Studies on the interrelationship between the syntheses of noradrenaline and metaraminol

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Br. J. Pharmac. (1970), 39, 167-174. Studies n the interrelatinship between the syntheses f nradrenaline and metaraminl G.. JOHNSON AN

1 Br. J. Pharmac. (1970), 39, Studies n the interrelatinship between the syntheses f nradrenaline and metaraminl G.. JOHNSON AND T. A. PUGSLY Department f Pharmaclgy, University f Trnt, Trnt 5, Canada Summary 1. xperiments were cnducted t determine the influence f the rate f nradrenaline synthesis n the cnversin f alpha-methyl-m-tyrsine t metaraminl. 2. Male Wistar rats, g, were placed int fur grups and treated with (1) alpha-methyl-p-tyrsine methyl ester, 250 mg/kg; (2) DL-alpha-methylm-tyrsine, 400 mg/kg; (3) alpha-methyl-p-tyrsine methyl ester, 250 mg/kg plus DL-alpha-methyl-m-tyrsine, 400 mg/kg; r (4) an equivalent vlume f injectin vehicle. All slutins were injected intraperitneally. 3. Immediately after treatment half f the rats were transferred t 40 C with the remaining animals being kept at 270 C. 4. The rats were killed 4, 8 and 12 h after injectin, the brains, hearts, spleens and adrenals remved and analysed fr adrenaline, nradrenaline, metaraminl and alpha-methyl-m-tyramine. 5. In virtually all cases, bth during rest (270 C) and sympathetic stress (40 C), treatment f the rats with alpha-methyl-p-tyrsine methyl ester increased the amunt f metaraminl frmed frm alpha-methyl-m-tyrsine. The nly rgan nt cntaining increased quantities f metaraminl in the presence f alpha-methyl-p-tyrsine methyl ester was the adrenals, taken frm the rats kept at 270 C. Adrenals remved frm the cld-expsed rats cntained mre metaraminl when alpha-methyl-p-tyrsine methyl ester was cmbined with alpha-methyl-m-tyrsine than when alpha-methyl-m-tyrsine was used alne. 6. These results demnstrate that the inhibitin f nradrenaline synthesis, by treatment with the tyrsine hydrxylase inhibitr alpha-methyl-p-tyrsine methyl ester, increased the cnversin f alpha-methyl-m-tyrsine t metaraminl. It is cncluded that inhibiting the frmatin f dpa allwed increased amunts f alpha-methyl-m-tyrsine t enter the bisynthetic pathway. These results supprt the false sympathetic transmitter cncept advanced fr metaraminl. Intrductin Metaraminl accumulates in sympathetic nerves replacing, t a large extent, the endgenus nradrenaline (Shre, Busfield & Alpers, 1964; Anden, 1964). Metaraminl may be frmed frm administered alpha-methyl-m-tyrsine within sympathetic nerves (Carlssn & Lindqvist, 1962) and its synthesis is increased by

2 168 G.. Jhnsn and T. A. Pugsley sympathetic stimulatin (Jhnsn & Pugsley, 1968). Once bund within the nerves, metaraminl may be released by catechlamine releasing drugs r by in vitr (Crut, Alpers, Tatum & Shre, 1964) r in viv sympathetic stimulatin (Jhnsn & Mickle, 1966; Csta, Neff & Ngai, 1969). On the basis f this evidence it has been pstulated that metaraminl may functin as a false sympathetic transmitter. During the past few years experiments have been cnducted in ur labratry t study varius aspects f the false transmitter thery. Our results have been published in a series f articles (Jhnsn & Mickle, 1966; Jhnsn & Pugsley, 1968). The present paper is a cntinuatin f this prgramme. Bth nradrenaline and metaraminl share the same bisynthetic pathway. Because f this, a study was undertaken t determine the influence f the rate f nradrenaline synthesis n the cnversin f alpha-methyl-m-tyrsine t metaraminl. Cld expsure increases the rate f synthesis f bth metaraminl (Jhnsn & Pugsley, 1968) and nradrenaline (Oliveri & Stjarne, 1965). Use was made f this fact t study the interactin between nradrenaline and metaraminl synthesis under cnditins f sympathetic rest, 27 C, and stress, 40 C. Methds Male Wistar rats, g, btained frm the High Oaks Breeding Labratries, were used thrughut the study. The rats were divided int fur grups and kept at C fr at least 24 h befre the experiment. The grups were injected intraperitneally as fllws: grup 1, alpha-methyl-p-tyrsine methyl ester, 250 mg/kg; grup 2, DL-alpha-methyl-m-tyrsine, 400 mg/kg; grup 3, alpha-methylp-tyrsine methyl ester, 250 mg/kg plus DL-alpha-methyl-m-tyrsine, 400 mg/kg; grup 4, an equivalent vlume f injectin vehicle. Immediately after treatment half f the rats were transferred t 40 C and the remaining animals were kept at 270 C. The animals were placed in individual stainless steel cages. Fur, eight and twelve hurs after injectin the rats were killed and the brains, hearts, spleens and adrenals quickly remved. These tissues were hmgenized in 0-4 N perchlric acid and analysed fr adrenaline, nradrenaline (uler & Lishajk, 1961), metaraminl and alpha-methyl-m-tyramine (Pugsley & Jhnsn, 1968). Results The results f the tissue analyses are given in Tables 1, 2, 3 and 4 and Fig. 1. Alpha-methyl-p-tyrsine methyl ester prduced a marked fall in the nradrenaline levels in the hearts and brains with the initial effect being greater in the cld-expsed rats (P<0-01). The drug caused a smaller fall in the nradrenaline stres in the spleen. The catechlamine levels in the adrenals fell nly slightly at 270 C. At 40 C the effect f alpha-methyl-p-tyrsine methyl ester n the adrenals was greater. Alpha-methyl-m-tyrsine lwered the tissue levels f nradrenaline in the hearts, spleens and brains (P<0-01 at 4 h). The catechlamine depleting actin f the drug was greater than that f the ester f the para ismer. The catechlamine stres in the adrenals fell slightly. Accmpanying the fall in nradrenaline, the administratin f alpha-methyl-m-tyrsine prduced high levels f metaraminl and alpha-methyl-m-tyramine in the hearts, spleens and brains. The initial

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5 Alpha-methyl-p-tyrsine and metaraminl frmatin 171 levels f metaraminl in the brains and hearts were higher in the rats kept at 40 C (P<005). In the spleen the initial levels were higher in the rats kept at 270 C (P<0-01). Twelve hurs after treatment spleens remved frm the cldexpsed animals cntained cnsiderably mre metaraminl than did spleens taken frm the warm-rm rats. Metaraminl and alpha-methyl-m-tyramine were als fund in the adrenals f bth grups f rats. Treatment f the rats with alpha-methyl-p-tyrsine methyl ester increased the amunt f metaraminl frmed frm alpha-methyl-m-tyrsine. On ccasin the increased frmatin f metaraminl by rats given bth drugs was nt immediately seen; fr example brain 4 h sample at 40 C and heart 4 h sample at 270 C. Hwever, when these tissues were remved either 8 r 12 h after injectin, the ptentiating actin f alpha-methyl-p-tyrsine methyl ester was seen. The nly rgan nt cntaining increased quantities f metaraminl in the presence f alphamethyl-p-tyrsine methyl ester was the adrenals. Adrenals remved frm the rats kept at 27 C cntained less metaraminl when alpha-methyl-m-tyrsine was cmbined with alpha-methyl-p-tyrsine methyl ester than when alpha-methyl-m-tyrsine was given alne. Treatment f the cld-expsed rats with bth drugs increased the amunt f metaraminl fund in the adrenals. 04 C - 3 _: F A Il I *0 c C 1! 410 C -a._ 1I 0.u Hurs after iniectin FIG. 1. Cncentratins f metaraminl in brains (A), hearts (B), adrenals (C) and spleens (D) f rats after the administratin f alpha-methyl-m-tyrsine (-), and alpha-methyl-m-tyrsine plus alpha-methyl-p-tyrsine (- -- -), at 27' C (DI]) r 4' C (U). Pltted values are means f fur determinatins.

6 172 G.. Jhnsn and T. A. Pugsley Discussin The cncept f metaraminl as a false sympathetic transmitter is based n the evidence that it replaces nradrenaline within sympathetic nerves and is subject t release by sympathetic stimulatin (Crut et al., 1964; Jhnsn & Mickle, 1966). Metaraminl may be synthesized within sympathetic nerves frm previusly administered alpha-methyl-nm-tyrsine (Carlssn & Lindqvist, 1962). vidence f this was seen in ur study. Fllwing the injectin f alpha-methyl-mn-tyrsine, all tissues analysed cntained high cncentratins f bth alpha-methyl-m-tyramine and metaraminl. Nradrenaline stres fell as the catechlamine was replaced by metaraminl. Cnsiderable wrk has been published n varius aspects f the interactins between nradrenaline and metaraminl (see Crut (1966) and Kpin (1968a, b) fr references). Hwever, in spite f the fact that the synthesis f metaraminl in sympathetic nerves utilizes tw enzymes nrmally emplyed in nradrenaline frmatin-namely, L-armatic amin-acid decarbxylase and dpamine-/3-xidase n wrk has appeared describing the influence f the rate f nradrenaline synthesis n the cnversin f alpha-methyl-in-tyrsine t metaraminl. The chemical alpha-methyl-p-tyrsine methyl ester is an effective inhibitr f tyrsine hydrxylase and has been used by numerus wrkers t inhibit nradrenaline synthesis. Alpha-methyl-p-tyrsine methyl ester was used as a tl t blck nradrenaline synthesis at a stage, the cnversin f tyrsine t dpa, that wuld nt impair the frmatin f metaraminl frm alpha-methyl-mn-tyrsine. The dse used in the present experiments, 250 mg/kg, was selected n the basis f studies by Crrdi & Hanssn (1966) and Crrdi & Malmfrs (1966), wh shwed an inhibitin f tyrsine hydrxylase prduced a fall in nradrenaline and dpamine tissue cncentratins. vidence f the effects f the alpha-methyl-ptyrsine methyl ester in ur study can be seen by the fall in nradrenaline tissue levels after its injectin. The effect was mre prnunced at 40 C, due t the increased turnver f nradrenaline in the cld. The inhibitin f nradrenaline synthesis increased the frmatin f metaraminl frm alpha-methyl-m-tyrsine. Brains, heart and spleens taken frm rats given bth alpha-methyl-m-tyrsine and alpha-methyl-p-tyrsine methyl ester cntained, in almst all cases, mre metaraminl than the crrespnding rgans remved frm the animals given nly alpha-methyl-m-tyrsine. Spectr, Sjerdsma & Udenfriend (1965) previusly reprted that alpha-methyl-p-tyrsine increased the incrpratin f dpa- H int tissue catechlamines. These wrkers cncluded that a decrease in the frmatin f endgenus dpa allwed the entry f the labelled analgue int the pathway with less dilutin. Our results suggest that inhibiting the frmatin f dpa allwed increased amunts f alpha-methyl-mtyrsine t enter the bisynthetic pathway. This infrmatin demnstrates a cmpetitin between the synthesis f nradrenaline and metaraminl and reinfrces the false transmitter cncept. Cld expsure increases nradrenaline turnver (Oliveri & Stjarne, 1965 ; Csta, Neff & Ngai, 1969) and can, fllwing the administratin f alpha-methyl-imtyrsine, increase the frmatin f metaraminl (Jhnsn & Pugsley, 1968). Fur hurs after treatment with alpha-methyl-mn-tyrsine higher levels f metaraminl were fund in the hearts and brains f the cld-expsed rats. The increased rate

7 Alpha-methyl-p-tyrsine and metaraminl frmatin 173 f metaraminl frmatin, seen at 40 C, was further elevated by alpha-methyl-ptyrsine treatment. It can be cncluded that, under cnditins f bth sympathetic rest r stress, the rate f metaraminl frmatin can be influenced by the amunt f tyrsine being cnverted t dpa. Inhibitin f tyrsine hydrxylase did nt increase the frmatin f metaraminl in the adrenals f the rats kept at 270 C. In fact, in this gland treatment with alpha-methyl-m-tyrsine alne prduced higher levels f metaraminl than alphamethyl-m-tyrsine and alpha-methyl-p-tyrsine cmbined. In the warm the turnver f catechlamines in the adrenals is depressed and this prbably accunts fr the failure f tyrsine hydrxylase inhibitin t increase metaraminl frmatin. In the cld, under cnditins f sympathetic stress and increased catechlamine turnver, alpha-methyl-p-tyrsine increased the frmatin f metaraminl in the adrenals. Althugh significant amunts f metaraminl were fund in the adrenals, they represented nly a small fractin f the nrmal catechlamine levels. The drug prduced nly a small fall in the nrmal stres f adrenaline and nradrenaline. One additinal pint deserves cmment. Cld expsure increased the frmatin f metaraminl in hearts and brains, with the increase being nted 4 h after injectin. Cld expsure did nt increase the frmatin f metaraminl by the spleen during the first 8 h after treatment. It has been a cnsistent bservatin in ur labratry that the release f nradrenaline in the spleen is relatively refractive t cld expsure. Because f this it is nt surprising t bserve that cld expsure prduced n immediate increase in metaraminl synthesis. Twelve hurs after treatment the spleens remved frm the cld-stressed rats cntained many times the levels f metaraminl fund in the animals kept at 27 C. This wrk was supprted by grant 1595 frm the Medical Research Cuncil f Canada. T. A. Pugsley is a recipient f a Medical Research Cuncil f Canada Studentship. Alphamethyl-p-tyrsine methyl ester (H 44/68) was kindly supplied by Dr. H. Crrdi, Hassle AB, Gthenburg, Sweden. RFRNCS ANDN, N.. (1964). On the mechanism f nradrenaline depletin by alpha-methyl-m-tyrsine and metaraminl. Acta pharmac. tx., 31, CARLSSON, A. & LTNDQVIST, M. (1962). In viv decarbxylatin f alpha-methyl-dpa and alphamethyl-meta-tyrsine. Acta physil. scand., 54, COSTA,., NFF, N. H. & NGAI, S. H. (1969). Regulatin f metaraminl efflux frm rat heart and salivary gland. Br. J. Pharmac. Chemther., 28, CORRODI, H. & HANSSON, K. (1966). Central effects f an inhibitr f tyrsine hydrxylatin. Psychpharmaclgy, 10, CORRODI, H. & MALMFORS, T. (1966). The effect f nerve activity n the depletin f the adrenergic transmitter by inhibitrs f nradrenaline synthesis. Acta physil. scand., 67, CROUT, J. R., ALPRS, H. S., TATuM,. L. & SHOR, P. A. (1964). Release f metaraminl (aramine) frm the heart by sympathetic nerve stimulatin. Science, N. Y., 145, CROUT, J. R. (1966). Substitute adrenergic transmitters. Circulatin Res., 18, Suppl. 1, ULR, U. S. V. & LISHAJKO, F. (1961). Imprved technique fr the flurmetric estimatin f catechlamines. Acta physil. scand., 51, JOHNSON, G.. & MICKL, D. (1966). The influence f cld expsure n the in viv release f metaraminl. Br. J. Pharmac. Chemther., 28, JOHNSON, G.. & PUGSLY, T. A. (1968). The frmatin and release f metaraminl during expsure t warm and cld envirnments. Br. J. Pharmac., 34, KOPIN, I. J. (1968a). False adrenergic transmitters. Ann. Rev. Pharmac., 8, KOPIN, I. J. (1968b). The influence f false adrenergic transmitters n adrenergic transmissin. Adrenergic Neurtransmissin, ed. Wlstenhlme, G.. W. & O'Cnnr, M., pp Lndn: J. A. Churchill.

174 G.. Jhnsn and T. A. Pugsley OLIVRIO, A. & STJXRN, L. (1965). Acceleratin f nradrenaline turnver in muse heart by cld expsure. Life Sci., Oxfrd, 4, 2330-2343. PUGSLY, T. A. & JOHNSON, G.. (1968).

8 174 G.. Jhnsn and T. A. Pugsley OLIVRIO, A. & STJXRN, L. (1965). Acceleratin f nradrenaline turnver in muse heart by cld expsure. Life Sci., Oxfrd, 4, PUGSLY, T. A. & JOHNSON, G.. (1968). Mdified methd fr the estimatin f metaraminl and alpha-methyl-m-tyramine. J. Pharm. Pharmac., 20, SHOR, P. A., BUSFILD, D. & ALPRS, H. S. (1964). Binding and release f metaraminl: mechanism f nrepinephrine depletin by alpha-methyl-m-tyrsine and related agents. J. Pharmac. exp. Ther., 146, SPCTOR, S., SJORDSMA, A. & UDNFRIND, S. (1965). Blckade f endgenus nrepinephrine synthesis by alpha-methyltyrsine and inhibitr f tyrsine hydrxylase. J. Pharmac. exp. Ther., 147, (Received January 9, 1970)

A Plasma Humoral Factor of Extrarenal Origin Causing Release of Reninlike Activity in Hypotensive Dogs

A Plasma Humoral Factor of Extrarenal Origin Causing Release of Reninlike Activity in Hypotensive Dogs A Plasma Humral Factr f Extrarenal Origin Causing Release f Reninlike Activity in Hyptensive Dgs By E. De Vit, C. Wilsn, R. E. Shipley, R. P. Miller, and B. L. Mrtx ABSTRACT Plasma reninlike activity significantly