Tobacco Documents Online

SURFACE RNPLICAS OF PULMONARY ENDOTHELIAL Cti1.LS IN CULTURE Although numerous enzymes are known to esisl on the surface of pul- monary endolhelial cells, their topographical d'utribution is unknown. De- scribcd here is a simple method which uses an unmodified critical point drying apparatus and a high vacuum freczestch unit to prepare surface rcplicas of pulmonary endothelial cells. Thin lechsiqu, should be applicable to all eelis in monolayer culture. F-ndothelial eeW Srowe on cover slips or Slass slides were washed free of the culture medium, dted, dehydrated, and critical point dried. A 8alzer s freeze-etch unit wu used to prepare surface replicas. These have certain advantages over other types of preparations in Iha1 they can be examined with the resolving power of Ihe transmission electron microscope d and they can be used to esamine the aurfaw of whole cells. This technique may prove to be particularly useful for mapping specific enzyn.es, reccplors, and cell surface factors with the aid of markers attached either to specific antibodies or to subslances such as /ecliss that bind specifically to surface Rroups The ppearance of pulmonary endothelial cells in surface replicas is dc:cribed as a first step toward establishing a basis for such studies. Hart, M. A. and Ryan, U. S. TiUfue 6 ('.ll 1l)()):441-449, 1978. Other support: U. S. Public Health Service and the John A. Hartford Foundation, Inc. From the Department of Medicine. I/nivenily of Miami School of Medicine, Miami, Fla. SPECIFIC METAHOI IC ACTIVITIPS OF PULMONARY ENI)4)TTIELIAI. CELLS This brkf overview of lung metabolism focuses on interactions of the lung with circulating substances that ue processed actively by pulmonary endothelial cells. It is now knows that Lhe lunp are capable of sekclively processing a large number of subslasces of very different chemical types, in- eluding biogenic amines, adenine nucleotides, proslaglandins, polypeptides, drup, and lipids. Furthermore, pulmonary endothelium may contain enzymes and other agents active in coagulation and antiooagulation reactions. One enzyme of 1he pulmonary endothelial celis, angiotensintonverting enzyme, is capable of Inactivating bradykinin and of forming angiolensins 11 and 111. In addition to their specific metabolic ac/ivities. Ihese cells may also Iwsseu en- cymes Inhibitors such as .,-macrokarbulin. Although endolhelium of other vascular beds has similar or ldenlical properties, the pulmonary crKlothelium is uniquely silualed, between the central venous and systemic artrria/ circu- laliun., to affect specific functions of the lungs and specific functions of dis- lant organs. Thus, kinins and some pro.laglandins and biogenic amines do not survive passage through the lungs and do not enter systemic arteria( blood. In contrast. the quantities of angiotensins 11 and III which reach peripheral organs and glands via the systemic arterial circulation are probahly primarily dclcrmined by pulmonary endothelium. Rr'ln, 11 S and Ryan, I W. I In: S.nden, C. L. er d. (eds.): pulmowary ALrrooAase and E'ltheled CeQr (16th Ann. Hanford Biol. Symp.), National Technical Information Center, Energy Research A Devetoprnest Administration Seriea 43. U.S. Dept. of Commerce, Springfkld, Va., 1977, pp. 11S-14s0. Other awp'ort: U. S. Public Health Serviee, Hartford Foundation and the Heart Assocutan of Palm Beach Couoty; Fla. From the Papanicolaou Cancer Research Institute and Deparlment of Modicine. University of Miami School of Medieim, Miami, Fla. PULMONARY EtNI>ryfllF.l.IAL CELLS: KININASL311 AND OTIINR PL'PTIDB HYDROLASP. ENZYMES The present study was started /o (ocus attention on the fact that kinisrs 11 is not the only peplide invdved (n the processing of circulating bradykinis and angiotensin I by intacl lungs. While aniliolerria I aod bradykinin disappear during a single passage through the pulmonary eirculstion, these polypeptide Iwrmones are not lakea up by the lungs but are hydrolyzed by enzymes on or near the luminal surface of the pulmonary endothelial cells. Kininase 11 (aniliotensintonverting eszyme), a dipeptidyl earboaypeplidaae with tM capacity of inactivating bradykinin and of converting anRiotcnsis I to ila potent lower homoloRtrc, selliotensia 11, is described in detail here. Ilowever, the lungs contain other enzymes that sre capable of inactivating bradykisia and of metabolizing anpolensin I to yield products other than angiotensin 11. These also are likely to be situated on the lumissl surface of endothelial ulls. Two of the eszymes are capable of removing the N-terminal amino acid reaidues of bradykinin and angioteosin 1. For bradykinin, the removal of the N-terminal residue yields biologically inactive products. However, the removal of the aapartyl residue of ansiolessis I yidda a precursor of anRioleosM 11/. Interestingly esuubh, close observation of kininoe 11 shows that this enzyme eae aho convert des-Asp'-angioknsis I to angiolensin 111, a compound aves more active thas angiotessin 11 is stimulating the secretion of aldosteroae. la wmmary, this p.per. In adefNios to considering the apernative tates of theas polypeplides, attempts to n.ssess the selectivity of action of kiainaae 11. Thi, enzyme while acting as a dipeptidyl carboaypeptid..e can, in fad, hydrolyze a wide variety of polypeptide substrates. However, the relative aflinity of the enzyme for different substrates varies enorsnously, suggesting Iha/ this type of selective action may be of physiological importasoe. Ryan,1. W., Rym, U. S. sod Chiu, A. T. Is: Sanden, C. 1.. et a/. (eds ): rrlnwnory M.rropbage, .nd EOlr11e7Jaf Crlb (16th Ann. Hanford Ilial. Symp.)t National Technical Information C<oter, Energy Research & Developrnent Administration Series 4), U. S. Dept. of C.ommeree, Spriagfield, Va., 1977, pp. 141-14e. OtRsr arp'orrr U. S. Public Health Servioe, Ilarttord Foundation and the Heart Association of Palm Beach County, Fla. F om the P,panicolaou Cancer Research Institute; Ikpartment of Medkine. University of Miami School of Medkine, Miami, Fla ; and the Medical ('olkge of Virginia, Richmond. 38 39

11UMORAL CONTROL OF ARTERIAL BLOOD PRESSURE: A ROI.@ FOR TH8 LUNG? White the primary function of the hmp Is the eschante of tua. between blood aod dr, the past 10 yean have nhorre that the lungs can sekclively process a wide range of hortnonea, prohormooes, and other escitatory asents that have direct or Indirect eRects on the trystank arterial blood pressure. It is known now, for instance, bow bradylinie and antiolensin I are rm:tabolized at the cellular, subcellular, and molecvlar Ieveh. More speciAcalty, recent studies have defined in detail bow a.w Mmg enzyme, antiotemin-converting enzyme, can participate in the diwieatb" of a hypoknsive hormore, brady- tinin, while forming antiotensin 11, oea of the most polent hypertensone agents known. Tris enzyme Is found along the hu+inat surface of the endothelid cells lining both the larte pulnsonary vessels and Ihs capillaries. As blood passes through the pulmonary vascular bed, aegioteosin<onverting enzyme is washed with its owe aubslrates ia a cwntirNwrrly eowioR liquid phase, and the reaction products are released direcdy Into tlt" systemic arterial clrculation. The enzyme is found in vascular beda other due that of the lunp, but only the lungs export the reaction product, angjolerssin 11, to the systemic ecrculation. Also, the lungs cannot detrade angioleruie 11, which ie.ures its avail.biliry for export. These features suggest a role for aediotensin<oevertint enzyme in blood pressure homeostasis, but the detailed rehtiorohips of the luop to humoral control of the arterial blood prsrurc remain to be defloed. Ryae, /. W. and Ryew, U. S. C.rdlovarcrfa. Aledk/ne 7:S)1-3)7 rr pactbn, 1971. Other st.'p~rt: U. S. Public Ilcalth Service, John A. Hartford Foundatioo, American Ileart Association, and Ileart Association of Palm Beach County. Fla. From the Department of Modkine. University of Miami School of Medicine, Miami, Fla. NEW SUBSTRATES FOR TIIE RADIOASSAY OF ANGIOTENSIN CONVERTING ENZYME OF ENDOTHF.LIAL CELLS IN CULTURE Three simple acylated tripeptidea were synthesized as substrates for angiotensin converting enzyme in order to provide the meaos to measure Its activity in cultured endothelial ee0s. Specifically, these preparations were p-I'Hlhenroyl-Pro-Phe-Art-OI( (11), p-1•H/benzoyl-Phe-Ala-Pro-OII (1) and p-I'Hlbennoyl-0Iy-His-Ltu-OH (111). Substrate I haa the lowest Km (12.5 rM) and is the most sensitive for assay. The angiolensie converting enzyme content of 10' cells can be measured afler a)0-minute Incubation period al )7' C. TLe rad'aactive reaction product (s separated from the substrate by estraction of the acidified reaction misture with an organic solvent. Tlrc rate of product formation is then quantified by liquid scintillation counting of the organic phase. Substrate 111 can also be used for this aaay, but requires longer in- cubalion OJ MNns/ and high uit concentrations (0.75 M Na,SO.). Sub- suate 11 is rwrt y.ec rfic and rs hydrolyzed by more than one aufothelial cell enrymc Ryan. J. W., Chung, A., Martio, L C., and Ryan. U. S. Tiuue d Cell 10( )):335-362, 1971. From the Department of Medicine. University of Miami Schoo( of Medkin., Miami, Fla. A SIMPLE RADIOASSAY FOR ANOIOTENSIN-CONVERTINO ENZYME This paper describea a radio.s..y method in which p-/stllbenzoy/gly- cylRlycyltlycine ('11-labeled hippuMtlycyltlycine) is used as substrate for aegiolensiotonvertind enzyme. Since enzyme activity is measured in lerms of the rate of release of 'H-labcled hippurate, prior di.lysis of serum sueplea and Iiaue homoteeatn is not rcquired. TUe product in separable from the wrbsurate by eatrauion of acidified Kaction mislurea with ethyl acetate. Tbis way was used to measure angiotensin<onverting enzyme activity In sera of mae, rat and guinea pig, and in homogenates of rat kidney and guinea pig bn=. To further test the specificity of the assay, serum samples (from healthy vdunteen, p.tients with active aareoidosis and guinea pip) were examined for their ability to form radioactive products other than hippursic. In each esperiment, all radioactivity was associated with hippurate and unhydrolysed substrate. The major advantages of this assay over those used previously are simplicity and rapidity. For eaneple assay results for human serum anSio- leesiatonvertieR enzyme can be obtained withi. 1.5 hour. of receipt of serum samples. Within the limits /ested here, the assay appears to be specific. llowever, interference by hitherto unrecognized enzymes of abnormal sera must be ruled out. I Ryaa, ). W. et J. (Ryae, U. S.) dloc/kndrd lournal 167:S01-SW, 1977. From tbe Department of Medkine. University of Miami School of Medkin., Minol, Fla. 111. Ne.rt .nd CJrcul.Hon PURIFICATION AND CIIARACTP.RITATTON OF 1.ECfININ:CIfO1.P.STP.RO1. ACYI.TRANSFERASP. The purification of kcithin:chokaterol acyllraederase has previously been reported. The newer method described here, however, yields a preparalion purified 16,000-fold and free of high density lipoprotein and albumin. In coo- trast with the product previously obtained in this laboratory, which was only hdf as pure and contained two maior proteins, the one obtained by this newer technique showed on/y one band upon polyacrylamide tc) electrophoresis. SDS- uree polyacrylamale tel electrophoresis and ialKlectr/c f.x:using, and one arc in immuoodiRusion against a toat anliserum preparatioo. The enzyme was 40 41

determined to be a tlycoprolein with a molecular weight of about 70,D00 and a pl of ).7-1.0. Oustow, E., Varma, K. Q, and Soloo, L. A. Scandlnovian lorrnd o/ Clinkd 1 LaAorarory Investigation )6(Sltppl 150) :1-S, 1978. OtAer.rpportr U.S. Public Health Service. From the 11pid Rexarcb Laboratory. Department of Medicine, Temple Uoi- versity Ilealth Scknces Center, Philadelpbh. RPLATIONSIIIP BITiWIiPN 111011 DENSITY LIPOPROTEINS AND 111P. RAl EOP fN VITRO SERUM CNOLESTEROL ESfI?RIFICATION Mounting evidence suggests that the Incidence of coronary heart disease is inversely relaled to the concentration of high densily lipoprolein cholesterol (IIDLl'). Also, the plasma concentration of HDL-C is foveraely related to the body pool of cholesterol suggesting that HDL protects against the development of coronary heart disease by transporting tissue cholesterol to other ortane (liver) for escretion. In addition, HDL has been shown to be the substrate of choice for kcilhinchokslerol acyllransferase (LCAT), the enzyme responsible for the esterilkation of human plasma cholesterol. This study of geriatric aub- tects attempts to determine any relationships between LCAT. NDL-C and the rate of esterifkation which might indicate /hat the enzyme i activity is one of the factors involved in IIDL's supposed antialherosclerotie effect. The rate of cholesterol eslerifkalioo and the IIDL-C concentrations were measured in vitro in serum samples from nine men and 18 women whose age ranged from 74 to 95. 7he resulls clearly show Ihat asymptomalic geriatric subjects can have lower HD1.C levels than those considered normal in persons under 60. Un- especledly, however, the biochemical data also reveal that the cholesterol esteri(kation reaction rate is more rapid In sera containing ku than 40 mt/d) Nl)1.-C than In samples with more than 40 mt/dl. No correlation appeared between cholesterol eslerifkation and a proleia(s) which can produce an immune tamma globulin Ihat completely inhibits in vitro serum cholesterol e.terilkalion. These rewlts are discussed with reference to the presumed re- lationships between HDI.C and atherosclerosis. So1o0, L. A. sod Varma, K. O. Standlnavian Journal of Cl/nkd • Laboratory Fnvenltarion 38(Suppl. ISO): 72•76, 1978. OtAer support: t/ S. Public Health Service. From Temple lloiversity Ilospital and School of Medicine. Philadelphia. IMMUNI)t (N.1('AI 1•VAI IlA t U)N t)P 1CA 1' 1)1'Fll'IFN('Y Ibn saN1r aurmpu Io rv.lu.lt the nsture uf Ihc 1('A1 dcftciency en• c+.umcit.1 m i..• s.,b/c.is hy intan1 nf an immun,kIJlusion ud tmuronumhi bition technique, utilj;int two different antibodiea obtaine;l from two diRereat toats to test a highly purifled LCAT preparation against LCAT-0eOcknt sera Tbe reaction of identily between normal aerum, defkient sera and purified LCAT suggested the presence of a catalytically inactive en:ytne in the da- lkient sera. In normal serum, excas antibody inhibi/ed 50% of the LCAT activity. One of the antibodies faikd to inhibit the enzyme when the Wt system contained escess deficient serum, seemingly supporting the above eon- cluiwn. The other antibody preparation, however, inhibited LCAT activity even in antigen escess. The precipitin lines observed in MnmunodiRwiqo do oot seem to represent serum LCAT. In view, of the higher 1i1er of antibody observed In immunoinhibilion esperimenta with this antibody, a question eow arises as to whether immunoinhibiuon by the antibody will also be abdiahed at lower ralios of antibody to defkienl serum. , Varma, K. (1., SofoQ, 1.. A. and Frohlkh, 1. Scandlnavian Jorrrnal of Cqnkal d Laboratory Investigation )ti(Suppl. 1S0): 6-11, 1978. From the Lipid Research Laboralories, Department of Medicine. Temple Uni- versity. Philadelphia; sod the Dep.rtment of Palholoty. University of British Columbia, Vancouver, Canada. a TNE INHIBITORY EFFECT OP 7-KETOCHOLESTEROL ON Tt1E CHOLESTEROL UPTAKE BY THE ARTERIAL WALL Previous esperimenls have ahown that 7-ketoehoksterol inhibits the up- take of cholesterol by the artery in .itro, but In vlvo Inhibition proved difikvlt to demonstrate. This report eaamina the mechanisms underlying this in- hibilory action on cholesterol uptake by the arterial wall. The sorus of hepaleclami:ed rabbits injected with sohrbilized 7-ketochokuerol failed to show In vivo inhibition of choksterol uptake. Neither wu choksterol uptake inhibited when rabbit carotid arteries were perfused /n vitro with pla.mm pooled fronm hepaleclomizcd animals injecled with solubilited 7-ketochokslerol. llowever, porcine coronary arteries demonstrated dgnifkant cholesterol uptake inhibition when they were perfused wilh iaologoua plasma containing par- ticulate 7-kelochoksterol. These resulta demonNrale that only the p.rticulate fraction of 7-ketochoksterol inhibits cholesterol uptake by the arterial wall. llrus. the possibility that the inhibition may not occur at the arterial wall but results from a physical Inleraclion between particulate 7-kelochoksterol sod e/wkslerul in the perfusale must be considered. Sarma, 1. S. M. and elng, R, l. Journal of Motecular and Cellular Cardiology 10:197-201, 1979. Other support: lhe Hoover Foundation. • From the Iluntintlon Memorial Ilospilal and lluntinti.•. Insthute of Applial Medical Reseaich. ('alilmnia lnstilute of 'Iechnoluty, 1'asadcns, and the University of Suulhern ('ablornia, I os Anteks. 42 43

1 l3FFHCT OF H1GH DENSITY LIPOPROTFJNS ON THE CHOI.PSI'(iROL UPf AKN BY ISOI.ATED PIO CORONARY ARTERIES In this series of eight esperimenu dealiog with the effect of added high density lipoproteins (11D1.) on chokaterol uplake, pig coronary arteries were perfused In vitro with isologous plasma containing sll-I,2-choksterol with and without added 11D1. from the sarne species, and the cholesterol up- take by the arteries was measured. Raulta showed that the arteries perfused in the presence of added HOL took up sijaificantly kss cholesterol than did the controls. Cholesterol uptake by Ihe cootrol group of arteries averaged 180 :t 34 n nwles/g, whereas tbe arteries pertused in the presence of added Ill)1. took up only an average of Ilt :t 2 a rnoks/l. The difference between the two values was significant (p<0.001). While one possible reason for this observaNon may be that HDL direcdy inter(eres with the entry of chokstero) intn the arterial t'ssue, a second possibility is that lID1. is able to promote cmua of cholesterol from the tissue. There is some supporting evidence for thia, and from this standpoint the results presented here, which co.nplement epiJen,iological and cell culture uudies by olhen, seem to flt into the general hyprwhesis that HDIL promotes efllux of cholesterol from tissue. Sarma, 1. S. M.. Tschurtschenthakr, O. V. and Iint. R. l. Artery 4()):214-22), 1978. OOther support: Hoover Foundatioo. Prom the Uoiversity of Southern California School of Medkine, 1d Angeles; Huntington Memorial llospital, California Institute of Technology and the Huntington Institute for Applied Medical Research. Pasadena. ('OMPARATTVEi E?FFE('iS UP ('HRONI(' FTIIANO1. AND AC@TALUIiHYDII FXPl)SURP ON MY(X'ARUTAL FUNCIION IN RATS In order to develop an esperimental model that would permit separate monitoring of the effects of ethanol and acelddehyde (one of its mclabolita) on cardiac performance. two metabolic inhibilors, 4-methylpyrazok (4-MP) and pargyline (PAR). were used to iodepeodently raise the blood levels of ethanol and acetaldehyde respectively. Rats who received as much as 36% of their total daily caloric supply (rom ethanol for a period o( three to (our weeks were sacrificed after injection wilh eilher saline or one of the in- hibiton. Blood samples were measured for their ethanol and acclaldehyde kvels. Tbe milochondrial respiration. /n vJtro contractility of Rlycuinated heart muscle fibers and myocardial protein synthesis were determined. Accord- ing to the results, either 4-MP or PAR administered in addition to ethanol damaged mitochrondrial respiration and myocardial protein synthesis more severely than did akohol ingestion alone. These data illustrate that both acetaWehyde and ethanol are capable of damaging the myocardium. and that high concentrations of either substance can severely aflecl its metabolism. Wei.haar, R, Bcrruglia, S, Ashikawa, K.. Sarma, 1. S M., and 8ins. R. I. 1'hr lourn,d o/C'Lni.ulPharnru,.rloRy 1R(R39):177 )R7, 1978. 44 Other support: U. S. Public Health Service and the Hoover Foundation. From the tluolioglon Institute of Applied Medical Research and Iluetiogloa Memorial Hoapital, Pnadena; the University of Southern Cali(oroia, Los Angeles; and the California lmtitute of Technology. Pasadena. CARDIAC INDEX AND INCIDENCE OF HEART FAILURB CBILS Because there appears to be a relationship betweea pulmonary blood (bw and the presence of iron-ladea macrophapn is bronchial washiop, the percentage of these ulla was assessed in the apwurn of heart disease paticou before their cardiac index was measured. An inverse relationship was found between the percentage of these qcatkd heart failure cells ia aputura amcan and the cardiac indes of the palienla, teprdkr of the (ype of cardiac illness. The highest proprxlion (73%) of theae cells was found in association with the lowest cardiac indes, or I Ii1er/min/aq m of body wr(aee area (BSA). In eoa- trast, when the cardiac indes was around 3 liter/miNaq rrs BSA, less than 2% of the macrophaRes contained iron, which is consistent with previous obaervr /ions in healthy controls. However, widely scattered and seemingly uordaled values were obtained at intermediate cardiac index kvels, presumably reflecting individual variations in the degree of activity of the aubjects' reliculoendothelial system. I Friedmao-Mor, Z., Cha(on, l., Turodor(, H. and Orkin. L R. Archfrei oJ Pathology .nd lALporatory 1/edlclnne 102:41t-119, 1978. From the Departmenu of Anesthaiology, Albert Einstein College of Mediciec, The Brona, and New York Univenity Medical Center, New York. TRACHEOBRONCIIIAL CYT'OLOOIC CIIANGES AND ABNORMAL SERUM HEME PIGMENTS IN HEMORRIIA(7IC SHOCK Among the pathophyaiologie and metabolie aberrations caused by hna- onhajic ahock are lysis of erylhrocyles and degradation of free serum bemo- Rlobin into abnormal heme piRmenls which are believed to be toxic. /1 had previously been shown that significantly elevated numbers of iron-laden his- tiocyta, as demonstrated by the Prussian-blue staining melhod, appear ia the tracheobronchial secrelions of patients in hemorrhagic shock and, during cardiopulmonary bypass, in those undergoing open-heart surgery. Because the hisliocyles may ingest abnormal heeneyiRments as part of a defense mechanism, this study altcmpu to correlate the percentage of iron-laden ulls in Ihe tracheobronchial sccretions of patients in hemorrhagic shock with the presence of hemoglobin degradalion products in their serum, as delermined by acanning spectrophotometry. In general, there were fewer iron-laden hisliocytq when hemoglobin de1gradatinn was advanced Ihan either In the absence of abKormal heme pigrncnls or when Ihere were only minor degrees of dcRradatiun. Ihcse observations demonstrate the presence of abnormal heme pigmcols in the act-um 45

I I of humans in hemorrhagic shock, and suggest that few of the degradation products of hemoglobin remain in circulation when the degree of reticuloen- dothelial response is high. lAus, the dala preaeoted here probably reflect the reticuloendothelial prouss that eliminatea toxic products accumulated in the circulation during low-flow sutea. It is also pouible, however, that the resulta actually represent the liberation of myoglobin or of enzymes, such as cyto- chromes, perosidase, catalase and othen, that incorporate heme molecules into the circulation. Friedman-Mot, 7.., CAalon, I., 7Lrsdorf, H., and Orkin, l.. R. The Iournal of Trauma 17(11):/29-i31, 1977. From the Departments of Anesthesiology of New York University Medical ('enter, New York, and of the Albert Plmteio College of Medicine, 'Ihe Bronx. N. Y. ABNORMAL Ifl?MP.PROTEIN PATTERNS IN HEMORRIIAGIC SI(OCK Abnormal heme pigments have been described earlier in the aera of animats bled to hemorrhagic shock Ifere, electrophoretie studies have been run on the sera of patients in hemorrhagic shock in an attempt to identify abnormal heme pigments and to relate the appearance of some of these compounds to rnor- tality rates. Subjects for this study included 10 consenting adults who were suAerinR from hemorrhagic shock. In addition, blood umpka were obtained from: (1) five hcalthy volunteers, (2) three 21-day-old specimens of bank blood, and (3) two healthy patienu anesthetiud but not in shock. Results showed that the accumulalion of inetabolilea, caused by the impedcd eircula- tiun, degraded free hemoglobin into herne pigmeota, and their concen- Iralion then reached a level that esceeded the normal henre<arryinR capacity of aerum prolelna. All patienu In shock bad distinct bands e/ varying iotensity in the benzidine-stained strip. Nina patients had haptoglohin-hemo- globin bands, live patients also had a hemopeain-heme band, and four patients had an additional melhemdbumin band. 'il+ere were no deaths associated with the presence of haptoSlobin-hemo6lobia aloee In serum. However, as shock deepened and mortality roae, hemope.in-heme and mcthemalbumin appeared. The highest mortality rate (4 out of 3 cases) was found when bcah herno- pe.in-heme and methemalbumin were present. 1t seems, therefore, that ad- minntration of additional serum proteins to increase Ihe binding capacity of the blood might reduce the toxic effect of heme. Fricdman-Mot, Z., CAafon. I., Oorstein, P., Tbrndorf, It., ('huba, 1. V., and Orkin, L. R: TAe lor.rnal o/ Trauma 1s(2):104-107, 1978. Wnm the Ikpartmtnts of Anesthesioluty and Pathnlaaty. New York llnivenity f.lydu.l ('tater, New Yurt, and 1he 1)epartment of Anesthesiology. Albert I.n.tr~n I~..Ilete a•l MrJia ~nt 1 ht Ilr.m• N V 46 ROLE OF N1OH-f(IOI-ECUI.AR-WEIOHT KININOGFN IN SURFACP,-BINDING AND ACTIVATION OF COAGULATION FACTOR XI AND PREKALLIKREIN It is known that high-moletular-weight (M,) kininolien is a cofactor for the activation of Factor XI, prekallikrein and HaReman facwr, but several hypothescs exist in explanation of Ihis functional role. This study demonatrata that one mechanian by which high M, kininoRen acts as a cofactor for the activation of both Factor XI and prekallikrein in plasma is to bind both these molecules to the aurface where they are activated by surface-bound activated Hageman /actor. Experimenlally, when rs'1-Fac/or XI and r'al-prc- kallikrein were added 1o kaolin-activated nornril human plasma and plasmaa deficient in high M, kininoRen ad IlaReman faetor, reaulta showed that high M, kininotien was essential tor normal binding and cleavage of both Factor XI and prekallikreia on the kaoli• .urface, whereas Ilalleman factor was eucntid tor the cleavage but not for the binding. In normal plaama, tiA% of the activated Factor XI remained surface-bound, but 80% of the kallikreie did not. These findings are consistent with the hypothesis that. In the initial phase ol contact activation, high M, kininogen links both Factor XI and prekallikrein to the esposed surface where they are activated by wr/ace-bound activated Hageman factor. Once aetivated, the Factor XI molecules remain localized at the aite of activation,-in contrast to the kallikrein mokcuks that are found largely in the surrounding plasma. Wiuin., R. C., Bouma, l!. N., CocAraose. C. Q., and Oriflin, 1. H. proceedings of the National Academy of Sciences of the Unlted Srates of Arneriea 74(10) :16)6-4610, 1977. Ot/Ler .r pprt t National Instituta of Health. From the Department of Immunopathobp, Scripps Clinic and Reaearci Foundation, La 1o11a, Cal. SURFACE AND FLUID PHASE ACTIVITIES OF TWO FORMS OP A(?IVATED IIAOEMAN FACTOR PRODUCED DURIN(7 CONTACT ACTIVATION OF PLASMA This report describes the ability of the two forms of activated Harsna, factor (HF,) produced during contact activation of plasma to activate prf kailikrein and lactor XI. The infhrence of the wrf.ce on the cleavage of lactor XI and prekallikrein, and the distribution of these proteins between the sur- face and fluid phase compartments before and after their interaction with HF, were the particular fackm considered. In these e.periroenta, the 110,000 mokeular weight Iwo<hain enzyme'Ihat remains bound to Ihe sur/ace, termed r1IF„ was capable of cleaving surface-bound prekallikrein and /actor XI. Factor X/ remained bound to the wrfloce, while prekallikrein and kallikrein rapidly entered the supernate. A 2e,000 molecular wei6ht single-chain mok- eule released fu,m the surface dutinR contact activation, termed 0 IfP„ split prekallikrein. Inrt nnt factor X1. 'lhis reaction occurred whlthcr the substrate was surface brwrnd or in solution. lhcse data suggest that Intrinsic coagulation resulting (rons the activation of (actor Xt is  localized event thal 47

occurs at the site of contact activation and is caused by the action of a-HF,. On the other hand, kinin generalioo and flbrinolysis resulting from the forma- tion of kallikrein can be initiated either by r1117. at the contact activation site or by fi-HFs throughout the plasma. Theae two activities are kuaranteed further dissemination by kallikrein i rapid dissociation from the surface. Revak, S. D., Cochrane, C. G.. Bouma, B. N., and Orifflo, l. H. The lourrwi of Ei perimenrd Altdicinr 147:719-729. 1978. Other auprortr U. S. Public lie.h6 Service. From the tkpartment of Immunopatho{og,y, Scripps Clink and Research Foundation. I.a lolla, C.I. 1111? INTI?RA(T1ON OF rs-I-PRO'iE1NASt31NHIBITOR WI111 StrRINE PROi'EINASBS About 10% by weight of the proteins ie human plasma are known to io- bibit proteolytic enzymes. 7lrese proteim may have either a very narrow range of inhibitory specificity or very little specificity. While the major plasma pro/einax inhibitor. .-I-proteinase inhibilor (e-1-PI), previously known as .-1-antitryp.in, has a broad range of speeificity, it cannot inactivate certain enzymn. llws, in spite of the very high .-1-P) concentration in plasma. It seems certain that ..2-macroglobulin is more significant in the regulation of prnlcolysis by all types of enzymes of this clasa. 1lre potential role of ~t-PI in the regulation ol the coagulation process hu been a controvcnial subject for many years. If it were found to inhibit thrombin, it might have some significance In the regulation of thrombin activity in vivo. Newer procedures have made it possible to isolate .-1-PI from antithromlwn III activity and have allowed several conclusions to be drawn. Although the tole of this Inhibitor in eoagulation is probably minimal, based on its high concentration In plasma, its concentration in the acute phase state aod its ability to inactivate thrombin, a supporting role in Ihis process cannot be entirely dismisxd. 'ilre development of powerful synthetic peptide iahNtors provides new methods for studying the inhibitory activity of pluma proteiaues. Trerls, !.. Matheaon, N. and lohaaon. D. In: l.uodblad, R. L., Penlon, 1. W., 11. and Mann. K. 0. (ods.): CJrrmtnry .nd eiolory of ThromAin, Ann Arbor, Michigan: Ann Arlwr Science Pub- tlshers, 1977, pp. 431-440. Ot6er support: Natiooat Institutes of Hedth. From the Department of Biochemistry. TUe Univcnity of ()corgia, Athens. CONTRIBITTION OF CARI)IOP111.MONARY BARORF.('EPTORti ' TO '1111'. ('ON IRI )I. O1' 77I1i K 11)NI!Y The electrophysinlogic behavior of many of the eardiopulrnonary baro- receplnrs /us bccn eslen.ively e.ammed and a sreat deal is knr.wn abrwt 48 I the determinants of; their discharge frequency. However, their rok In the orpnism is not aa well understood. Whik evidence indicatea tbat receptors in the cardiopulmonary region ean, under certain eircumatances, eaert a pro- found influence on renal sympalhetic nerve activity, renal vascular resistance and renin rekase, it is difficult to prove etperimeotally that these receptors .r. Involved in the cohtrol of blood volume. Hrrwever, the available evidence in- dicatea that cardiopulmonary receptors with vagal aRerenta eaert a took inhibilion on both renal nerve activity and on renin release. The magnitude o( this inhibition appears directly related to changes in blood volume. Atriat r well as ventricular receptors can infhacnce the aecretioa of renin. Cardio- pulmonary receptors with vagal aRereota may also reAealy modulate renal prostaglaodia secretiorr. There is preliminary evidence suggesting that cardio- pulmonary receptors with .ympathelie aRerenta can inAuence renal nerve acGvity. le this paper, the limitations of previous studiea are outlined and a direction for future studies is suggested. PiaaRy, it is tooch.ded tbat alteratioe. in cardiopulmonary vapl aAerenl input and tbe resulting chantea in renal nerve activity and ie reain release are appropriate tot Iha maintenance of blood volume bomeo.tasi.. TArner, A(. D. Pe4radon rroceedi'nngs )7(S):1209-1217, 1978. Other support: National ImtitWa of Health. Prom the Department of Physiobsy and Biopbraica, Mayo Clink and Mayo Foundation, Rochester, Mim. IV. Neuroph.rntatology and Pky.iotosy DISCRIMINATIVE STIMUI.US PROPERTIBS OP NIaOT1NE AND NICOTINE-RELATED COMPOUNDS Nicotine, one of the drup moat widely used by man, has been observed to have very apecifk and sensitive discriminative stimulus (DS) properties 'The DS approach, which has been very useful in studying the mechanism of action of nicotine, measures the subjects' response, oot to a specific effect of the drug on behavior, but to the presence of the drug within the CNS. In thV Investigation, a series of eaperimenta was Initiated which studied the See- eraliralkm of the nicotine ()S to chemical analogues of nicoline arwl to various eompounds purporaed to harAe similar behavinral effecta. Usinl the DS behavioral paradigm, rats were trained to discriminate between the US eflecta of nicotine (200 y.g/kg, s.c.) aod saline using a two-kver, food-reinforced operant procedure (VI 15 sec.). Teo diQerent compounds were evaluated in rats trained to discriminate nicotine over a wide ranje of doses and .cron both subcutanern.s and intUavenlrieular routes of administration Oidy on. w.npound. )-pyridytmdhylpyrrolwline, produced significant nicotinclike ac- tivity and, although the experimental animals perceived this compound u it 49

they were given nicotine, it possessed only one-seventh the potency of nicotine. The niccNine stimulus did not generalize to compounds such as lobeline, colinine, or d amphctamine. One of the most striking results of these studies was the specifkity of the nicotine DS. indicating that Ihis paradigm mav provide a unique approach to studying the structure-aclivity relationships oC the be- havioral eflecls of nicoline. Rozt.-rans. l. A. er d. In: Biittig, K. (ed ): Sellaviora/ EQects o/ Nfcodne, B..el: Karger, 9979. pp. 70-82. From the Department of PhannacoloRy, Medical College of Virginia, Richmond. A('UMI'ARISON OF NI('OTINB AND STRUC7URAI.LY RCI.ATI'.f) COMPUUNI)S AS UISCRIMINATIVE S71MUU lhe discriminative cue produced by oiootine Injeetion seems to depend upon the central rather than the peripheral effect of this agent, and recent aludres have suggested that Ihis centrally produced cue occurs at N<holineraie receptors hut not at muscarinic (M) reuplors. Thua, relatively specific stim- ulus cflecls are involved Seven compounds were eaamined for generalization to the nimulus effects of nicotine, in order to investigate whether structural similarity elreru an analogous central cue and to elucidate the chemical con- flguratron crucial to its pnxhrctnm Of the compounds lesled, only )-pyridyl- mc/hylpyrndidine ( 1-PMI') generalized to the stimulus effect of nicotine. Pquivaknt nkoline-like responscs were obtained with tlx) pg/kg. a dose ap- prosimately four limes that used for the original nicotine discrimination. Tlre IiD„ for )-PMP was atxwt five times that of nicotine. None of the carn- pounds signifkantly blocked the nicotine cue. Nowever, the nicotine-like cue produced by 800 pg of 3-PMP was effectively blocked by meeamylamine but not by heaamethonium or by alrupine. llrerefore, 3-PMP appears to cause generalization to the nicotine cue by acting on central nicotinic cholino receplors, as was previously reported for the nieotine discriminative stimulus. The I:D„ for the blocking action of ineesmylamine on the stimulus effects of 3-PMP (s00 pg/kg) and of nicotine (200 M6/kg) was 0.32 and 0.20 mg/kIl, respec/ively. The use of this discrimination technique for comparing structurally related compounds is an effective as well as speeifSe technique. Chance, W. T.. Kallman, M. F)., Roseccauu, /. A., and Spencer. R. M. Drkish lorrnaf o/ Phannaroloty 6):609 616, 1978. From the Department of Pharmacolop, Medical College of Virginia. Rich- mond. A DESCRIPI-1t7N OF' TIIP. NI('OTINP, ST7MULl1S ANI) l I:STS OI' fIS (ll?NIiRAI /IA fl()N 10 AMF'IIHa AMINN. ihis aeries of c.pe+imenii was designed to investigate the discriroinalive slirmdus prrqemes uf nwulrne and ro detcrmine the degree of kencraluatton 50 of he n'r.otine stime)lus effects to those of d-amphetamioe. In each of three separate experiments, the subjoct's performance was assessed wioR a twokver operant procedure with liquid food reinforcement. In the nrst study, rats wero trained to discriminale between various dosea of nicotine (100, 200, or 400 pVkt) and saline under a VI-(S a schedule of reiniorcement. The second study investigated the time-0urat'an parameter of the nicotine atimulus by mooiloring discrimination between 40 pg/kg of nicotiae and saline under three different schedules of reinforcement. Oeneraliralioo of the nicotine stim- ulus (400 pg/kg) to the stimulue effects of several daes of d-amphctamioa (60, 120, 210, 480. and 720 pg/kg) was investiRalod is the third study. Re- wlts of /he dose-gencralisdion and Gmeduratios studies showed that the sensitivity of the rats to the nicotine cue wr directly related to the training dose under the VI-IS s scheduk. Allhoujh response ratta differed across the scheduka of reinforcement, the rat: sensitivity to the stimulus eflects of nico- tine was not aflected. l ack of complete Renerafaation of the nko/ine stimulus to d-amphetaminc indicated that the rals did not perceive those two drup as the same. In addition to these e.perimenlal findinp, this paper presenla sev- eral techniques useful for proper evaluation of the stimulus properties of Iea1 compounds. Chance, W. T., Murtin, D., Krynock, O. M., and Rorecranu, l. A. psycAophnmardosr SS:19-26, 1977. From the Department of Pharmacology, Medical College of Virginia, Rich- mood. I CONVERSION BETWEEN CONFIGURATIONAL STATES OP THt3 MUSCARINIC RECEPTOR IN RAT BRAIN AccordinR to these eaperimenh, reductive alkylalion of neural mem- branes by N-ethyl makimide (NEM) converts muscarinic acetylcholine ro- eeplors from a stale of low Io one of high aAinity for receptor allonists. This treatment does not affect the inleractions of mu.carinie antagonists with Ihe receptor. The NEM treatment also increases the proportion of rat tekncephaloa high aRonisl aA'inily recepoa from 34.2 to 53.4% of the total receptor popu- lalion; and it decreases the potential esrb.mykholine inhibition d)-quinu- clidinyl benzilale binding from a K, of 1.2 a 10-• to 6.9 a 10- •. These re- sults seem to support  previously proposed theoretical rnodel, which suggests the e.islence of two populalions of receplon with different aR~nities for agoo- ists. In lhis model, the anlagonisls supposedly bind to either receptor with a uniformly high a0inity, and the binding of a6onista and sulagr.niats is com- pelilive arwf mutually esclusive. , Aronatam, R. S., Hosa, W. and Abood. f.. G. European lournaf o/ PAarmocology 16:279-2l2, 1977. Other support: U. S. Public Nealth Seryice. ~ From the Center (ur Brain Research, University of Rochesler Scfwr,t of Mcdi- eino aod Dentistry. Rochester. N. Y. SI

INFLUGNCE OP SUI-FIIYDRYL REAGENTS AND IIP.AVY METAIS ON 111E ft1NCTIONAI. STATE OP TNE MUSCARINIC ACE(1f1.CHOLINE RECEPTOR IN RAT BRAIN Rat brain membranes contain several mobcular groups, )ocatei both within and contiguous to the receptor bindinR aite, that react with wl!hydryl reaaenls to influence muaearinic aeetykholine receptor binding. Oa! com- pound, pahbromercuribensoate (PCMB), reacla with a group(s) wi hie or under the allosteric control of the seeeptor binding aite to inhibit both atonist and antagonist binding. Receptor lipsda (agoniats or antapnists) will protect receptors from PCMB inactivation, but ths inhibitory process can r.lso be reversed by subsequent Irea/ment with organic sulfhydryla. Through the re- duclive alkylatioa of neurd membranes. Ndhy/makimide (NNM ) a n pre- vent much of the antagonist binding inhibition by subsequent PCMB trei.tment, but not that of the a4onist, suuestiaR that 11 la a mercuribenzoate, a ad not an elhylmaleimide residue within the bindiol sNe that interferes with receptnr binding. lfie NP.M treatment increases ajoeid binding by convertio` low agonist af8nity receptors to a.tate of high a6onial affinity, while their high dlinity for antagonists remains the same. The ability of NEM to Increase agonist tfinity is enhanced by (he presence of agonins, but not anutonisu, during the trealrnent. Low concentrationa of PCMB abolish the NPM ability to suA.equenlly increase agonist biodinL even when concentrations of receptor ligands sufficiently high to saturate the binding aites are included during Ihe P('MB treatment so as to protect the binding site. This suggests that NPM influences agonist binding through interaction with a group contiguous to the receptor binding site. 1)ependin6 on their concentrations heavy metal ions (Zn' •, C'd• •, C'o" and Mn•' ), which appear to interact with all of the NIiM- and PCMB-reaclive moeities, either increase or decrease agonist bind- ing, or decrease both agonist and antagonist binding. Different brain regiom contain membrane receptors that vary in distribution from the high- and low-atonist-a(finNy forms. The high-aflonist-afRnity muscarinic recepton are more numerous in the brain atem than in the kkncephalon, which contains more than the hippocampus. Roupton from different areas of the brain also have differing af/initiea (or sul(hydryl reagents and heavy metals. Aronstam, R. S.. Abood. L. G. and llow W. A(olerular Pharwrerolosy 14:S7S-SR6,1978. Other support: National Institutes of Health. From the Center for Brain Research, University of Rochester School of Medi- cine and Ikntatry, Rochester. N. Y. FNIIAN('1?MFNf (H cIPIAfI? HINI)INCI BY VAR1OtIS MOI P.(-111 AR 1ORMti (NU/•11OSPIIA I lt)YI 1FRINN ANI) INIIIBI I ION BY O1F11'R lINM1UkAtl 1) 111'1115 Nrr.e h..ue n t6..oth1 1.. .•.nr.m an opiate recelNot ealsUug as a prl+. lem •41d 1•h.•yh.dq..J. mrud•~.ur un.plca u( whwh phosphaedyherme. or i other acidic Iipid,'i. possibly an Integral component. Consequently, a wWe variety o( saturated and unsaturated phospholipida and fatty acids were ex- amined for their effect on the slereoepecifk opiate binding of rat brain Irynaptk rnembrane preparations. In fact, addition of acidic pho.photipids aiginific.ntfy enhanced opiate binding. Except for phosphatidyl.erine, "ic piwxpholipiaM containing a polyunsaturated acyl group were actually inhibitory aa were neu- tral pho.pholipids derived from brain. The CIe:0,22:6 form of phosphatidyl- aerine derived (tom synaptic membranes enhanced opiate binding by as mueh a 45%. Unsaturated fatty acids were highly inhibitory with tha degree of Inhibition related to the degree of uns.turation. Pho.photipue A and C wera inhibitory and the effect of A could not be prevented by albumin or overwme by the addition of phosphatidylserine. The use of dinilrodilluorobeorene, a eross-linkial agent, showed that the pho.phatidyLerine of synaptic roembranea could be preferentially associated with membrane protein. lAe enhancing effect of phosphatidylserine diminished with an increase is the degree of croas-linkiog. A6ood,L.G.rtalA elochinrka rt 8loohy.lra Acra 466:31-62, 1977. Other aupportr U. S. Public Health Service. Prom the Center for Brain Research and the Department of BiochemWry. University of Rochester Medical Center. Rochester, N. Y. I PHOSPNOI.IPID CNANOES IN SYNAPTIC MEMBRANES BY LIPOLYTIC ENZYMFS AND SI/BSEQUENT RESTORATION OF OPIATE BINDING WITN PIIOSPNATIDYLSERINE This study of the role of phosphalMyl.erinu in the xterempeciflc opiale binding to neural roembrana provides new evidel.oe in support of the thesis that phoaphalidylserine b an important, it not srrential, component of the opiate receptor. Among the signiflcant findings here .re the observations that: (1) exposure of mernbranes to phosphatidyhterine decarboxylase dimis- iahes opiate binding; (2) addition of phosphatidyberine following tredme.t with either phospholipase A, or Triton X-100 (inhibitors) wiB partially n- qore opiate binding: and (3) addition of phosphatidylserine following pptlal inhibition of opiate binding by treatment with phosphatidylserine decarboxyl..s will restore activity. lhe binding of 'It-naloxone, an opiate antagoniat, Is simi- lar to that of agonists in iU behavior towards phoaphAipaaea and pho.phatidyl- serine; however, binding of naltrexone, also an antagonisl, is far less respawr- sive. It is concluded thal the phosQhalidylserine associaled with the opiate receptor is the C1d:0,22:6 diacyl form, which is closely associated with protein. Abood, L. 0. at a1. eloch/mica er Stophy.ka Actts 3)0aS-46, 1975. Other support: 11 . S Public Flcatth Service. From the ('enter of Brain Research and Departmenl of Bwuhemistry. Unl- versity of Rochester Medicd ('enter, Rochester. N. Y. 52 411

i t HUMAN PLACENTAI. CHOI.INP.RO)C SYSTEM : STIMULATION- SECRETION COUPLINO FOR REI.EASE OP ACETYI.CHOl.INB FROM ISOLATED PLACENTAL VILLUS It has beta suggested that the permeability of the placental villus is coo- trolkd by acetylcholine (M:'h). According to a working hypotheuis. A/:'h iu released from the villu..timulating a eholinergk receptor on the nyncytiotro- phobtast, which kad. Io changes Ia the permeability of the trophob utic mem- braee; these changes are coupled to 1Se transport of nutrients acroas the trophoblut. Here, tbe conditions under whki ACb is rekaaed from the iso- lated placental villus were studied as a Anl step toward obtainiag evidence for this working model of stimulatios-ACA aecretion-tranaport coupling. Tha Molaled altus incubated Ie a muaele bath containing Kreb.-Ringer bkarbo- eale bufler apontaneously released )3 pmol/R/min of ACh Into /'x medium in a manner that was linear with 1ime. Tbere was no release ot .\('h In the absence of Ca" from the medium. With an increase of buAa G' • concen- tration from 2.4 to 4.61 mM, or with the addition of L-okotine (ftl rM) to the balh. Ihe rate of ACh release rose to 53 and 47 pmol/g/min, respectively. In the absence of Ca", however, nicoline did sot have any effe:t on ACb release. Atropine lowered both the rate of apootaneoui ACh rek ue and ib aicotine-induoed one, u did coeaine, a Car • antagonist; but d luhocurarioe (30 rM) did not aRect either rate. Tha presence of depolariziag concentra- tions of potassium In the medium (16-63 mM) also increased the nAe of ACh release. According to these dala, (1) the release of ACh requires tirc presence of ionic calcium in the ealernal medium; (2) Ca' • ions funcliou n a link between the stimulation of ACh and its Anal release; and (3) as ir.dicated by the effect o( nicotine on the placental release of ACh. there is a muscarink type of cholincrgic receptor in the ayncytiotrophoblast. Olubadiwo,1. O. and Ssrrry, d. Y. R. The Journal o/ rAornmroloty ond E, perLnentd Tl4eraperrlcs 204(2) :4l)-413, 1978. Other er.rport: U. S. Public Health Service. Prom the Dep.rtment of Pharmaooim, Vaaderbih University School of Medicine, Nashvilk, Teea. 711E INFI.UENCI'. OF CHOLINP.ROIC e1.OCKADB ON T11E UPfAK!? UF mAMINOI.SOHI('YRIC ACID BY ISULATI'.1) HUMAN PIaCI'NTAI. VILLI One of the major functions of placental trophoblast is nutrient transfer from the maternal blood to the fetal circulation. In view of the location of acetylcholine ( A('h )•chnline acelyltransferase (('hA ) in the placenta and the relea+e of A(-h into the maternal hkxxl, it has been suggested that A('h may stimulate a cholinertrc receplor un the Iropht.bla.1 and nNdula/e the placental tran.purt ut iom nd nutrienls In the e.periments dex'rihed here, n- 1"(-) 54 r I amiaoiaobrdyric aoid (AIS) wr used to aner the amino add uptak.e qnlnm ia human placenta and to measure the tramport efficiency of this active up- take system. Since it was not possible to obtain ACh-free human placer:ul villus, the effects o( agents which block ACb at various typea of cholinergic receptors and the effectc of antidroline.lerara on arnirw acid uptake by human placental villu. were studied ia this attempt to exptaia their antiRrowth effects. These studies inchded eholinergic bkockade by: (a) eaoeas e>togeaoua A(1, (b) massive release of endogeeao ACh by dcotiae, and (c) sparing eado- Renously released ACh by inhibitinR eholioeNer>t.e with pho.pholioe; also studied were the cholioergic blockade of: (d) trw.carieic reeeptor using atropine. (e) oiootink receptor of paRlionk type u.inR mecamylamioe, a.d (f) nicotinic receptor of newonw.cular-iunctbn type uaiag d-tubocyrarise. tinder all oondilioes eacept (a) and (t), Ih. AIB uptak. wr depressed by cholioergic blockade. '(beae ob.ervatio.n imply thN eadogenoualy reka.ed ACh eahibiu a mu.cariaic effect on placental villua and facilitates the tranr- port of amino acids. Blockade of the muacarinic receptor resulu in a deprea- lioo of the AIB uptake, a state which could, after a long period of lime, produce a retardation of fetal Rrowth. Rowep, P. P. aad Sartry, I. Y. R. Toakolop and Applied rA.rnrardop 13:79-9), 1976. O/her wrprtt U. S. Public Health Service. Frorn the Department o1 Ph.rmaeoloRy, Vanderbilt University School of Medi- ~ cioe, Na.hville, Tem. EFFECTS OF NICOTINE AND COCAINE ON THE RELEASE OF ACETYLCHOLINB PROM ISOLATED HUMAN PLACENTAL VIW The placental villu., which r devoid of neurnw.l innervation, w a.rw as an information wruw about the prooa. of aoetykholina (ACJt) rekau trom noA-nervou. Iitrues. With thi. In tnMtd, isolated human placental del were .u.perded i. Kreb'a bicarbonate bu//er ud the ACh relea.ed into 68 medium was analysed by gas chromatography. 71u .pontanear release of ACh into the medium was linear with time and was 35 pewl/R/min. Against this base line, K', Ca's. oocaine, and akotine were investigated for their inAuence on the pattera of ACh reka.e. Coeaiam In dwn of 290 and 560 rM decreased the spontaneous reka.e of ACh to 23 and 17 pmol/R/min, respoo tively. Raising the Ca' • concentration ia /he Kreb'. bicarbonate buffer (rom 2.34 to 4.64 mM or adding niootiee (SR rM) to the balh. Increased the rau of release of ACh to 53 and 47 pmol/R/min, respectively. Cocaine decreased the rate of release of ACh even in the presence of Ca' • or nicotine. Ilikh concentrations of K• increared A('h release in the presence of Ca• • bul It had no effect on A('h release in the absence of Ca ••; In fact,. A(lr was never released when Ca" was absent in the medium. Nicoline, which ie- creased both initial output and the rate of release of placental A('h, did not stimulate release of placental ACh In the absence of eaternal Ca•'. Cocaine 33

{ i depressed both of the observed nkotine-induced increases. These observa- lions indieale that (1) nkotine-ioduced increase in placental ACh is mediated through mu.carinic receptors. (2) nicotine iocreases The release of ACh by possibly facilitating the cell entry of external Ca•', and (3) cocaine de- pressa nkoline-induced incre..e in ACh rekue, possibly by depressing Ca •• entry into the oeli- Sanry, 0. V. lt., Olubadewo, l. O. and Boefirq, P. H. Arch/res /nternatfonales dt pAarmarotjn.A7lk tf dt TAlraple 229(1) :23-36, 1977. Oth...wpp.rt: U. S. Public Health larvb., From The Departmenb of Pharmaooiogy, and Obstetrics and Gynecology. VanderbiU U.ivenity Scbool of Medicir, NnhrilM, Tenn. HUMAN PI.ACENTAL CHOI.INB ACETYLTRANSFERASE. NATURE AND MOLECULAR ASPP-CTS OP THE INHIBITION BY IODO- AND BROMOACETYICHOLINFS /lalogenaled acetykholioes and other cholins oqen owe their high serum dtiolinnterase and nicotinic receptors aRinities so the eleclron dirplacement uused by the (nfluence of an Nubflitucnt. Organic reactioo studics have demonstrated that halogenalion produces sleric hindrance, and the s.me ef- fecl has been shown to have a significant role (n the hydrolysis of halogenated acy) groups by acelykholioesleraae and in their interaction at muacarinic re- eep/on. Their structural similarity to acetyp"C)choline (A('h), a product of the choline aoetyllransfer..e (ChA) reaction, mates the halogeru.acelyl- cholioes' ability to inhibit ChA and the nature of their inhibitions par/kularly important. The present inveqiption describes studies of halodenated acetyl- cbolina as inhibitors of human placental ChA. The resulting data support The formation of 1en.ry, inlermediates (CoA-ChA•IACh or CoA•ChA BrACh), during the reaction of placental ChA. The quaternary ammonium head and the positive carbo.yk.rbon do appear to contribute to Ihe inhibition of ChA by IACh and BrACh. An irreversible component of inhibitios (20-30%) ob- tained with hatogea-subatituled aoolykholines or lodo- and bromoacetnles can be explained by the possible: (1) formation of R<arbo><ymethylalyd t:'hA (where R- -0-, -S- or -N-), which is less active than The original ChA: (2) formation of Scarbotymethylated CoA, which is slowly released from the enzyme surface; or ()) eowvenion of part of the enzyme 10 the Inactive - N -earboaymethylated form. Ilcndenon. O. 1. and Sarrry, d. V. R. QloeAemkd tA.nnarolop 27:1331-1310, 197t<. OtAwr supportr U S Public Health Service. Nrt.m rhe lkparlmrnl of Nham.c.,luRy. Vanderbilt linivenity School of Medi crne. N..hville, lenn tt, r RELAT1ONSNIPS BETWEEN CHEMICAL STRUCTURE AND INHIBITION OF HUMAN PLACENTAL CHLORINE ACETYL- TRANSFERASE BY KETO ANALOOSOF ACETYLCHOLINE The /n rlro syolhesis of aeetykhdine ia achieved through a coupled aysl.m Involving an acetate-activatin6 entyme, aoelyl ooentryme A synlhetase, as,d an ensyme, cholioe aeetyltranaferase, that couples the activated acelata to choline. An inhibitor of the Anal step iu this synthetic pathway would, lherm- fore, be a valuable qent for studying cholinergic mech.nirna. le this aeries of esperiments, aeven teto analop of aoctykholine wero synthesized aed evaluated as lnA/blto.s o/ hrtrnae p/rantd cllo4nr aceryirrnwr/erara. Also tested was their capacity to inhibit horse serum dfolinesterasn aed to s14me late cholinergie receptors in the longitudinal muscle of the psinca pig. Of these aubNances, (2-bensoykthyl) Munnwoium chloride (compound VII). with an Ir, of 3 x 10-• M, was The most potent and sebclive bhibitor of choline acetyl(ransfer..e. Ito muacarink aad nieolinie sUmulatory activity wr quite low; so also was its cholinealerase-inhibilory activity. A study of Ih. Mruclure-activity, relatronahiys demonstrated three requirements for the i.hibi- tion of choline aeelyltramferaae by the altylaminoethyl eaten or their oocre- apooding quaternary aremordum derivatives: (1) a lermind eatioak head on the amine eod of the molecuk; (2) the ability to delocalize (or stabilize) a partial negative charge on The acyl end, and (3) a leaving group on The a-car- boa a1 the acyl end. Compound VII fulfills t.early all theae conditions. It has (1) a cationic terminal, a site for an electron acceptor interactlo.; (2) a. aryl moiety for hydrophobic and electron donor contributions; aod (3) a positive carbon atom adjacent to the bente.e rin6, due to the presence of The carboayl group, which interacts witL Me .udoophilic residue on the .nsyme. Cbalut.edi, A. K., Rowell, P. P. and Sarrry, B. V. R. lowrnd o/ pArnracertkaf Sclences 67(3):637-660, 1978. Other aupport: U. S. Publk Health Service. From the Departmenl of Pharmaooiogy, VanderbiN University Scbobl of Maal- dne, Nashville, Ten.. + (2-BENZOYLETIIYL)TRIMETHYLAMMONIUM CHIARIDE: A NEW, SELECTIVE AND STABI.E 1NIIIBITOR OP HUMAN PLACENTAL CHOLINE ACE7YLTRANSFERASE Since the discovery of .fyrYlpyridinea as the Ant poteM iahiblton of choline acelyltransfcrase (ChA), much effort has been devoted to the dovelop• ment of new and better ChA inhibiton of various types. Thr rrcport describa the propenks of a newly synthesized eompound, (2-benwytethyl)Irimethy4 ammonium chloride (BETA), a keto analog of acerykholine, which h s1abM aod meets The known structural requirements for ChA Inhibition. It was foued to be a selecrive, polenl, and noocompetitive InhibNor of (rA with respect to both of its sutntrates, acctylfoA and choline, with an 1„ of 3.1 x 10 a. Inhibition was rapid and slowly reversible, with 50% of the ('hA activity 57 -