Renomedullary Vasodepressor Substance, Medullin:

Transcription

1 Renmedullary Vasdepressr Substance, Medullin: ISOLATION, CHEMICAL CHARACTERIZATION AND PHYSIOLOGICAL PROPERTIES By James B. Lee, M.D., Benjamin G. Cvin, M.D., Ph.D., Bertil H. Takman, F.L., and Emil R. Smith, Ph.D. With the technical assistance f Marilyn Mazze The cncept that the kidney may pssess antihypertensive endcrine activity in varius frms f experimental hypertensin " 3 has stimulated investigatins int the presence and lcalizatin f renal antihypertensive substances. Althugh vasdepressr substances have been fund in extracts f whle kidney and kidney crtex, 4 - s little evidence is available t suggest a physilgical rle in bld pressure regulatin. On the ther hand, Muirhead et al. 6 have shwn that extracts f renal medulla prevent the develpment f renprival hypertensin which therwise ccurs in the bilaterally nephrectmized animal." Althugh the principle active in the preventin f renprival hypertensin pssessed antihypertensive activity in established experimental hypertensin, n vasdepressr effect was bserved in intact nrmtensive animals. 8 Recently, crude hmgenates f renal me- Frm the Metablic Research Labratry, St. Vincent Research Fundatin, St. Vincent Hspital, Wrcester, Massachusetts. Supprted in part by Grants AM , 02, 03 frm the Natinal Institutes f Health, U. S. Public Health Service; the Wrcester Chapter, Massachusetts Heart Assciatin; Astra Pharmaceutical Cmpany, and the St. Vincent Research Fundatin. Presented in part at the Fall meeting f the American Physilgical Sciety, Brwn University, September 0, 964, and at the Annual Meeting f the American Cllege f Clinical Pharmaclgy and Chemtherapy, New Yrk, Octber 29, 964. Preliminary reprts f these studies appeared in Clin. Res. 2: 79, 964; Clin. Res. 2: 254, 964; and the Physilgist 7: 88, 964. Accepted fr publicatin December 8, 964. dulla have been shwn t prduce sustained bld pressure depressin in the nrmtensive, vagtmized, pentlinium-treated rat. 9 Althugh the material respnsible fr the vasdepressr effect is dialyzable, ethanl sluble, and f relatively lw mlecular weight, little is knwn f its chemical and physilgical prperties. Mrever, althugh bld pressure depressin has been bserved with rabbit, rat, pig, and human renmedullary extracts, 9 the mechanism f the sustained depressr effect is bscure. Thus the present studies were undertaken t () islate and characterize renmedullary vasdepressr substance(s), (2) determine the mechanism f their hyptensive actin, (3) investigate their activity n nnvascular smth muscle, and (4) cmpare the chemical and physilgical prperties f renmedullary vasdepressr substance(s) and prstaglandin E-l (PGE -). The results suggest that sustained lwering f bld pressure by extracts f renal medulla is the result f at least tw unsaturated acidic lipids which are clsely related derivatives f prstanic acid. One substance is a hithert unknwn, highly unsaturated cmpund with ptent vasdepressr effects, but with relatively weak activity in stimulating nnvascular smth muscle. This material has been called medullin in cntrast t the secnd renmedullary cmpund which has been identified tentatively as prstaglandin E-l (PGE-). Medullin appears t exert its hyptensive actin by a direct effect n peripheral arteriles resulting in peripheral vasdilatin and lwered peripheral resistance with a Circulatin Research, Vl. XVII, July 96} 57

2 58 LEE, COVINO, TAKMAN, SMITH cmpensatry increase f heart rate and cardiac utput. Methds CHEMICAL STUDIES Crude Hmgenates Specific chemical methds relating t the purificatin, islatin, and chemical characteristics f renmedullary depressr substance are described under the apprpriate heading in the sectin n Results. Either fresh r quick-frzen rabbit medulla was the surce material fr all chemical and physilgical experiments. Fr studies with fresh medulla, male albin rabbits f the New Zealand/Flemish strain, weighing between 3 and 4 kg, were killed by neck strke fllwed by cartid exsanguinatin. The kidneys were rapidly remved, decapsulated, and placed n ice-cld petri dishes fr dissectin. Each kidney was quartered, and the medulla separated frm the crtex by dissectin thrugh the crtical-medullary junctin. The medulla represented abut 80% inner medulla (papilla) and 20% uter medulla. Pled medullary tissue was hmgenized in ice-cld M Na 2 HPO 4 ( g medulla/2 ml Na 2 HPO 4 ), the hmgenate centrifuged t remve cellular debris, and the supernate (cntaining vasdepressr activity) utilized fr further prcedures. Fr experiments with quickfrzen tissue, cmmercially available predissected medulla* was thawed and hmgenized in M Na 2 HPO 4 by a glass hand-type hmgenizer r, with large amunts f tissue, by means f a Waring blender. Hmgenates f fresh r quickfrzen rabbit medulla were assayed fr depressr activity by injectin int the jugular vein f the pentbarbital-anaesthetized, vagtmized, pentlinium-treated rat as previusly described." Mean cartid bld pressure was recrded kymgraphically. Figure A shws that sustained renmedullary vasdepressr activity was present in the crude hmgenate f bth fresh and quick-frzen rabbit medulla. In rder t determine its ethanl slubility, abslute alchl was added t the crude hmgenate t a final cncentratin f 80%. After centrifugatin, the ethanl supernate was evaprated t dryness and the residue restred t the riginal hmgenate vlume with M Na 2 - HPO 4. Figure IB illustrates that the depressr material f bth fresh and quick-frzen medulla was ethanl-sluble. In additin, the quick-frzen depressr substance(s) were dialyzable, f lw mlecular weight, and did nt pssess the chemical and rapid-acting depressr characteristics f nucletides, findings previusly bserved with *Pel-Freeze Bilgicals Inc., Rgers, Arkansas. A. CRUDE MEDULLARY EXTRACT (0.25 ml i.v. MEDULLA : FRESH FROZEN MEDULLA : B. 80 X ETHANOL EXTRACT (0.25 ml i.r.*) FRESH FIGURE Depressin f rat bld pressure by (A) a crude hmgenate ( g medulla/2 ml M Na 2 HPO, t ) and (B) an equivalent cncentratin f an 80% ethanl extract f fresh and frzen rabbit medulla. -ksee text fr experimental details. sustained vasdepressr material frm fresh rabbit medulla. 0 Therefre, it appeared that renmedullary depressr substance(s) present in the fresh medulla, were well preserved in cmmercially available frzen tissue. Except as nted, experiments were carried ut with frzen medulla because f the greater amunt f readily available material. Clumn Chrmatgraphy Clumn chrmatgraphy f crude ethanl extracts f medulla was perfrmed with diethylaminethyl ether-cellulse (DEAE-cellulse). Fr preparatin f the clumn, 60 g DEAE-cellulse were sprinkled n 0. N NaOH, allwed t settle, and the supernate decanted. The resin was washed with distilled water, the ph adjusted t 4.5 with 0 N HC, and allwed t settle. The supernate was decanted and the DEAE-cellulse washed with water, resuspended in M Na 2 HPO 4 buffer and pured int a 60 x 5 cm glass clumn under 00 cm water pressure (height f DEAE-cellulse apprximately 30 cm). The clumn was washed repeatedly with M Na 2 HPO 4 until the eluate ph was 7.8 t 8.2. Apprximately 25 t 35 ml f medullary extract were placed n the clumn and eluted at 20 C by gradient elutin: (A) M Na,- HPO 4) (B) 0.2 M Na 2 HPO 4 /0.08 M NaH 2 PO 4) and (C).0 M NaH 2 PO 4. Gradient elutin was accmplished by a tandem arrangement f bttles cntaining slutins A, B, and C placed abut

3 MEDULLIN: CHEMICAL AND PHYSIOLOGICAL PROPERTIES 59 fur feet abve the clumn head in rder t insure adequate eluate flw. Ten-ml eluate fractins were cllected in an autmatic fractin cllectr with a ttal eluate vlume f 800 t 000 ml. After passage f apprximately 300 ml f M Na 2 HPO 4 buffer (ph 8), the initial eluate (00 t 50 ml, pssessing n depressr activity) appeared as a deep yellw band assciated with a rise in ph frm 8 t 0. Depressr material was recvered during passage f the next 250 t 350 ml. Cessatin f elutin f depressr activity was assciated with a decrease in eluate ph frm 0 t 6; n subsequent elutin f depressr substance was bserved. Table illustrates the recvery f rganic slids in a representative small scale experiment utilizing nly M Na 2 HPO 4 elutin; it is evident that sustained depressr material was recvered in fractins 40 t 52 with the exclusin f abut 90% f the rganic slids initially placed n the clumn. A similar tenfld purificatin was achieved in larger scale experiments utilizing gradient elutin. Thin-Layer Chrmatgraphy Thin-layer chrmatgraphy f purified depressr substance was emplyed utilizing a Desaga- Brinkman apparatus. Glass plates, 20 x 5 cm (analytical) r 20 x 20 cm (preparative), were cated with 250 fj, r 500 /x, respectively, f a slurry f 30 g silica gel G in 65 ml distilled water. In certain experiments, 2% silver nitrate was incrprated int the silica gel. The plates were allwed t dry in rm air fr fifteen minutes, activated at 00 F fr ne hur, and stred in a dessicatr. Material was sptted with Hamiltn syringes (50 ^.liters), dried in a cld air stream, and placed in sealed, filter paperlined glass tanks cntaining the apprpriate slvents. All rganic slvents fr extractin and partitin as well as thin-layer chrmatgraphy were spectr-grade and redistilled befre use. The slvent systems used were: I. Trimethylpentane 20 Isprpyl alchl 40 Acetic acid II. Benzene 80 Dixane 80 Acetic acid 4 III. Ethyl acetate 0 Acetic acid 30 Methanl 35 2, 2, 4 trimethylpentane 0 Water 00 IV. Petrleum ether 90 Diethyl ether 0 Acetic acid V. Chlrfrm 65 Methanl 25 Acetic acid 8 Water 4 VI. Ethyl acetate 60 Methanl 3 Water 00 System I was adpted frm Enerth, 0 systems II and III frm Green and Samuelssn, 3 system IV frm Vgel et al., 2 and system V frm Skipski et al. 3 Systems III and VI were allwed t equilibrate fr ne hur and the less plar phase used. The plates were allwed t develp by the ascending technique t 0 cm at 20 C with a ttal develpment time f 20 t 40 minutes. After develpment, the plates were allwed t dry and visualized by ultravilet light, TABLE Recvery f Sustained Medullary Depressr Activity by DEAE-Cellulse Clumn Chrmatgraphy'' Initial n clumn Eluate number M Na 2 HPO 4 Ttal n clumn Ttal ff clumn Sustained depressr activity Pled vlume ml Dry weight mg Ash weight mg Organic slids mg 82 'Clumn: 7 g DEAE-cellulse packed under 00 cm water pressure. Clumn height, 25 cm; width, 3 cm. 80% ethanl extract f 7 g wet wt medulla dried under nitrgen and recnstituted t 6.0 ml with.005 M Na 2 HPO ml n clumn. Elutin:.005 M Na 2 HPO 4 ; #65: 0 ml eluate fractins pled t 30 ml as indicated

4 LEE, COVINO, TAXMAN, SMITH idine vaprs, charring fllwing 50% H0SO4 spray, r by spraying with 0% phsphmlybdic acid in 95% ethanl and heating at 25 C fr 20 minutes. The spts were remved with a spatula int an ultrafine sintered glass funnel, and the material eluted with 3 ml x 3 f methanl. Since the bilgically active substances culd nt be cmpletely extracted with less plar rganic slvents frm a dried methanl residue, the methanpl was evaprated t abut ml, diluted t 0 ml with distilled water, acidified with hydrchlric acid, and extracted five times with chlrfrm, benzene, r diethyl ether (v:v). The rganic phases were washed with distilled water until neutral and evaprated t a small vlume. This extract was used fr rechrmatgraphy r, in the case f the pure material, fr determinatin f chemical and physilgical prperties. CARDIOVASCULAR STUDIES Intact Animals In the first series f experiments, the direct effect f renmedullary depressr substance and prstaglandin E-l (PGE-)* n peripheral resistance was determined in ten dgs by measuring the change in femral bld flw and arterial pressure fllwing intra-arterial injectin f these cmpunds. Adult mngrel dgs were anaesthetized with pentbarbital sdium (30 mg/kg iv) and the left femral artery was cannulated fr the measurement f bld pressure by means f a pressure transducer. The right cartid and right femral arteries were then expsed and cannulated, s that bld flw was diverted by means f plyethylene tubing frm the cartid artery thrugh a Shipley-Wilsn rtameter int the distal end f the femral artery. This prvided a cntinuus measurement f bld flw t ne hind limb f the dg. Simultaneus recrdings f the bld pressure and bld flw were made n a Sanbrn tw-channel recrder. Frm these measurements, peripheral resistance was calculated: PBU (peripheral resistance units) = mean bld pressure, mm Hg bld flw, ml/min After cntrl bservatins, either renmedullary depressr substance r PGE- was injected int the femral artery while femral arterial bld flw and bld pressure were measured cntinuusly. In a secnd series f experiments, the effect f renmedullary depressr substance and PGE- n artic bld pressure and cardiac utput was * Fr these studies, crystalline PGE- was generusly supplied by Prfessr Sune Bergstrm f the Karlinska Institute, Stckhlm, Sweden. determined in a grup f ten dgs. A catheter was passed int the arta by way f the left femral artery, and artic bld pressure measured by means f'a pressure transducer. The right cartid artery was cannulated fr the purpse f btaining bld samples fr measurements f cardiac utput accrding t the dye dilutin technique. 4 Indcyanine green* was injected intravenusly via the jugular vein, and bld cncentratin curves btained by means f a Gilsn densitmeter. Ttal peripheral resistance (TPR) was calculated: TPR (ttal peripheral resistance) = mean artic bld pressure, mm Hg cardiac utput, ml/min After cntrl measurements f cardiac utput and bld pressure, either renmedullary depressr substance r PGE- was administered thrugh a catheter in the jugular vein. At the time f maximal hyptensive effect, indcyanine green was administered intravenusly fr repeat determinatin f cardiac utput. Islated Heart and Artic Strips The direct effect f renmedullary depressr substance n the islated rabbit heart was studied in a grup f seven rabbits. Rabbits were stunned by neck strke, and the heart remved and placed in a beaker f aerated Krebs-Ringer bicarbnate buffer. The arta was cannulated and attached t a Carver-Andersn crnary perfusin apparatus. The hearts were perfused with Krebs-Ringer bicarbnate buffer (glucse 0 ITIM), aerated with 95% O 3 t 5% CO 2, and maintained at a cnstant temperature f 37 C. In rder t measure cardiac cntractility, a ligature was passed thrugh the apex f the left ventricle and attached, in turn, t a Grass frce displacement transducer; cntractile frce was then recrded by means f a Sanbrn recrder. Injectins f depressr substance were made int the perfusin fluid thrugh the artic cannula in such a fashin that dilutin f depressr activity by the perfusin fluid was minimal. The effect f 0.5 ml (50 /xg) f renmedullary depressr substance n heart rate and mycardial cntractility was cmpared with the effect f 0.5 ml f % ptassium chlride and 0.5 ml f % calcium chlride. Fr studies n islated artic strips, rabbits were killed by neck strke and sectins f thracic arta were remved, cut int helical strips, and suspended in a 0-ml bath cntaining xygenated Tyrde slutin. NONVASCULAR SMOOTH MUSCLE STUDIES Nnvascular smth muscle studies were per- *Cardi-green, Hynsn, VVestctt, and Dunning Inc., Baltimre, Maryland.

5 MEDULLIN: CHEMICAL AND PHYSIOLOGICAL PROPERTIES 6 frmed utilizing islated segments f rabbit jejunum; rat dudenum, stmach, uterus, bladder, vas deferens, seminal vesicle; and terminal guinea pig ileum. With the exceptin f islated uteri, the tissues were suspended in 0 ml f xygenated Tyrde slutin at either 30 r 37 C. Uteri were remved frm rats which received 500 / A g/kg f stilbesterl, sc, 8 hurs befre use and were suspended in 0 ml f Jaln's slutin at 37 C. All tissues were allwed t equilibrate with the media fr ne hur befre use; istnic cntractins were recrded at ne gram resting tensin and eightfld magnificatin. Results CHEMICAL STUDIES Acidic Lipid Prperties Previus bservatins had established that renmedullary vasdepressr substance(s) were dialyzable, ethanl sluble, f relatively lw mlecular weight (< 4500 ml wt), and resistant t a mixture f peptide hydrlases. 0 In additin, they pssessed chemical and physilgical characteristics distinct frm thse f renmedullary nucletides which prduce shrt-acting vasdepressr effects." 9 In rder t determine whether the active material was lipid sluble, fresh medullary slices were prepared with a Stadie-Riggs micrtme and g f slices placed in each f tw 40-ml capped test tubes cntaining 20 ml chlrfrm-methanl (2:, v:v). They were allwed t stand vernight and the tissue was then remved and discarded. Fur PHASE mm Hg PHASE mmhr, O 0 CHLOROFORM n in 5 0 min 5 0 A. ACIDIFIED (0. Z5 ml i.v. *) 8. ALKALINIZED CHLOROFORM 5 20 m FIGURE 2 0 m mhg ACIO-METHANOL min 5 0 f 0.25 ml i.v. ; ALKALINE-METHAN0L TiHq 0-0 ^ _ - 0in Chlrfrm-methanl extract f fresh medullary tissue. Depressin f rat bld pressure by (A) chlrfrm phase f acidified lipid extract and (B) alkalinemethanl phase f alkalinized lipid extract f fresh medullary slices. +See text fr experimental details. Circulatin Research, Vl. XVII, July 96} A. ACIDIFIED (0.25 ml i. v.* ) PHASE : CHLOROFORM ACID-METHANOL mm Hg 0 -r nin min B. ALKALINIZED (0.25 ml. i.v. * ) CHLOROFORM FIGURI 3 ALKALINE-METHANOL Chlrfrm-methanl extract f purified renmedullary depressr substance. Depressin f rat bld pressure by (A) chlrfrm phase f acidified lipid extract and (B) alkaline-methanl phase f alkalinized lipid extract f purified renmedullary depressr substance(s). irsee text fr experimental details. ml 0.08 N H2SO4 were added t ne chlrfrm extract and fur ml 0.0 N NaOH t the secnd extract. The tubes were inverted slwly and allwed t stand accrding t the methd f Bragdn. 5 They were then centrifuged and the acid and alkaline aqueus phases (ph 3 and 9 respectively) aspirated, dried under nitrgen t a vlume f 2 ml, and neutralized fr assay in the pentliniumtreated rat. The chlrfrm phase was als dried and the residue redisslved in 2 ml 0.2 M sdium phsphate buffer (ph 8) fr assay. Figure 2A illustrates that the vasdepressr substance(s) partitined almst cmpletely int chlrfrm frm an acidmethanl phase. Hwever, in an alkalinized chlrfrm-methanl extract, mst f the vasdepressr activity was recvered in the alkaline-methanl phase (fig. 2B). In rder t cmpare the lipid slubility f vasdepressr substance(s) extracted directly frm fresh medullary tissue with that f purified depressr substance(s), ml f eluate, after DEAE-cellulse chrmatgraphy, was injected frcefully int each f tw tubes cntaining chlrfrm-methanl (2:, v:v). The chlrfrm-methanl extracts were acidified and alkalinized as described with fresh medulla. Figure 3A reveals purified depressr

6 62 LEE, COVINO, TAKMAN, SMITH i* Ph«M: Mrclcum Eth«r Chi Organic m -2 A^IMOM* Organic activity was recvered in the rganic phase frm acidified chlrfrm-methanl, and in the alkaline-methanl phase frm alkalinized chlrfrm-methanl (fig. 3B), findings similar t a direct lipid extract f fresh medulla. Figure 4 illustrates the effect f ph n the partitin f purified vasdepressr substance (s) between aqueus and certain rganic phases. It is evident that partitin int petrleum ether is relatively minimal at all hydrgen in cncentratins, while partitin int benzene, chlrfrm, and ethyl acetate is increased by a prgressive reductin in ph. Althugh partitin int benzene, chlrfrm, and ethyl acetate is almst cmplete at ph t 2, recvery f depressr activity in the rganic phase at any given higher ph appears t be greater as the plarity f the rganic phase increases (ethyl acetate > chlrfrm > benzene). These studies indicate that the substance(s) respnsible fr sustained lwering f bld pressure are relatively plar acidic lipids. The acidic lipid sluble residue which remained after evapratin f benzene, chlrfrm, and ethyl acetate appeared as a yellw il. Depressr activity culd be recvered frm this material after extractin with saturated NaHCO 3, 5% Na 2 CO 3, as well as N NaOH. Purificatin Figure 5 illustrates schematically the preliminary purificatin prcedures which were based n the knwn ethanl sluble, strngly acidic lipid characteristics f renmedullary depressr substance. An 80% ethanl extract f 3 t 5 kg f wet weight medulla was distilled in vacu t 2 t 3 liters. After adjustment f the ph t 7.5, the ethanl extract was partitined 3X against petrleum ether (v:v) and the petrleum ether discarded. The aqueus-ethanl phase was then distilled in vacu t 300 t 400 ml (phase I extract) and clumn chrmatgraphed n DEAE-cellulse by gradient elutin as described in the sectin under Methds. The pled DEAE eluate was acidified and extracted with benzene (chlrfrm r ethyl acetate) and reduced t a small vlume. Frm 3 t 4 kg wet weight frzen medulla, 00 t 50 mg f ily material were recvered, which was extremely active when assayed fr depressr effects. This material was redisslved in buffer 5.7 Aqu«u* Ethyl FIGURE 4 Effect f ph n the partitin f purified renmedullary depressr substance(s) between varius rganic and aqueus phases. Circulatin Research, Vl. XVII, July 963

7 MEDULLIN: CHEMICAL AND PHYSIOLOGICAL PROPERTIES 63 PREPARATION OF MEDULLIN FROM FROZEN RABBIT MEDULLA 4 kg WET WEIGHT MEDULLA HOMOGENIZED IN.005 M N2 HP04 (I00ml/200g MEDULLA) CENTRIFUGED 0,000» G. SEDIMENT DISCARDED SUPERNATE + ABSOLUTE ETHANOL (FINAL CONCENTRATION 80%) FILTERED, 8 PRECIPITATE DISCARDED SUPERNATE DISTILLED IN VACUO TO 2-3 L. AT 35 C ph ADJUSTED TO 7.5 EXTRACTED 3» WITH PETROLEUM ETHER PET. ETHER PHASE DISCARDED (NO DEPRESSOR ACTIVITY) PHASE I AQUEOUS-ETHANOL PHASE DISTILLED IN VACUO TO ml COLUMN CHROMATOGRAPHED : DEAE-CELLULOSE, GRADIENT ELUTION AQUEOUS ELUATE DISCARDED (NO DEPRESSOR ACTIVITY) ELUATE CONTAINING DEPRESSOR ACTIVITY POOLED ACIDIFIED TO ph -2 EXTRACTED 3 x WITH BENZENE, CHLOROFORM OR DIETHYL ETHER I ORGANIC PHASE DISTILLED IN VACUO TO 0 ml YELLOW OIL (00-50 mg) PHASE H ORGANIC PHASE DRIED UNDER NITROGEN REDISSOLVED IN PHOSPHATE BUFFER (00/ig/ml) MAXIMUM RAT DEPRESSOR EFFECT : 5ju.g FIGURE 5 THIN LAYER PHASE IS PURE MEDULLIN CHROMATOGRAPHY (3-5 mg) MAXIMUM RAT DEPRESSOR EFFECT : IOO-3OOmji.g Schematic representatin f sequential steps utilized in the purificatin and islatin f renmedullary depressr substance (medullin). fr physilgical studies (phase II extract) r was utilized directly as the free acid fr islatin f the pure material (medullin) by thin-layer chrmatgraphy (phase III). Thin-Layer Chrmatgraphic Islatin Initial bservatins revealed that vasdepressr activity was recvered nly at the rigin in a neutral lipid slvent system (system IV). N activity was bserved in areas crrespnding t chlesterl, chlesterl esters, free fatty acids, diglycerides, r triglycerides. In a slvent system utilized fr phsphlipid separatin (system V), vasdepressr material was recvered nly at the slvent frnt. These experiments suggested that renmedullary depressr substance(s) did nt appear t be an unsubstituted fatty acid r phsphlipid with thin-layer mbilities which resemble knwn serum neutral lipids r phsphlipids. Fr the islatin f depressr substances, a cmbinatin f tw successive thin-layer chrmatgraphic systems were emplyed. Figure 6 illustrates the results f chrmatgraphy in system I f phase III extract, prepared by extractin f acidified, pled DEAE-eluate with benzene. Depressr activity was recvered nly in spt 3 which was an intensely idine-staining, nnflurescent spt with an Rf f In system II, the Rf f spt 3 was 0.57 t 0.60, which is represented as spt 4 in figure 7. In the initial chrmatgram, n depressr activity was bserved in ther spts, unless phase III extract was prepared by extractin f pled DEAE-eluate with chlrfrm r ethyl acetate rather than benzene. Figure 7 illustrates the results f chrmatgraphy in system II under such cnditins. As nted, spt 4 (Rf = 0.57 t 0.60) was an intensely idine-staining spt Circulatin Research, Vl. XVII, July 96}

8 64 LEE, COVINO, TAKMAN, SMITH with marked vasdepressr activity which crrespnded t spt 3 in system I (fig. 6). In additin, marked depressr activity was als bserved in spt 2, with very weak activity present in spt ; these were less mbile, nnflurescing spts with weak idine-staining prperties. The ability t stimulate islated rabbit jejunum was an additinal bilgical activity present in spts and 2, and t a lesser extent in spt 4. It appeared therefre, that rabbit medulla pssessed at least tw acidic lipids capable f lwering f bld pressure and stimulating nnvascular smth muscle, while a third cmpund displayed nly nnvascular smth muscle stimulating prperties. In the present study, the material f primary interest was the relatively mbile, intensely idine-staining substance Rf ldin* Vapr U-V Flur*M«nc«Dvpresir Activity Spt Number s.f * * - * 3 O.35 _ 2 r.-»*«> OJO FIGURE 6 Thin-layer chrmatgraphy f phase renmedullary extract prepared by extractin f acidified pled DEAE-eluate with benzene. System I. MEDULLIN - COMPOUND 2 COMPOUND I -»0T Rf O DEPRESSOR ACTIVITY - JEJUNAL STIM IODINE VAPOR - U-V FLUOR. - _ OR. FIGURE 7 Thin-layer chrmatgraphy f phase HI renmedullary extract prepared by extractin f acidified pled DEAE-eluate with chlrfrm. System II. PMA: phsjihmlybdic acid.

9 MEDULLIN: CHEMICAL AND PHYSIOLOGICAL PROPERTIES 65 (spt 3, system I; spt 4, system II) with marked depressr, but relatively weak intestinal smth muscle stimulating prperties. This material has been designated "medullin." Fr cmparative purpses, the mre plar substances are referred t as cmpunds and 2, utilizing spts and 2, respectively, f the chrmatgram in figure 7 as reference. The Rf values f these cmpunds are summarized in table 2. Ultravilet and Infrared Spectra *8) The ultravilet (UV) absrptin f pure medullin (57 /^tg/ml methanl) is shwn as the slid line in figure 8A. A very weak maximum is evident at 2780 t 2800A. The dashed line (fig. 8A) illustrates the UV spectrum f a slutin f 5 fig medullin in ml 96% H2SO4, which was allwed t stand at 22 C fr fur hurs. Tw strng maxima were bserved at 2580 t 2600A and at 3200 t 3220A, with an inflexin pint at ap , A. -, B. f ' WAVELENGTH (millimicrns) Free acid +96% H.SO. Free acid + NOH at 00* C Free acid acidified after NOH f 00'C WAVELENGTH (millimicrns) FIGURE 8 Ultravilet spectra f (A) medullin (slid line) and chrmphres derived after treatment with 96% H^O^ (dashed line); (B) fllwing heat treatment with 0.0 N NaOH befre (slid line), and after additin f 3 N HCl (dashed line). Perkin-Elmer spectrphtmeter, mdel 202. Circulatin Research. Vl. XVII, July :! CM" II "*" WAVELENGTH (micrns) FIGURE 9 Infrared spectrum f medidlin (3.5 mg in ml chlrfrm) Perkin-Elmer Infracrd 37B. prximately 3600A. Althugh nt shwn, a third maximum was present in the visible range assciated with the develpment f yellw clr during the perid f acid treatment. The slid line in figure 8B shws the UV spectrum f 55 fig medullin fllwing treatment with ml f 0.0 N NaOH fr ne hur at 00 C; a strng absrptin at 2800 t 280A was evident. After treatment f this slutin with excess 3 N HCl, the previusly bserved maximum at 2800A was n lnger evident, and a weak maximum appeared at 3280 t 330A (dashed line, fig. 8B). Figure 9 illustrates an infrared spectrum btained n a slutin f medullin in chlrfrm (3.5 mg/ml). The fllwing characteristic peaks were present: 3470(w), 2890(s), 2820(m),700(s),40(m),and972(m)cm-. In additin t the well-defined peak at 700, an inflexin pint was present at apprximately 725 cm-. Cmprisn with Prstaglandin The chemical, chrmatgraphic, and spectral prperties f medullin clsely resembled thse f a recently islated series f bilgically active acidic lipids called prstaglandins E(PGE). Althugh medullin appears clsely related t these substances, figure 0 illustrates imprtant chrmatgraphic differences between these cmpunds. It is evident that medullin has a significantly greater mbility than PGE-, a characteristic which has been fund in a variety f slvent systems (table 2). In certain experiments, 2% silver nitrate was incrprated int the silica gel, cnditins E-l

10 66 LEE, COVINO, TAKMAN, SMITH TABLE 2 Rf Values f Medullin and Prstaglandin E-l (PGE-) Slvent system i n II* in in* Medullin.4 (.35.48).60 (.57.62) PGE-.29 (.25.32).43 (.40.45) Cmpund 2.29 (.26.32).42 (.38.45) Cmpund.26 (.22.30).75 *With 2% silver nitrate. Numbers in parentheses indicate the range between leading and trailing edges f the spts. S.F.- NUdullin- POI- - Or. FIGURE 0 Rf.4O-.45 ledin* Vapr MM.25-.3O» Cmparisn f 50 /ig f medullin and PGE- in slvent system I. The dark bands tward the frnt represent slvent-air interface reactins which were unusually prnunced in this chrmatgram. Spts identified by heating after spraying with 0% phsphmlybdic acid. Nte decreased intensity f medullin in cmparisn t PGE-. Previus expsure t idine vaprs revealed a greater intensity in medullin than PGE-. which separate derivatives f PGE and prstaglandins F (PGF) accrding t the degree f unsaturatin. Medullin cnsistently ran as a single spt in a mre mbile psitin than PGE, cnfirming the hmgeneity f the islated material. Since all knwn unsaturated cmpunds f PGE and PGF are less mbile than PGE-, it is evident that the mre mbile unsaturated medullin represents an unknwn althugh clsely related cmpund. In additin t its characteristic mbility, medullin was much mre intensely idine-staining than PGE-. In cntrast t medullin, medullary cmpund-2 displayed weak idine-staining characteristics and greater smth musclestimulating ability (fig. 7). In additin, cmpund-2 revealed identical chrmatgraphic mbilities as PGE- in several slvent systems, with and withut added silver nitrate (table 2). CARDIOVASCULAR STUDIES Intact Animals Peripheral bld flw studies were carried ut in eight dgs with partially purified medullin (phase II extract),* and in tw dgs with chrmatgraphically pure medullin and PGE-. Additinal studies were nt pssible with pure medullin and PGE- because f the limited amunt f available material. Figure shws a typical respnse fllwing the intra-arterial injectin f 50 /ig f partially purified medullin. Femral bld flw rse prmptly, fllwed by a slight reductin f arterial bld pressure. The average changes in mean arterial bld pressure, * "Partially purified" medullin cntained several substances in additin t medullin. Individually, hwever, nne f these substances pssessed bilgic activity when islated by thin-layer chrmatgraphy (fig. 6). Observatins f bilgical activity with partially purified medullin, therefre, represent nly the activity assciated with chrmatgraphically pure medullin.

11 MEDULLIN: CHEMICAL AND PHYSIOLOGICAL PROPERTIES 67 5 CO s s i 5 ft * (0. 3 < r* «^ 3 O.2 Si * Is artia 0, cent nge &* n Pi inje * u c CJ c Per c chan c Pst..2 njecl c O i a. 5 u c J..2 t S P injec "3 c U 00 CD 00 CD CO 7 t- CD t t> - i d V in Oi. i l I ( t S " ssur a terial H r-i 00 CO CO ( +l + CO C5 s in CD c r ( d V in CO CD i f s UlU _ 5 in + i t < { t f ( i! + Ol s q i i 00 CO i O5 Ol CO CO CO CB CO CD c CD i i i d V ! i Ol in < i 0 nee, g 5 3. Per CO T 8 00 i + Ol d + a 2 vali _e I ired fco E =,2 S 2 x S I «.= 5 bld flw, and peripheral resistance fllwing the intra-arterial injectin f partially purified medullin, pure medullin, and PGE- are presented in table 3. The predminant effect was a significant increase f femral arterial bld flw accmpanied by a slight reductin f arterial bld pressure. The relatinship between these changes f bld flw and bld pressure was such that the calculated resistance f the peripheral vasculature decreased significantly. The nly difference between partially purified medullin, pure medullin, and PGE- was ne f ptency, i.e., 0.5 fig f pure medullin r PGE- was equivalent t 50 fig f partially purified medullin. An attempt was made t btain a dseeffect relatinship between intra-arterial injectins f medullin and arterial bld flw. This was nt pssible in all cases since, in sme dgs, the arterial pressure failed t return t the pre-injectin cntrl level after repeated administratin f medullin. Hwever, in several animals, the bld pressure remained sufficiently cnstant thrughut the experimental prcedure t permit a dse respnse study. Figure 2 shws the relatinship between varying dses f partially purified medullin and femral arterial bld flw in a representative experiment. It is evident that the increase f bld flw was directly prprtinal t the dse injected until maximum flw ccurred. The systemic hemdynamic effects f partially purified medullin were evaluated in an 50 BLOOD 00 FLOW ml /mln BLOOD PRESSURE 00 mm. H, 50yUg I.A. FIGURE I sec Effect f medullin n femral arterial bld flw and bld pressure in the dg, hind limb (phase II extract). I.A: intra-arterial.

12 68 LEE, COVINO, TAKMAN, SMITH additinal series f experiments n eight dgs. The mean values fr artic pressure, cardiac utput, and ttal peripheral resistance, befre and after administratin f partially purified medullin, are shwn in figure 3. In all eight animals, there was a significant decrease in mean artic pressure which persisted fr 0 t 7 minutes befre returning t cntrl levels. The decrease in mean artic pressure was almst exclusively the result f a fall in diastlic pressure which decreased an average f 40 ± 6 mm Hg. The reductin f systlic pressure was statistically insignificant and the net result was a marked increase f pulse pressure. At the time f maximum decrease in mean artic pressure, a 30% increase in cardiac utput was bserved. Thus, the ttal peripheral resistance decreased significantly in all eight experiments. An average increase in heart rate f 8.5 ± 5.0 beats/ min was als bserved after treatment with partially purified medullin. Hwever, the acceleratin f heart rate ccurred after the establishment f maximal bld pressure depressin suggesting a tachycardia f reflex rigin. Frm the heart rate and cardiac utput data, strke vlume was calculated and fund t increase an average f 2.2 ± 0.6 ml/beat fllwing injectin f partially purified medullin. A cmparisn f the systemic hemdynamic effects f partially purified medullin with pure medullin and PGE- is shwn in table 4. OOSE («g> FIGURE 2 Dse respnse f femral arterial bld flw increase in the dg hind limb. Maximal bld flw increment ccurred at abut 00 ng f partially purified medullin (phase II extract). Representative experiment. 20Or. MEAN SYSTOLIC DIASTOLIC PULSE PRESSURE CARDIAC OUTPUT TOTAL PERIPHERAL RESISTANCE CONTROL EJ POST-INJECTION (JOO^g. i.t.l FIGURE 3 Hemdynamic effects f medullin in the dg. Phase II extract. n = 8. See text fr experimental details. In these studies, the initial mean, systlic, and diastlic pressures were cnsiderably higher than in experiments with partially purified medullin. Injectin f pure medullin r PGE- under these cnditins reduced systlic pressure frm 76 t 5 mm Hg. Again, hwever, the decrease in diastlic bld pressure was greater and pulse pressure increased. As in the experiments with partially purified medullin, there was an increase in cardiac utput with a resultant 26 t 30% decrease in ttal peripheral resistances. N differences were bserved between pure medullin and PGE-. The nly difference between the pure cmpunds and partially purified medullin was ne f ptency: 0 jug f pure medullin elicited respnses similar t that prduced by 300 /ttg f partially purified medullin. Figure 4 illustrates the dse respnse relatinship between pure medullin and PGE- n bld pressure reductin. Althugh the number f experiments with pure medullin r PGE- were limited by the amunt f material available, it wuld appear that maximum hyptensive effect with bth cmpunds

13 m O Z a X O (7 73 I TABLE 4 Effect f Partially Purified Medullin, Pure Medullin, and PGE- n Artic Pressure, Cardiac Output, and Ttal Peripheral Resistance in the Intact Dg Cntrl Partially purified medullin (300 y.g m) n = S Mean artic pressure, mm Hg 43 ± 7 Systlic pressure 6 ± 6 Dinstlic pressure 26 ± 5 Pulse pressure 34 ± 2 Cardiac utput, liters/min 2.20 ± 0.27 Ttal peripheral resistance, PRU ± 0.0 Pure medullin (0 ng ivj n 2 Mean artic pressure, mm Hg Systlic pressure Dinstlic pressure Pulse pressure Cardiac utput, liters/min Ttal peripheral resistance, vnu Pstinjectin 20 ±6 55 ±6 86 ±7 69 ± ± ± Mean difference -23 ±4-6 ±6-40 ±6 +35 ± ± ± Per cent change P* <0.0 >0.3 <0.0 <0.0 <0.0 <0.0 PGE- (0 Mg iv; n = 2 Mean artic pressure, mm Hg Systlic pressure Diastlic pressure Pulse pressure Cardiac utput, liters/min Ttal peripheral resistance, pnu *P: significance determined by Student's t test utilizing paired data analysis

14 70 LEE, COVINO, TAKMAN, SMITH Dse-repnse relatinship between pure medullin and PGE- n mean artic bld pressure reductin, n 2. ccurred at a dse between 0 and 50 /xg (.0t5.0/*g/kg). Islated Hearts and Artic Strips Studies were made n the islated rabbit heart and islated artic strips. Table 5 illustrates that n change in either heart rate r cntractile frce was bserved fllwing the direct injectin f 0.5 ml f partially purified medullin int the artic cannula f the islated rabbit heart. On the ther hand, 0.5 ml f % calcium chlride prduced a mean increase in amphtude f cntractin f 28.3 ± 3.4 mm, while 0.5 ml f % ptassium chlride resulted in a mean decrease f 8.4 ± 2.3 mm. Typical respnses f the islated rabbit heart are shwn in figure 5. High cncentratins f crude medullary extract prduced very weak cntractins f islated artic strips. Hwever, pure medullin at cncentratins f /Ag/ml prduced n stimulatin f such strips, nr did it result in relaxatin f strips partially cntracted with either nrepinephrine r angitensin. NONVASCULAR SMOOTH MUSCLE STUDIES Because f the direct effect f these renmedullary substances n vascular smth muscle, their effect n nnvascular smth muscle was examined. Crude ethanl extracts f rabbit medulla, pssessing depressr activity, prduced strng cntractins f islated segments f rabbit jejunum, and rat dudenum, stmach, and uterus; absent r weak cntractins were bserved with terminal guinea pig ileum, rat bladder, vas deferens, and seminal vesicle. It appeared that the islated rabbit jejunum was particularly respnsive t medullary extracts and this preparatin was subsequently emplyed fr assaying the nnvascular smth muscle stimulating activity. Figure 6 shws the results when crude medullary extracts were clumn-chrmatgraphed n DEAE-cellulse as previusly described and the eluate fractins assayed fr bth depressr and intestinal smth musclestimulating activity. Althugh bth hyptensive and nnvascular smth muscle-stimulating activities were bserved in fractins 27 t 60, it is apparent that cnsiderable intestinal smth muscle-stimulating activity withut assciated depressr activity was initially eluted in fractins 9 t 25. Fractins 27 t TABLE 5 Effect f Medullin n the Islated Perfused Rabbit Heart Heart rate, beats/min Amplitude f cntractin, mm Cntrl 98 ± ± 3.7 Pstinjectin* 98 ± ± 3.6 Perfusin media: Krebs-Ringer bicarbnate buffer, glucse 0 HIM, 37 C aerated with 95% O 2-5% CO.,. n = 7, with SEM. *50 ng medullin injected int the artic cannula.

15 MEDULLIN: CHEMICAL AND PHYSIOLOGICAL PROPERTIES mm mm r t 0.5 ml 20 '^*$~~ 0-' /.K^_ 0^""A" ' MEDULLIN POTASSIUM CHLORIDE»*'»' 0.5 ml (I KCI FIGURE IS Effect f partially purified medullin (phase II extract) and ptassium chlride n cntractile frce and heart rate in the islated perfused rabbit heart. Perfusin media: Krebs-Ringer bicarbnate buffer, glucse 0 mm. Gas phase: 95% O s -5% CO e. Typical experiment. 60 were pled, acidified, extracted with chlrfrm, and the rganic phase subjected t thin-layer chrmatgraphy. As previusly indicated (fig. 7), nnvascular smth muscle-stimulating activity was present in three chrmatgraphically distinct substances: cmpund, cmpund 2, and, t a much lesser extent, medullin. Cncentratin-effect curves fr a crude ethanl extract f rabbit medulla, medullin, PGE-, and acetylchline are shwn in figure 7. The typical respnse f rabbit jejunum t these substances is shwn t cnsist f an initial phase f rapid cntractin fllwed by prgressive relaxatin. A cmparisn f the ptency f medullin, PGE-, and acetylchline reveals that the activity f PGE- was equal t acetylchline, as evidenced by superimpsable cncentratin-effect curves. On the ther hand, medullin, a cmpund equal t PGE- in vasdepressr activity, was apprximately l/50th as ptent as PGE- in prducing stimulatin f nnvascular smth muscle. Analysis f the relative ptencies f pure medullin and relatively crude renmedullary Circulatin Research, Vl. XV, July 965 extract (phase I) indicates a purificatin f apprximately ne-hundredfld. Discussin Original studies 9 in 963 revealed that crude extracts f rabbit, rat, pig, and human renal medulla cntained substance(s) which resulted in a sustained lwering f bld pressure when injected int the vagtmized, pentbarbital-anaesthetized, pentlinium-treated rat. The present study demnstrates that the depressr activity f such crude hmgenates is sluble in the lipid phase after direct lipid extractin f medullary tissue r acidified renmedullary extract, an bservatin recently reprted by Hickler et al. 0 In additin, such fractins pssessed nnvascular smth muscle-stimulating prperties. Assay f the eluate fractins after clumn chrmatgraphy revealed differing eluate recvery f vasdepressr and nnvascular smth muscle-stimulating activities suggesting the presence f mre than ne active substance, each with differential effects upn bld pressure and nnvascular smth muscle. After final islatin by thin-layer chrmatgraphy, three substances were fund t be principally respnsible fr the vasdepressr and nnvascular smth muscle-stimulating prperties f the crude hmgenate. These substances have been designated, "medullin," "cmpund," and "cmpund TUBE NUMBER FIGURE 6 Eluate recvery f intestinal smth muscle-stimulating and vasdepressr activity after gradient elutin f crude medullary extract n DEAE-cellulse. 0 ml eluate fractins.

16 72 LEE, COVINO, TAKMAN, SMITH FIGURE 7 Cncentratin-effect curves f the stimulatin f islated rabbit jejunum by crude renmedullary extract, pure medullin, PGE-, and acetylchline. Insets shw typical respnses f jejunal smth muscle t these cmpunds. W: remval f test material frm medium. 2." All three substances stimulate intestinal smth muscle, but nly medullin and cmpund 2 pssess significant activity in lwering bld pressure. At the present time, there are several classes f naturally ccurring acidic lipids which are knwn t stimulate either nnvascular smth muscle and/r lwer bld pressure. "Darmstff," extracted frm the intestine is prbably acetalphsphatidic acid; this cmpund stimulates intestinal smth muscle, but pssesses little depressr activity. 7 G-acid, 8 and hemlytic acid, 9 frm human plasma, are straight chain unsaturated mncarbxylic acids withut hydrxyl r ket grups and are prbably A-3-ctadecenic and cis-a--ctadecenic acid (cis-vaccenic acid) respectively. 20 Slw reacting substance A 2 frm guinea pig lung prduces nly slw cntractin f islated nnvascular smth muscle. The mst well-defined grup f lipid sluble acids which pssess vasdepressr and nnvascular smth muscle activity is a recently islated series f cmpunds referred t as prstaglandins. Of the varius knwn bilgically active acidic lipids, medullin and cmpunds and 2 appear t belng t this general class f cmpunds. The vasdepressr and smth muscle-stimulating activities assciated with prstaglandins were first independently described in human seminal fluid and sheep vesicular glands by Gldblatt 22 ' 23 and vn Euler. 24 In further investigatins, the activity was fund t depend n an acidic lipid, 24 " 27 that was named prstaglandin by vn Euler. 25 Mre recently, Bergstrbm et al. have islated and identified a series f such cmpunds 282 " including PGE-, PGE-2, and PGE-3, 80 S which structurally are unsaturated, hydrxylated, ketnic derivatives f the parent 20 carbn, five-membered ring cmpund, prstanic acid. Figure 8 illustrates the structure f PGE- which is a carbxylic acid cntaining a cyclpentanne ring (C8-2), tw hydrxyl grups (Cll, 5), ne ket grup (C9), and ne trans duble band (C3-4). Reductin f the ket grup in the PGE cmpunds results in the frmatin f PGF-, PGF-2, and PGF-3, 3-32 each f which may exist in tw stere-ismeric frms. The widespread ccurrence f such cmpunds is evidenced by their islatin frm such surces as sheep seminal vesicles, 24 ' 2S sheep and pig lung, 33 human and sheep seminal plasma, bvine brain, 30 calf thymus, 37 human menstrual fluid, 38 and sheep iris. 39 The PGE cmpunds pssess bth vasdepressr and nnvascular smth musclestimulating activity while the PGF substances prduce nly nnvascular smth muscle stimulatin. CHEMICAL PROPERTIES OF MEDULLIN Of die three bilgically-active cmpunds islated frm rabbit renal medulla in the present study, the cmpund f majr interest was medullin. Medullin was bserved t be an ethanl sluble cmpund which was recvered in the rganic phase by direct lipid extractin frm an acid aqueus phase, but nly incmpletely frm an alkaline aqueus phase. In additin, medullin was extracted by saturated NaHCO 3 and 5% Na 2 CO 3, as

17 MEDULLIN: CHEMICAL AND PHYSIOLOGICAL PROPERTIES ie 7 CH 3 -CH 2 -CH 2 -CH 2 H H I CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -COOH HO '' NS'^ PROSTAGLANDIN E - ( l5-dihydrxy-9 -ket-prst -3--enic acid) (fig.8) FIGURE 8 Chemical structure f prstaglandin E-l. well as N NaOH, suggesting that it is a relatively strng acidic lipid. Medullin was islated by thin-layer chrmatgraphy in a variety f slvent systems, with and withut silver nitrate, verifying the hmgeneity f the islated material. An infrared spectrum f pure medullin shws the presence f carbnyl (C = O) and methylene (-CH 2 -) grups, with evidence fr the existence f hydrxyl grups (-OH) and trans ethylene bnds ( > C = C < ), features in cmmn with the structural cmpsitin f prstaglandins. Cmparing the UV spectrum f medullin in sulfuric acid with similar spectra f prstaglandins E 29 and prstaglandins F 28 reveals a striking spectrphtmetric similarity between medullin and prstaglandins E, bth f which have maximal absrptin at 2600A and 3200A with an inflexin pint at 3600A this is in cntrast t prstaglandins F which display a single maximal absrptin at apprximately 3000A. Furthermre, a cmparisn between the spectra btained frm heattreated alkaline slutins f medullin and prstaglandins E-l 34 shw similar absrptin maxima at apprximately 2800A. The chrmphre frmed after treatment with alkali is believed t be the result f a dienine frmed by dehydratin and ismerizatin f a duble bnd intrduced between C8 and C2 (fig. 8 ). 37 In additin, acid treatment f these slutins results in disappearance f the maxima at 2800A and appearance f a Circulatin Research, Vl. XVU, July 96} new maximum at 3280A fr bth medullin and prstaglandin E-l. These studies btained n derivatives f medullin and PGE- suggest identity f majr structural cmpnents in the tw mlecules. Althugh the chemical prperties and cmpsitin f medullin and PGE- reveal clse structural relatinship, imprtant differences exist which prve clearly that the tw cmpunds are nt identical: () Medullin reveals a cnsistently greater mbility n thinlayer chrmatgraphy in cmparisn t the less mbile PGE-, PGE-2, and PGE-3. (2) Medullin stains much mre intensely with idine vaprs than equal amunts f PGE-. (3) PGE- in ethanl pssesses n characteristic absrptin at higher wavelengths, while a methanlic slutin f medullin displays a weak but definite absrptin maximum at A. (4) Medullin is apprximately fifty times weaker than PGE- in stimulating nnvascular smth muscle. Thus, the available evidence indicates that medullin is a derivative f prstanic acid which pssesses a basic structural similarity t PGE- but appears t be a mre unsaturated cmpund with fewer plar (i.e., hydrxyl) cmpnents. In additin t medullin, tw additinal bilgically active acidic lipids were islated frm renmedullary extracts. The first substance (cmpund 2) displayed weak idinestaining characteristics and prduced vasdepressin and nnvascular smth muscle

18 74 LEE, COVINO, TAKMAN, SMITH stimulatin cmparable in grade t PGE-. Since the thin-layer chrmatgraphic mbilities f cmpund 2 and PGE- were identical in a variety f slvent systems, with and withut added silver nitrate, this substance has been tentatively identified as PGE-. The third islate (cmpund ) was less mbile than PGE n thin-layer chrmatgraphic separatin, and revealed strng nn vascular smth muscle-stimulating activity, but absent t weak vasdepressr prperties, characteristics cmmn t PGF. PHYSIOLOGICAL PROPERTIES OF MEDULLIN Althugh the ability f medullin t reduce bld pressure in the pentlinium-treated rat suggests that its actin is independent f ganglinic blckade, little infrmatin has been available n the mechanism f its vasdepressin. The present study was dne in rder t determine whether the hyptensive actin f medullin was cardiac r peripheral in rigin. The results indicate that the hyptensive actin f this substance is due t a direct vasdilatr effect n peripheral arteriles, since a significant decrease was bserved bth in the peripheral resistance f the dg hind limb fllwing intra-arterial injectin f vaspressr substance, and in the ttal peripheral resistance f the dg fllwing intravenus injectin f this material. In additin, diastlic bld pressure reductin exceeded systlic, which indicates again a direct actin f medullin n peripheral resistance vessels. The hyptensive actin f medullin des nt appear t invlve depressin f cardiac rate r cntractility, because cardiac utput increased significantly and simultaneusly with the maximum reductin f bld pressure. Mrever, studies n the islated rabbit heart failed t demnstrate any significant change in cardiac rate r cntractile frce, supprting the cnclusin that renmeduljary depressr substance is devid f a negative intrpic and chrntrpic actin. Althugh n direct measurements f the actin f medullin n veins were btained, it appears unlikely that any imprtant vendilatin was prduced. If a significant degree f vendilatin was present, pling f bld in peripheral veins wuld be expected with reductin f venus return and a subsequent decrease f cardiac utput. Actually, a rise in cardiac utput and strke vlume ccurred at the time f maximal bld pressure depressin, suggesting increased venus return, which is mst prbably due t the delivery f a greater vlume f bld t the venus bed by the dilated arteriles. It wuld appear, therefre, that the hyptensive actin f medullin is attributable t a direct dilatr effect n the smth muscle f peripheral arteriles, resulting in a lwered ttal peripheral resistance and a subsequent decrease f diastlic and mean arterial bld pressure. Cardiac utput is prbably increased, with a reflex increase in heart rate and a rise in strke vlume secndary t increased venus return. Prstaglandins have previusly been shwn t increase bld flw t the islated hind limb f frgs 26 and cats 40 withut a discernible effect n the cntractile frce f the islated frg heart, 20 suggesting a mechanism f vasdepressin similar t that f medullin. By cntrast, infusins f PGE int nrmal human beings were fund t decrease cardiac utput as well as lwer bld pressure. 4 In the present study, hwever, the vasdepressr effects f PGE- were indistinguishable frm thse f medullin, since vasdepressin fllwing administratin f PGE- was the result f a decrease in peripheral resistance and was nt assciated widi a demnstrable reductin f cardiac utput. Furthermre, a cmparisn f the dse respnse curves f medullin and PGE-, in lwering artic bld pressure, revealed n difference in ptency between the tw substances. The nly bserved bilgical difference between medullin and PGE- was the relatively weak stimulatin f nnvascular smth muscle by medullin, which was in cntrast t the relatively strng acetylchline-like effect f PGE-. The "prtective" rle f intact functining renal mass in preventin f varius frms f experimental renal hypertensin (partial renal artery cclusin, chrnic cmpressin f renal

19 MEDULLIN: CHEMICAL AND PHYSIOLOGICAL PROPERTIES 75 parenchyma, and bilateral nephrectmy) is well established, and is illustrated by the difficulty f creating experimental hypertensin by prcedures invlving ne kidney withut remval f an ppsite intact functining kidney. In additin, it has been shwn that remval f bth kidneys prduces renprival hypertensin, 7 which bviusly cannt be ascribed t a renal renin-angitensin mechanism. Intrductin f a nrmally perfused kidney, 2 explantatin f renmedullary tissue, 4 - and the ral administratin f renmedullary extracts 0 t bilaterally nephrectmized animals have been shwn by Muirhead et al. t prevent the develpment f such renprival hypertensin. Furthermre, in human hypertensin and in chrnic established experimental hypertensin resulting frm renal cmpressin 43 r renal artery ligatin, pressr agents have nt been detected cnsistently in amunts sufficient t accunt fr the bserved degree f bld pressure elevatin. These bservatins suggest that certain states f hypertensin may result frm a deficiency f renal vasdepressr substance(s), rather than slely frm a relative increase in renal pressr mechanisms. In the present study, the islatin f medullin demnstrates that the renal medulla cntains a ptent, naturallyccurring vasdepressr substance which lwers bld pressure by direct peripheral arterilar vasdilatin, thus prviding further evidence fr the renal depressr hypthesis. The bservatins that prstaglandin cmpunds are widely distributed in many tissues suggests that, in additin t pssible systemic effects, they may exert a lcal hrmne-like activity n vascular resistance and bld flw. Since medullin is lcalized primarily in renal medulla, intrarenal cntrl f medullary rather than crtical bld flw wuld be expected if such lcal functinal activity exists. Whether medullin r related cmpunds are active in intrarenal regulatin f medullary bld flw and whether they pssess a physilgical rle in systemic bld pressure regulatin will have t await much additinal investigatin. Summary The chemical and physilgical prperties f renmedullary depressr substances were investigated utilizing the nrmtensive, vagtmized, pentlinium-treated rat fr assay. By a cmbinatin f slvent extractin, clumn chrmatgraphy, and thin-layer chrmatgraphy, three bilgically active acidic lipids were islated frm rabbit renal medulla. The first cmpund, called medullin, is an acidic lipid with ptent vasdepressr and relatively weak nnvascular smth muscle-stimulating prperties. Ultravilet analysis revealed spectra clsely resembling that f prstaglandin E-l (PGE-). Infrared analysis shwed the presence f carbnyl, methylene, and hydrxyl grups in additin t trans ethylene bnds. Althugh clsely resembling PGE-, medullin appears t be a mre unsaturated carbxylic lipid with less plar (hydrxyl) grups. The hyptensive actin f medullin is attributable t a direct effect n peripheral arterilar beds withut cardiac depressin, because intra-arterial and intravenus injectins f medullin reduced peripheral resistance markedly and increased cardiac utput simultaneusly. In additin, n effect n cardiac rate r cntractility was bserved in the islated rabbit heart preparatin. Cmparative studies f medullin and PGE- indicated that the hyptensive activity f these cmpunds is similar with respect t ptency and mechanism f actin. The nly bilgical difference between crystalline PGE- and medullin was a fiftyfld greater stimulatin f nnvascular smth muscle by PGE- in cmparisn t medullin. A secnd bilgically active acidic lipid islated frm rabbit medulla was tentatively identified as PGE-, and was fund t pssess bth vasdepressr and nnvascular smth muscle-stimulating prperties. The third islate frm rabbit medulla pssessed ptent intestinal stimulating activity but weak t absent vasdepressr effects, prperties similar t thse f prstaglandin F cmpunds. Acknwledgment The authrs express their appreciatin f the generus gift f crystalline prstaglandin E-l frm Pr-

20 76 LEE, COVINO, TAXMAN, SMITH fessr Sune Bergstrm f the Karlinska Institute, Stckhlm, Sweden. In additin, the authrs express their appreciatin f the technical assistance f Mr. Peter Rachwal, Mr. Marc Hurwitz, Miss Marie Cper, Mr. Gerge Krnberg, Mr. Elias Meymaris, Miss Linda Carlsn, Mrs. Carl Tivnan, Mr. Jhn Mc- Mrrw, and the secretarial assistance f Mrs. Natalie demichele and Miss Alice Haddad. References. GROLLMAN, A., MUIHHEAD, E. E., AND VANATTA, J.: Rle f the kidney in pathgenesis f hypertensin as determined by a study f the effects f bilateral nephrectmy and ther experimental prcedures n the bld pressure f the dg. Am. J. Physil. 57: 2, MUIHHEAD, E. E., STIRMAN, J. A., LESCH, W., AND JONES, F.: The reductin f pstnephrectmy hypertensin by renal hmtransplant. Surg. Gynecl. Obstet. 03: 673, TOTH, T., AND BARTFAI, J.: The antihypertensive endcrine functin f the kidney. Clin. Sci. 20: 307, SOKABE, H., AND GROLLMAN, A.: Lcalizatin f bld pressure regulating and erythrpietic functins in rat kidney. Am. J. Physil. 203: 99, GORDON, D. B.: Nature f the depressr agent in extracts f rabbit kidneys. Am. J. Physil. 96: 340, MUIRHEAD, E. E., JONES, F., AND STIRMAN, J. A.: Antihypertensive prperty in renprival hypertensin f extract frm renal medulla. J. Lab. Clin. Med. 56: 67, BRAUN-MENENDEZ, E., AND VON EULER, U. S.: Hypertensin after bilateral nephrectmy in the rat. Nature 60: 905, MUIRHEAD, E. E., AND KOSINSKI, M.: Renal medulla and renprival hypertensin. Circulatin Res. : 674, LEE, J. B., HICKLER, R. B., SARAVIS, C. A., AND THORN, G. W.: Sustained depressr effect f renal medullary extract in the nrmtensive rat. Circulatin Res. 3: 359, ENEROTH, P.: Thin-layer chrmatgraphy f bile acids. J. Lipid Res. 4:, GREEN, K., AND SAMUELSSON, B.: Prstaglandins and related factrs XIX. Thin-layer chrmatgraphy f prstaglandins. J. Lipid Res. 5: 7, VOCEL, W. C, DOIZAKI, W. M., AND ZlEVE, L.: Rapid thin-layer chrmatgraphic separatin f phsphlipids and neutral lipids f serum. J. Lipid Res. 3: 38, SKIPSKI, V. P., PETERSON, R. F., AND BARCLAY, M.: Separatin f phsphatidyl ethanlamine, phsphatidyl serine and ther phsphlipids by thin-layer chrmatgraphy. J. Lipid Res. 3: 467, Fx, I. J., BROOKER, L. G. S., HESELTINE, D. W., ESSEX, H. E., AND WOOD, E. H.: A tricarbcyanine dye fr cntinuus recrding f dilutin curves in whle bld independent f variatins in bld xygen saturatin. Prc. Staff Meetings May Clinic 32: 478, BRAGDON, J. H.: In Lipids and the Sterid Hrmnes in Clinical Medicine, ed. by F. W. Sunderman, and F. W. Sunderman, Jr. Philadelphia, J. B. Lippinctt Cmpany, 960, p HICKLER, R. B., LAULER, D. P., SARAVIS, C. A., VAGNUCCI, A. I., STEINER, G., AND THORN, G. W.: Vasdepressr lipid frm renal medulla. Can. Med. Assc. J. 90: 280, VOCT, W.: The chemical nature f Darmstff. J. Physil. 37: 54, GABR, Y.: Observatins n a substance in human plasma which gives a slw cntractin f guinea pig gut in vitr. Brit. J. Pharmacl. : 93, LASER, H., AND FRIEDMANN, E.: Crystalline haemlytic substance frm nrmal bld. Nature 56: 507, MORTON, I. D., AND TODD, A. R.: The hemlytic acid present in hrse brain.. Purificatin and identificatin as cis-ctadec--enic acid. Bichem. J. 47: 327, BROCKLEHURST, W. E.: Occurrence f an unidentified substance during anaphylactic shck in cavy lung. J. Physil. 20: 6 P, GOLDBLATT, M. W.: A depressr substance in seminal fluid. Chem. and Ind. (Lndn) 52: 056, GOLDBLATT, M. W.: Prperties f human seminal plasma. J. Physil. 84: 208, VON EULER, U. S.: Zur Kenntnis der pharmaklgischen Wirkungen vn nativsekreten und extrakten mannlicher accessrischer Geschlectsdriisen. Arch, exptl. Pathl. Pharmakl. 75: 78, VON EULER, U. S.: Uber die spezifische blutdriicksenkende Substanz des menschlichen Prstata und Samenglasensekretes. Klin. Wchschr. 4: 82, VON EULER, U. S.: On the specific vas-dilating and plain muscle stimulating substances frm accessry genital glands in man and certain animals (prstaglandin and vesiglandin). J. Physil. 88: 23, VON EULER, U. S.: Weitere Untersuchungen iiber Prstaglandin die physilgisch aktive Substanz gewisser Genitaldriisen. Skand. Arch. Physil. 8: 65, BERCSTROM, S., AND SJOVALL, J.: The islatin f prstaglandin F frm sheep prstate glands. Acta Chem. Scand. 4: 693, BERGSTROM, S., AND SJOVALL, J.: The islatin