Contraction of guinea pig trachea by epidermal

Contraction of guinea pig trachea by epidermal growth factor - urogastrone P. PATEL,H. ITOH,K. LEDERIS, AND M. B. HCBLLENBERG' i?ndocrine Research Group, Depcsrtme~aEof Pharmacology and Ther~ilpeutiss,FacuE~p:of Medicine, University sf Calgary, Calgary, Alta., Canada E N 4 N l

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Renmin University of China on 05/27/13 For personal use only.

Received Mack 2. 1988 PATEE,P o ,ITOH,H., LEDERIS, K . , and HBLLENBERG, M. D. 1988. Contraction of guinea pig trachea by epidermal growth factor - urogastrone. Can. J . Physiol. P h m a c o l . 66: 1308- T 3 12. TOevaluate further the action of epidermal growth factor- urogastrone (EGF-URO) in smooth muscle systems, we examined the effect of the peptide on guinea pig tracheal strips. The cumulative addition of EGF-UWO to the organ bath resulted in a concentration-dependent tonic contraction without tachyphylaxis. The half-maximal contraction was obtained at 13 9 3 ng/rnl EGF-URO (2 nM). The maximum contraction at 100 ng/rnL approached 60% of that induced by 1 p M histamine. No significant difference in the EGF-URO-induced contraction was observed in the presence or absence of a functional epithelium. Beincubation with 1 pM indomethacin for 20 min abolished the action of EGF-URB. The contractile effect of EGF-URO was not affected by yohimbine, propranolol, atropine, tetrodotoxin, and esculetin. However, mepacrine caused inhibition by 37 k 7% (mean L SEM for ra = 3). VeraparniX (10 pM) inhibited the EGF-induced response by 62 +s 5% (mean & %EMfor n = 4); the response was also absent in Ca-free (1 mM EGTA) buffer. However, the response was restored after the readdition of calcium. Our results suggest that EGF-URB can modulate tracheal smooth ~nusclecontractility via a cyclooxygenase product and raise the possibility that EGF-URO might play a role in controlling pulmonary smooth muscle tone in vivo.

PATEE,B . , ITOH,H., LEDERIS,K . , et HQELENBERG, kl. D. 1988. Contraction of guinea pig trachea by epidermal growth factor - urogastrone. Can. J. Physiol. Phamacol. 66 : 1308- 1312. Pour Cvaluer davantage I'action du facteur de croissance tpidermiqaae urogastrone (EGF-URO) dans les systkmes B muscles Bisses, nous avons exmink l'effet du peptide sur des bandes trachkales de cobaye. L'addition cumulative d'EGF-URO A la prkpxation a rQuEtC en une contraction eonique concentration-dipendante sans tachyphylaxie. La contraction demi-maximale a CtC obtenaae B 13 + 3 ng/mL d9EGF-URO (2 nM). La contraction maximale B 100 ng/mL ktiait e n v i r ~ n60% de celle induite par 1 p M d'histamine. On n'a observC aucune diffkrence significative dans la contraction induite par 19EGF-URO en prksence et en I'absence d'un Cpithelium fomctionnel. Une pr6incubatiasn avec I FM d'indomCthacine pendant 20 min a aboli I'actioam de I'EGF-URO. L'effet contractile de l'EGF-URO n9apas Ctk affect6 par la yohimbine, le propranolol, I'atropine. la tktrodotoxine et I'esculttine. Toueefois, Ba mkcaprine a produit une inhibition de 36 2 7% ((rt-ETpour n = 3). Le vCrapmil(10 pM) a inhibk la riponse induite par BH'EGF de 42 L- 5% (AET pour n = 4); la rkponse a aussi 6tC absente dans un tampon (1 m M d9EGTA) sans Ca. Toutefois, la rCpsnse s'est retablie a p e s la &addition de calcium. Nos rdsultats saggitrent qaae I'EGF-UR0 peut modifier %acontractilitk daa muscle lisse trachkal via un produit de cyclooxygCnase; ils augmentent aussi la possibilitk que l'EGF-LTR0 puisse jouer un r6le en contr6Eant le tonus du muscle lisse pulmonaire in vivo. [Traduit par la revue]

Introduction Epidermal growth factor - urogastrone (EGF-URB), a 53 residue single-chain polypeptide of about 60-00Da, was first isolated from mouse submaxilay glands (Cohen 1972) and from human urine (Gregory et a&.1977). EGF-URB has been studied extensively for its effects on cellular proliferation and ow a variety of parameters associated with cell growth (Carpenter and Cohen 1979; HolHenberg 1979; Carpenter 188 1). The ability sf EGF-URO to regulate blood Wow in vivo (Gan eta!. 1987a) and to modulate the contractility of isolated arterial strips in vitro (Murarnats~et ak. 1985; Berk et wH. 1985; Muramatsu et al. 1986;Gan et aH. 1987b ) has been reported. Murmatsu eta&. (1985) observed that the EGF-URB-induced contraction of rat ileocolie artery preparations in vitro was abolished by indomethacin, thereby implicating a role for prostaglandins in the contractile effect of EGF-URO. The ability of EGF-URO to stimulate prostaglandin production has been observed in a variety of tissues, including the perfused rat stomach (Chiba et al. 1982), a mouse fibrosxcsma cell line (Hiratat et al. 1985), and cultured osteoblastic cells (Yokota et al, 1986). Since receptors for EGP-URB are known to be present in smooth muscle-containing tissues other than the vascaalature (Bhagava et al. 19'39; Hsfmann et aH. 1984; Mukku and Staracel 1985; G d n e r et al. 1987), our own work has begun to focus on the action of EGF-URO on nonvascular smooth muscle-containing ' ~ u t h o rto whom correspondence should be addressed.

preparations (Muramatsu et a / . 1988) and on the possible involvement of the cyclooxygenase pathway. In the work we report here, we describe the indomethacin-sensitive contractile effect of EGF-URO on isolated helical strips sf guinea pig trachea.

Methods isolation of tissue for bioassay Mde guinea pigs, weighing 250-350 g, were sacrificed by a blow on the head and were decapitated. The thorax was opened md the heart, lungs, and trachea were removed en bloc. The trachea was cleaned of adhering fat and connective tissue, and was cut spirally from the larynx to the carha (Constantine 1965). Helical tracheal strips (2.5 x 10 man) were suspended vertically in a plastic organ bath containing 4 Hnk of Krebs-Henseleit solution sf the following composition (d): NaC%, 115; KCB, 4.7; CaC12, 2.5; MgCB2, 1.%; N a C O 3 , 25.0; KH2P0,, 1.2; md dextrose, 10.0 in distilled deionized water. The bath medium was maintained at 37'C and was aerated before and during each experiment with a mixture of 95% O2 and 5% C02. Under these conditions, the pH 45.4) remained constant. A resting tension of 1 g was applied. This tension was found to be optimal for the contractile effect of histamine (1 (aM). Tissue was allowed to equilibrate for 68 min during which time the tissue was washed at 20-min intervals. The contractile responses were recorded isometrically though force-displacement transducers, After equilibration, the integrity of the preparation was evaluated by measuring the response to 1 kE%Ihistamine (this concentration of histamine yielded a response 50% of maximum). Drugs were added directly to the bath, and concentrations were calculated accordingly.


PATEE ET AL.

Reagents Mouse epidermal growth factor - urcsgastrone was isolated from fresh-frozen submaxillary glands of testosterone-treated male mice as previously described (Savage and Cohen 1972). Routinely, the peptide isolated in our laboratory by this method yields a single band upon gel electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels (15%) under reducing conditions and a single peak upon reverse-phase liquid chromatography (C- 18 column) using an acetonivile gradient in 0.1 % trifluoracetic acid. In a mitogenesis assay (human skin fibroblasts) EGF-URO prepared in our laboratory and stored frozen (0.2-1 mg/mL in 50 mM sodium bicarbonate) has a potency (ECS8)of approximately 0.25 n g / d (Hollenberg and Gregory 1980). This value is in accord with the potency reported for murine f3-EGF-URO or for murine a-EGF-UROthat has been stored frozen in buffer solutions (Matrisian et ark. 1982). The absolute concentration of EGF-URO in stock solutions, from which appropriate dilutions were added to the organ bath, was measured spectrophotornetrically (Hollenberg and Gregory 1980). All other reagents were purchased from the following suppliers: yohimbine, propranolol, atropine. tetrodotoxin, esculetin, mepacrine, histamine, and verapamil from Sigma Chemical Co., St. Louis, MO; and indomethacin from Merck Sharp & Dohme, Borval, Quk., Canada. The reagents, except for indomethacin, were dissolved in distilled water; indomethacin, dissolved in dimethyl sulfoxide (DMSO), was diluted so that the final DMSO concentration in the organ bath was 10.81%. This concentration of DMSO alone had no effect on the preparation.

Results Response of helical tracheal ,~tripsPO EGF-URO EGF-UWO produced a sustained contraction of the guinea pig tracheal preparation that began within 3-5 min and lasted for at least 1.5-2 h (Fig. 1). The magnitude of the maximal contractile response to EGF-URO (at 100 ng/mL) appeared to vary somewhat from one batch of animals to another (the same animal strain was ordered from different sources) with a range between 50 and 650 mg tension. However, there appeared to be less variability within a single experimental series using a single batch of animals; the normalization of data relative to the contractile action of an optimal concentration of EGF-URO (100 ng/mL) yielded a reliable basis for comparing observations obtained with different batches of animals in separate experimental series. The reasons for these variabilities in responsiveness are the sub~ectof a separate study. In general, the magnitude of the tissue response to histamine ( I pM) served as a reliable prognostic indicator of a good response to EGF-URO. In general, the response to the first test concentration of EGF-UWO was reproducibly found to be somewhat smaller (by about 10%) than the reproducible response to the second and subsequent test concentrations of EGF-URO. Therefore, the second contraction caused by EGF-URO was used either as the first response for concentration-response curves, or as a standard response (maximal contraction caused by 100ng/mL EGF-URO) for the evaluation of putative inhibitors of EGF-URO action. Upon cumulative administration, EGF-URO produced a concentration-dependent contraction (Fig. 2A). The concentration-response curve for EGFUWO (Fig. 2B) administered by a cumulative dosing regimen demonstrated a minimum effective concentration of 1-3 n g / d , a half-maximal contraction (ECSCD) at 13 9 3 ng/mL (2 nM, mean 9 SEM for n = Q), and a maximal contraction at 100-200 ng/mL. For the concentration-response curve, the contraction at 100 ng/rnL was taken as 100%. Although concentration-response curves were not concurrently measured using single concentrations, as was done in our previous work (Muramatsu et al. L985), the EGOobtained by the cumulative dosing

FIG. 1. Contractile response to EGF-URO: time course and effect of indomethacin. (A) A typical response to 180 ng/rnL EGF-URO (A, 130mg tension) is shown both before (left) and after (middle) pretreatment of the tissue for 20 min with 1 pM indomethacin (V). The response of the indomethacin-treated tissue to 1 pM histamine (right, 0, 2Wmg tension) is also shown. (B) lndorneehacin (1 pM, V) was added at a time when the tension generated by 100 ng/mL EGF-URO (A) had reached a maximum. (C) Prolonged response to 100 n g / d EGF-URO (A). Tension was maintained until the tissue was washed (W, mow).

regimen with the tracheal tissue was essentially the same as the value measured previously using arterial strips.

E e c r s of removal of epithelium To evaluate the possible participation of the epithelium in the response to EGF-URO, helical strips were prepared without the use of wire so as to preserve the epithelium, and the EGFURO-mediated response of these specially prepared strips was compared with the response of strips that had been rubbed on buffer-wetted filter paper to remove the epithelium. The presence or absence of a functional epithelium was assessed by determining the ratio of response to KC1 (50 mM) and histamine (1 pM). A change in this ratio of response has been used as an indicator of the removal of a functional epithelium (Barnes et ~ l . 1985). Since removal of the epithelium causes an increase in the sensitivity of guinea pig tracheal preparations to histamine, but not to KC1 (Goldie et ak. 1986; Murlas 1986), we anticipated that the ratio of response (KC1:histamine) to submaximal concentrations of these two agonists would decrease, as was observed (Table 1). As indicated in Table 1 , although removal of the endothelium decreased the KCl: histamine ratio of contractility, there was no significant effect on the contractile activity of EGF-URO. Calcium requirement As was observed in our previous study (Muramatsu rt al. 1988), no contractile effect of EGF-URO was observed if calcium was removed from the organ bath with concomitant EGTA chelation (Fig. 3B). Nonetheless, restoration of the calcium to the medium yielded a response in a preparation that had been nonresponsive when pre-exposed to EGF-URO (Fig. 3B). Re-exposure to verapamil (Fig. 3A: 10 min, 18 pM) also inhibited the EGF-URO response (by 6255%, mean k §EM for n = 4), although not as completely as did the total removal of calcium from the medium. Eflecis of indomethacin Since indomethacin blocked the contractile action sf EGFURO in a vascular smooth muscle preparation (Muramatsu et ak. 1985), we were interested in evaluating the effect of indomethacin on EGF-URO action in the tracheal strip prepara-

CAW. I . PHYSIBL. PHARMACBL. VBL. 66,1988 200

/

W

W

A

B W

W

/

Ce-free buffer 1 mM EGTA

W 4 0.20


A

A

5min

FIG.3. Response to EGF-URO in the presence of verapamil (A) and in calcium-free buffer (B). (A) A standard response to 100 n g / d EGF-URO (A) was measured and the tissue was then exposed for 10 min to 18 p,M verapamil (O). EGF-URO ( 1 0 ng/mL) was again added to the tissue bath (A). On average, verapamil reduced the contractiie effect of EGF-URO by 62 a 5% (n = 4). W, tissue wash. (B) After a standard response to 100 ng/mL EGF-URO (A) was measured, the tissue was washed (W) and incubated for 10 min in calcium-free buffer containing I d v l EGTA; 100 ng/mL EGF-URO was again added (A). Fifteen minutes after the addition of EGF-URO, the calcium concentration was restored by the addition of 3.5 mM CaC12 ( 0 ) .The ensuing contraction was monitored before the tissue was washed (W).

CUMULATIVE CONCENTRATION OF EGF-URB (ng/mb)

FIG.2. Graded response to cumulative additions of EGF-URO and concentration-response curve. (A) Increasing amounts of EGF-URO were added to the organ bath to achieve a final concentration (rag/mL) indicated by the numbers above each symbol (V). The tracing is representative of results obtained in six independent experiments. (B) Data from six independent experiments, like the one shown above (A), were pooled to construct a concentration-response curve. The tension generated by lWng/mL EGF-URO (270 k 60mg) was taken as 108% for the calculation of the percent contraction caused by other concentrations of EGF-URO. Data points represent the mean values for which the vertical bars represent the SEM for n = 6. TABLE1. Effect sf removal of epithelium Response to Epithelium Present Absent

EGF-URO (mg tension)

KC]: histamine 2.420.2 1.2+-0.1" -

911927 911931 -

-

-

-

-

-

NOTE:Paired spiral strips from five separate preparations were either retained intact or were rubbed on wetted filter paper to remove the epithelium. The contractile responses to histamine ( I FM), KC1 (50mM), and EGF-URO ( I 0 0 ng/rnL; I6.7nM) were then measured. The effectiveness of removal of the epithelium was monitored according to the reduction in the ratio of contractile respnsiveness to KC1 and histamine (relative response, KCl: histamine). * p < 0.05 for difference of KC1:histamine ratio between rubbed aSB$ intact preparations.

tion. Ow its own, indomethacin (1 pM) reduced the resting tension from I to about 0.4 g; this tension was readjusted to 1 g before evaluating the effect of indomethacin on EGF-URO action (Fig. 1). Pretreatment with indomethacin (1 FM)for 20 min cornpleaeHy abolished the contractile effect of EGF-URO (Fig. 1A). In contrast, the contractile response to histamine ( I pM) was still present, even though indomethacin has been observed to reduce the contractile action of histarnine at rnicromola concentrations (Orehek et a / . 1975). The inhibitory

action of indomethacin was observed whether added prior to EGF-URO or added at a time when maximum tension was generated in response to EGF-URO (Fig. IB).

Effects of receptor antagonists and other compounds on EGFURO-induced contractions A number of compounds were evaluated for their ability to modulate the contractile action of EGF-URO. All of the agents were added to the organ bath 10 min before the addition sf the next dose of EGF-UWO (100 ng/mL). The ensuing contraction was compared with the one measured in response to EGF-URO (100 ng/mL) administered prior to the treatment of the tissue with each test compound. Several potential receptor antagonists including atropine (10 bM) , propranolol ( 10 pM) , yohimbine (10 p ~ ) and , cyproheptading (10 pM) had no effect on EGFURO-mediated contraction. Tetmdotoxin (1 pM) was dso without effect. The phospholipase-inhibitory arachidonate release inhibitor, mepacadne ($0bM) showed a partial (34%) inhibition of EGF-URO-induced contraction; however, the lipoxygenase inhibitor, esculetin (1 pM) and the prostacycliw synthetase inhibitor, tranylcypromine Q 10 pM) , had no effect. Qualitative effects of prostaglsndi~as Preliminary experiments were done to determine which, if any of the prostaglandins, might mimic the effect of EGF-URO. In agreement with a previous report (Coleman and Kennedy B980), prostaglandin F2, (1 pM), like EGF-UWO, caused a sustained contraction of the helical strip. Ira contrast, in accord with previous observations (Coleman and Kennedy 1980) neither prostaglandin El nor E2 elicited a contractile response; both of these agents caused either w s change in resting tension or a modest relaxation (in four of seven trials for PGEI and three of six trials for PGG: data not shown). It was decided that concentration-response curves for these prostaglandins would not yield decisive information and thus, no further comparative experiments were done.

Discussion The main finding of our study was that in isolated helical strips of guinea pig trachea, EGF-URO caused a concentxationdependent (ECS0 ~2 wM) contractile response that requires extracellular calcium and that is completely abolished by


indomethacin. The response does not appear to require the presence of an intact epithelium and does not appear to be due to the release of other smooth muscle agonists, apart from the prostanoids. In many ways, this reponse reflects the one we have observed previously in isolated vascular smooth muscle strips, wherein EGF-URO causes an indomethacin-sensitive contraction, with an Ee5()in the nanomolar range. However, in one important respect, related to tissue desensitization, the responsiveness of the tracheal strips differs from the contractile response we have observed previously (Muramatsu et al. 1985). In the indomethacin-sensitive rat vascular preparation, we observed a transient response to EGF-URO (tension returned to baseline witkin 20 min in the continued presence of EGF-URO), and a rapid tachyphylaxis (no further resgasnse was generated by adding a second dose of EGF-URO cumulatively to the organ bath). In contrast, the tracheal preparation responded with a sustained increase in tension which was augmented by the cumulative addition of EGF-URO to the organ bath (Figs. 1 and 2). This response was comparable to the one reported previously by Berk et al. (1985) for a rat aortic preparation, and was analogous in some respects to the respnse reported by Gardner et al. (1989) for a mature rat uterus preparation. Unfortunately, we were unable to reproduce the observations of Berk et al. (1985) with rat aortic strips for purposes of comparison with our new results. Thus, as opposed to the vascular preparations in which the action of EGF-URO exhibits a rapid desensitization phenomenon, the tracheal preparation would appear to be an attractive system in which to study the prolonged time course of EGF-URO action at the biochemical level. Although prostaglandin release during histamine- or acetylcholine-induced contraction of guinea pig trachea has been observed previously (Grodzinska et al. 1975; Orehek et al. 1975), the released prostaglandins were not required for the contractile actions of these agonists, as was the case for the contractile action of EGF-UWO. In our work, we used a concentration of indomethacin corresponding to the "low dose" used in the previous studies (Orehek et al. 1975); this concentration should be relatively specific for the inhibition of cyclooxygenase. Under our conditions of assay (1 g tension) because neither PGEl nor PG& caused a contraction in the tracheal preparation, it would appear that prostaglandins of the E-series cannot account for the action of EGF-URO; the observed contractile effect of PGF2, would point to prostanoids of the F-series as likely candidates to mediate the contractile response. The involvement of prostaglandins in the action of several vasoactive polypeptides has been documented in other smooth muscle preparations (Gemitsen ct al. 1981 ; Manku and Horrobin 1976). Further analytical work will be required to explore this possibility for the action of EGF-URO in the tracheal preparation. Our data indicated that a functional epithelium was not required for the action of EGF-URO. Thus, the most likely target for the polypeptide would appear to be the smooth muscle element per se; receptors for EGF-URO have been previously detected in smooth muscle preparations (Bhargava et al. 1979; Hofmann et aE. 1984; Mu&u and Stance1 1985; Gardner et al. 1987). However, it is difficult to mle out the possibility that the EGF-URO-mediated release of prostaglandins from nonmuscle elements of the tracheal preparation may have caused the observed contractile response. Further work with isolated tracheal-derived smooth muscle cell cultures may be required to resolve this question. Extracellular calcium was clearly required for the EGF-

LRO-mediated response. The partial inhibition caused by the phospholipase-inhibitory compound, mepacrine, could suggest further that a calcium-flux-triggered phospholipase might participate in the prostanoid-mediated response triggered by EGF-URO. Previous work has established that EGF-URO can stimulate calcium uptake in a target like the A431 cell and that this calcium uptake may be linked to the activation of the phosphatidylinositol pathway (Sawyer and Cohen 1981; Moolenaar et al. 1986). It remains to be seen whether the simultaneous elevation of intracellular calcium, the activation of phospholipases (possibly both A2 and C), with the subsequent triggering of kinase C (Rasmussen et al. 1984;Baraban et al. 1985), and the synthesis of cyclooxygenase pathway products play a concerted role in the overall response of tracheal preparations to EGF-URO . Irrespective of the precise cellular target for the contractile action of EGF-URO (i.e., smooth muscle cell or another cell type) our data indicate that EGF-LTRO (and possibly other growth factors) can potentially regulate pulmonary smooth muscle tone in vivo. This action could possibly play a role in viva wherein EGF-URO or related substances (transforming growth factor-a) might be released either from tumours (DeLarco and Todaro 1998; Roberts and Spom 1985) or from embolus-trapped platelets (Oka and Orth 1983). Unfortunately, because of a lack of information concerning the levels of either EGF-URO or transforming growth factor-ar that might be reached in the pulmonary circulation, the true physiological significance of our observations remains to be determined. Nonetheless, continuing work in our laboratory is focused on the effects of EGF-URO in perfused intact lung preparations.

Acknowledgements This work was supported by term grants (to M.D.H. and K.L.) from the Medical Research Council of Canada. H.H. was supported by a fellowship from the Alberta Heritage Foundation for Medical Research. BARABAN, J. M., GOULD, W. J., PEROUTKA, S. J . , and SNYDER, S. H. 1985. Phorhol ester effects on neurotransrnission: interaction with neurotransmitters and calcium in smooth muscle. Proc. Natl. Acad. Sci. U.S.A. 82: 684-609. BARNES, P. J., CUSS,I?. M . , and PALMER, J. B. 1985. The effect of airway epithelium on smooth muscle contractility in bovine trachea. Br. J. Phmacol. 86: 685-691. BERK,B . C., %ROCK, T. A., WEBB, W. C., TAUBMAN, M. B., ATKINSON, w. J . , GIMBWONE, M. A., JR., and ALEXANDER, R. w. 1985. Epidermal growth factor, a vascula smooth muscle mitogen, induces rat aortic contraction. J. Clin. Invest. 75: 1983- 1086. BHARGAVA, G., WIFAS,L., and MAKMAN, M. H. 1939. Presence of epidermal growth factor receptors and influence of epidermal growth Pictor on proliferation and aging in cultured smooth muscle cells. J. Cell. Physiol. 100: 245-374. CARPENTER, G. 1981. Tissue growth factors. In Handbook of experimental pharmacology. Vol. 57. Edited by W. Baserga. Springer-Verlag, Berlin, Heidelberg, New York. pp. 89- 132. CARPENTER, G., and COHEN,S. 1979. Epidermal growth factor. Annu. Rev. Biochem. 48: 193-216. CHIBA, T., WIRATA, Y., TAMPNATO, T., KADOWAKI, S., MATSUKUWA, S., and FUJITA,T. 1982. Epidermal growth factor stimulates prostaglandin E release from isolated perfused rat stomach. Biochem. Biophys. Wes. Csmmun. 105: 370-374. COHEN, S. 1972. Epidermal growth factor. J. Invest. Dematol. 59: 13-16. COLEMAN, R. A., and KENNEDY, I. 1980. Contractile and relaxant actions of prostaglandins on guinea-pig isolated trachea. Br. J . Phmacol. 68: 533-539.


1312

CAN. J. PHYSHOL.

PHAWMACBL. VOL. 66, I988

CONSTANTINE, J. W. 1965. The spirally cut tracheal strip preparation. J. P h m . Pharmacol. 17: 384-385. DELARCO, J. E., and TODARO, 6. J. 1978. Growth factors from murine Natl. Acad. Sci. U.S.A. 75: sarcoma vims-transformed cells. ROC. 400 1-4005. AN, B. S., MACCANNELL, K. L., and HOLLENBERG, M. D. 1987a. Epidermal growth factor - urogastrone causes vasodilatation in the anaesthetized dog. J. CIin. Invest. 80: 199-206. GAN,B. S., HOLEENBERG, M. B., MACCANNELL, K. L., LEDERHS. K . , WINKEER, M. E., and DEWYNCK, R. 1987b. Distinct vascular actions of epidemal growth factor - urogastrone and transforming growth factor-a. J. P h m a c o l . Exp. Ther. 242: 331-337. GARDNER, R. M., LINGHAM,R. B., and STANCEL,G. M. 1987. Contractions of the isolated uterus stimulated by epidermal growth factor. FASEB. J. 1: 224-228. GERRITSEN, M. E., MORGAN, D. O., PARKS,T. H Z . , PRINTZ,M. P., and LEDERIS,K. 1981. A proposed role for prostaglandin in the moduIation of the relaxation response to urotensin 1 in isolated rat arteries. Prostaglandins, 22: 873-892. GOLDIE,R. G., PAPADIMHTMOU, J. M., PATERSQN, J . W., RIGBY,P. J., SELF,H.M, , and SPINA,B. 1986. Influence of the epithelium on responsiveness of guinea-pig isolated trachea to contractile and relaxant agonists. Br. J. P h m a c o l . 87: 5- 14. GREGORY, W.. BOWER,J. M., and WILLSHIRE, I. R. 1977. Urogastrone and epidermal growth factor. In Growth factors. Edi~edby K. W. Kastrup and J. H. Nielsen. Proc. FEBS Meet. 48: 75-84, GRODZHNSKA, L., PANCZENKO, B., and GWYGLEWSKI, R. J. 1975. Generation of prostaglandin E-like material by the guinea-pig trachea contracted by histamine. J. Pharrn. Pharmacol. 27: 88-91. MIRATA,Y.,UCWIHASHI, M., NAKASHIMA, H., FUSITA,T., and MATSUKURA, S. 1985. Stirnulatory effect of epidermal growth factor on prostaglandin E2 production in mouse fibrosarcoma cell line (HSDMIC1). Horn. Metab. Wes. 17: 448-458. HOFMANN, G. E., WAO,C. V., BARROWS, 6 . M., and SAN-FILIPPO, %. S. 1984. Binding sites for epidemd growth factor in human uterine tissues and leiomyomas. J . CBin. Endocrinol. Metab. 58: 880-884. HOLLENBERG, M. D.1979. Epidermal growth factor - rarogastrone: a polypeptide acquiring homonal status. Vitam. Horn. 37: 69- 110. HBLLENBERG, M. D., and GREGORY,H. 1980. Epidermal growth factor - urogastrone: biological activity and receptor binding of derivatives. MOB. P h m a c o l . 87: 3 14-320. MANKU,M. B., and ~ O R ~ O BD. I NF. , 8976. Endomethacin inhibits responses to all vasoconstrictors in the rat mesenteric vascular bed: restoration of responses by prostaglandin E2. ProstagIandins, 12: 369-376.

M A T R ~ ~ ~L.A M., N , LARSEN,B . R., FINCH!J. S . , and MAGUN. B. E. 1982. Further purification of epidermal growth factsr by high-performance liquid chromatography. Anal. Biochem. 625: 339-35 1 . MOOLENAAR, W. H., AERTS,R. J., TERTOBLEN, L. G. J., and BE LAAT,S. W. 1986. The epidermal growth factor-induced calcium signal in A431 cells. J. Biol. Chern. 261: 279-284. MUKKU, V. R.,and STANCEL, G. M. 1985. Receptors for epidermal growth factor in the rat uterus. Endocrinology (Baltimore), 617: 149- 154. MURAMATSU, I., HOLLENBERG, M. D., and LEDERIS,K. 1985. Vascular actions of epidermal growth factor - urogastrone: possible relationship to prostaglandin production. Can. J. Physiol, P h m a col. 63: 9%-999. 1986. Modulation by epidemal growth factor - urogastrone of contraction in isolated canine helical mesenteric merial strips. Can. J. Physiol. PhmacoB. 64:1561-1565. MURAMATSU, I., HTOH, H., LEDERIS,K., and HQLLENBEWG, M. B. 1988. Distinctive actions of epidermal growth factor - urogastrone in isolated smooth muscle preparations from guinea pig stomach: differential inhibition by indomethacin. J. Pharmacd . Exp. Ther. 245: 625-63 1 . MURLAS, C. 1986. Effects of mucosal removal on guinea-pig airway smooth muscle responsiveness. Clln. Sci. 79): 571 -575. OKA,Y.,and ~ R T HD., N. 1983. Human plasma epidermal growth factorlp-ursgastrone is associated with blood platelets. J. Clin. Invest. 72: 249-259. OREHEK,J . , DOUGLAS,9. S., amd B ~ U H U YA. S , 1W5. Contractile responses of the guinea-pig trachea in vitro: modification by prostaglandin synthesis-inhibitiing drugs. J. P h m a c o l . Exp. Ther. 194: 554-564. RASMUSSEN, H., FOWDEW, J., KOJHMA, I., and SCRIABHNE, A. 1984. TPA-induced contraction of isolated rabbit vascular smooth muscle. Biochem. Bioghys. Res. C o m u ~ a 122: . 776-780. ROBERTS,A. B., and SPORN,M. B. 1985. Transforming growth factors. Cancer SUPV,4: 683-705. SAVAGE, C. R., JR., and COHEN,S. 1972. Epidermal growth factor and a new derivative. J . Biol. Chem. 247: 7609-761 1 . SAWYER, S. T o ,and COHEN,S . 1981. Enhancement of calcium uptake and phosphatidylinositd turnover by epidermal growth factor in A43 1 cells. Biochemistry, 20: 6280-6286. YBKOTA, K . , KURIHARA, N., YOSHINO, T., ~ ~ ~ K M E G A M.W1986. A, Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-EI) by epidermal growth factor. J. Biol. Chem. 261: 15 410-15 415.