN lO ml ORGANBATH SUHIECTEDTO (a) O'O4 ml ETHYL ALCOHOL; (b) lO IIIg OF Pimelea EXTRALI IN O'O4 ml ETHYL ALCOHOLAND

(c) 10 mg Pimelea ExTRACr nv 0'04 ml ETHYL ALCOHOL WASHED OUT AFTER 6 min .T Point of introduction ; " wash out.

that spiral strips of bovine pulmonary vein do not show any marked reaction of 004 mlof ethyl alcohol, the solvent used . Part B shows that 10 mg of crude extract of Pimeleacaused a prolonged slow contraction after a latent period of 2-3 min, and part C showsthat this prolonged contraction could not be reversed by washing. The average height of Bat 16 min is 3~9 cm and of C, 2~8 cm with an average standard error of 021 cm. The differ-ence is significant (P < 0~0~ . All six tissues subjected to the toxin continued to contractuntil the limits of the lever system to which they were attached were reached.

DISCUSSION

Organ bath preparations of bovine pulmonary veins have been used previously instudies of bovine anaphylactic responses . The sensitivity of this preparation to SHT wascomparable to that obtained by EYRE (1970, 1971) . Response to SHT in these studiesafforded a means of discerning whether the tissue was functioning satisfactorily, and thushad not become damaged during storage and preparation.

CLARK (1971b) suggested that the pulmonary venule was involved in the pathogenesisof St . George disease and later (CLARK, 1973) produced indirect evidence that this was thecase by measuring pressure changes on each side of the cardiopulmonary vascular systemafter intravenous administration of plant extract . Thepulmonary venule of cattle is remark-able in its extremely muscular media (ALEXANDER and JENSEN, 1963). According to CLARK(1971b) this would account for the natural disease occurring only in cattle . CLARK (1973)produced indirect evidence that pointed to a "constriction of part of the pulmonary venoussystem" as being the mechanism of action of the toxin . The present experiments providedirect evidence that Pirnelea toxins) cause contraction of pulmonary vascular smoothmuscle.

The use of spiral strips ofbovine pulmonary vein as a bioassay of the toxins) in Pimeleapresents problems since the effect of the toxins) seems virtually irreversible . Consequently,a 2 x 2 bioassay against a standard cannot be carried out on a single preparation. Toovercome this, a number of preparations (3 or more) must be subjected to the standardand a similar number to the test extract . Alternatively, a threshold (all-or-none) responsemay be determined by serial dilutions of extract. This gives a less accurate comparison,

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and is time consuming ; nevertheless, the preparation of bovine pulmonary vein may beuseful for the bioassay ofthe toxins) in Pimelea spp.*

Acknowledgements-I thank Mr . W. R. KEU.Y for supplying the plant extract, Professor O. E. BUD7Z-OISENfor the use of the organ bath and laboratory facilities ; Dr. R. J. LAnn and Dr. J. M. Wirre for advice withthis project andmy wife for technical advice . This paper reports part ofa thesis submitted to the Universityof Queensland in 1973, and was supported by University funds.

REFERENCESA~xANneR, A. F. and IENSEN, R. (1963) Normal structure of bovine pulmonary vasculature . Am. J. vet .

Res. 24, 1083 .CnN'tEra.o, J. E. (1969) Does St . George disease occur in New South Wales cattle? Agrlc. Guz. N.S.W. 80,

418.CLARK, I. A. (1971a) St . George disease of cattle . Aust. vet . J. 47, 123.CLARK, I. A . (1971b)Anote on the pathogeneses of St . George disease of cattle. Aust . vet. J. 47, 285.CY.ARx, I. A. (1973) The pathogeneses of St . George disease of cattle. Res. vet. Sc1.14, 341 .DonsoN, M. E. (1965) A disease of cattle in South Australia resembling St . George disease. Arrst . Vet . J. 41,

65.EYRe,P. (1970) The Schultz-Dale reaction in bovine pulmonary smooth muscle. Br. J . Pharmac. 40, 166.E~,P. (1971) Pharmacology of bovine pulmonary vein anaphylaxis üt vitro . Br. J. Pharmac. 43, 166.HURST, E. 0942) The Poison Plants ofNew South Wales, pp . 287-294. Sydney : The Poison Plants Committee

ofN.S.W .KREa,H. A. and HENSELEIT, K . (1932) Urea formation in the animal body . Hoppe-Seyler :eZ. physiol . Chem .

210, 33 .MAUNDER, J. C. H. (1947) St . George disease of cattle . Aust . vet. J. 23, 153.McCLUR$, T. J. and FARROW, B. R. H. (1971) Chronic poisoning of cattle by desert rice flower (Pimelea

simplex) and its resemblance to St . George disease as seen in north western New South Wales. Rust . vet .J. 47, 100.

ROBERrs, H. B. and HEALY, P. J. (1971) Pimelea simplex and St . George disease of cattle. Aust. vet . J. 47,123 .RoseRrs, H. H., McCLURS, T. J., RrrcH>E, E., TAYLOR,W. C. and FResMArr, P. W. (1975) The isolation and

structureof the toxin of Pimelea simplex responsible for St . George disease ofcattle. Aust. vet. J. 51, 325.SeAwRraar, A. A. and FRANCLS, J. (1971) Peliosis hepatis-a specific liver lesion in St . George disease of

cattle . Arrst . rel. J. 47, 911 .

'Since submitting this paper Roa$Rts et al. (1975) have isolated a toxic principle which they have calledsimplexin.

roxtcorv 196 vor. t~