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NOTES
activity. It is probable, therefore that the effect of hypotonicity is due to directIy induced changes in ion gradients rather than to ATPase-induced changes. ,4lthough a reduction of 10%) in the [K+] of the bathing solution did not affect the response to oxytocin, a reduction in tonicity of the bathing solution and consequent entry of water into cells with their much higher [K'] could temporarily have a more marked effect on the K+ gradient and therefore on membrane polarity. Results of studies of the involvement of cyclic nucleotides in the response of neurohypophysia1 hornlone target tissues have been equivocal. Oxytocin has been found to be without efiect on adenyIate cyclase levels in mammary gland (Bousa 1977). From the presently described experiments, it is d e a r that the hypotonicity-induced enhancement of the response to oxytocin is not due to increased levels of CAMP or cGMP. It may be of some significance that co-administration of oxytocin with the cyclic nucleotides consistently resulted in diminished responses. It was considered possible that the effect of hypotonicity could be caused by increased levels of prostaglandins in the mammary gland, perhaps as a result of release of arachidonic acid from the membranes of the swollen cells. The administration of indomethacin together with oxytocin, or for up to 45-min peric~dsbefore oxytocin, was without effect on the hypotonicity response, or on the standard response. suggesting that prostaglandin synthesis is not a necessary component of either. Bentley and McEwen ( 1961 ) reported that cell swelling did not correlate with uterine sensitivity to oxytoci~~.
317
In a11 conditions studied, the responses sf the strip of mammary gland to acetylcholine and to oxytocin were altered similarly. This further supports the idea that the influence of the changed conditions is at a stage beyond hormone-receptor interaction. The use of hypotonic Tprode soIution for routine biological assay of oxytocin enhances the sensitivity of the isolated strip of lactating mouse mammary gland, as claimed (Roca rt cil. 1972). It does not increase the assay's seIectivity. Acknowledgment This work was supported by the Medical Research Council of Canada. BENTLEY, P. J . , and MCEWFN,E. 1961. The effects of ion changes in the rat uterus stimulated by oxytocin. Br. J. Pharmacol. 16,45-49. DANIEL, E. E. 1964. The iilterconnection between active tramport and contracture in uterine tissues. Can. J. Phy4iol. Pharmacol. 42, 453-495. Dousa, T. P. 1977. Cyclic nucleotides in the cellular action of neurcohypophywal hormones. Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, 1867-1871. FIFI~ITI, C., ROCA,R., MATTEI,A., MFLANDER, S.. GAROFALO, E., GIOIADE COCH.M. N.. and C o c t ~J., A. 1970. A sensitive and specific bioa\say for oxytocin. l'roc. Soc. Exp. Biol. Med. 133, I 155-1 157. MARKLL, H.V.,~ V A KJK. ,, and BRANDA. L. A. 1978. Oxytocin binding site4 in lactating rabbit rnamrriary gland. Can. J. Biochern. 56, 968-976. ROCA, R. A.. G ~ R O F A L E, O , G., GIOIADE COCH, M. N., AROCINA, hl., and C o c ~ r .J. A. 1972. Tmprovemcnt in the bioassay of oxytocin by the mou\e mammary glai-md iiz vitro. Proc. Soc. Exp. Biol. Med. 139, 1010-1012. SOLOFF,M. S., SCHROEDEII, B. T., CHAKRABOIITY, J., and P E A R L ~ I U ' I ~A.L RF. , 1977. Characterization of oxytocin reccptors in the uterus and nlamtnary gland. Fetf. Proc. Fed. Am. Soc. Exp. Biol. 36. 1861-1866.
Study on the influence of tyrosine deprotonation on the myotropic action of angiotensin 111 E. ESCIIER~ AND G. GUII,I,EMETTE~ Department ofPhysiology and I'lmarnzacology, Unir*ersityofSherbrookc Medical ,School, Slmerbrooke, P.Q.,Carlada J l H 5N4 Received June 2, 1978 ESCHER,E., and GUILLEMETTE, C;. 1979. Study on the influence of tyrosine deprotonation on the myotropic action of angiotensin 11. Can. J. Physiol. Pharmacol. 57, 317-320. The influence of phenolic tyrosine ionization in angiotensin 11 on the myotropis action of this peptide has been investigated in vitro on rabbit aortic strips. [Sar1, Tyr'langiotensi~~ ABBREVIA? IONS:AT, angiotensin 11; Amp. L-4'-aminophenylalaninc. 'This work was supported by the grant DG154 from the Medical Kesearch Council of Canada and an installation grant from the Conseil de la Recherche en Sante du Quebec, No. 770150. "uthor to whom correspondence should be addressed. Tart of M.Sc. thesis of G.G.
0008-4212/79/030317-04$01.00/0 @) 1979 National Research Couilcil of Canada/Conseil national de recherches du Canada
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CAN. J . PHYSIOE. PHARMACOL. VOE. 57, 1979
I1 and [Sar1, (4'-amino) Phe4]angi~tensinI1 (as a reference which cannot undergo the same ionization) were tested over a pH range from 6.8 to 9.0 and their activities compared. The results clezlrly indicate that angiotensin 11 with a deprotonated phenolic hydroxyl group on Tyr in position 4 is not the most active o r only active form ~f angiotensin 11.
ESCHER,E., et GL;ILI.IF,~IETTE, G. 1979. Study on the influence of tyrosine deprotonation on the myotropic action of angiotensin 11. Can. 5 . Physiol. Pharrnacol. 57, 3 17-320. L'influence d'ionisation phknolique de tyrosine en angiotensine I1 fut investiganke pour B'actiom myotropique in \*it/.ode ce peptide SLIP des bandelettcs d'norte de lapila. T.es analogues [Sar', Tyr4]angiotensine11et [Sar1, (4'-amino)Phe4]angistensine I1 (cornme rkfkrence qui ne pcut pas subir la m6me ionisation) furent testees ii p H variant entre 6.8 ct 9.0 et leurs actia~itescomparies. kes rbsultats indiq~aentclairernent que l'ungiotensine 11 dbprotonee sur- I'hydroxyl phholique de Tyr en position 4 n'est pas la seule forme active ou plus active de l'angiotensine 11.
Introduction The high activity of the octapeptide hormone A T (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) is strongly reduced orn any modification in the Tyri residue. We recently investigated the myotropic action of [Sari]AT analogues, modified in position 4 with several aromatic amino acids, on rabbit aortic strips. Our results suggested a dependence of the agonistic properties on the electronegativity of the substituted aromatic nucleus of position 4 (Escher, Nguyen, and Regoli 1978 ) . High electronegativity gave low affinity for the receptors on aortic smooth muscle and vice versa, only the electronegativity of Tyr did not fit the relation well. The deprotonated form of Tyr is leqs electronegative than Tyr itself and would fit the relation much bctter. Furthermore earlier experiments with A T at lzigher pH gave higher activities (Paladini pt al. 1963; Methot et (11. 1964); therefore, we suspected that the ionized form of Tyr might be the active species. However, Nakaie et al. (1977) recently reported experiments with monochlorinated [Tyri]AT and concluded that deprotonation of the phenolic hydroxyl of Tyr is even unfavorable. Unfortunately the introduction of a chlorine raises the electro~~egativity of the aromatic nezclells of Tyr4 and explains the reduced poteracy of this analogue. The experiments of Nakaie et al. were performed ~virh[Aspl]AT analogues on rat uterus and guinea pig ileum; our earlier expcriments were performed with [Sarl]AT analogues on rabbit aorta. Although the bioassays and the peptides were not the same, the values can be co~nparedwith some reserve. We therefore undertook experiments at a range of different pH values with the two peptides [Sar1, Tyrt]AT and the Amp-containing analogue [Sar1, Amp4]A% (Escher, Nguyen. Robert et al. 1978) (Fig. 1 ) . The phenolic function of Tyr has a dissociation constan; pK,, of 10.0 and the aromatic amino func-
FIG.I . Structure of the t w investigated ~ AT analogues.
tion of Amp a pKB of >> 14. This means that Amp does not deprotonatc at all in aqueous solutio~~s Since both compounds are identical with the exception of the -OH or the -NH2 group, any difference in conformation or biological activity would result from this molecular difference. Position 4 is not directly linked to the myotropic response release. This is mainly the function of position 8 (Regoli ct cal. 1 974). Because of this, i.e., the specificity of their action and great similarity of these two peptides, it is reasonable to assume that both have the same mode of action. If, as suspected, the deprotonated [Sar1, TyrYAT were the most or only active form, the activity would increase with higher deprotonation. Extreme pH values affect the tissues quite severely; therefore any change in AT activity could be seen only if a nonionizable AT analogue like [Sar1, Amp4]A% acts as internal reference. At a given pH the half-maximal doses (EDr,,,)of each peptide give a dose differential ( = E19,,(Tyr4)/ ED5,,(Amp4)). If any increase were to occur due to deprstonation, this dose differential should develop a pH dependence similar to that of Tyr deprotonation (Fig. 2).
Materials and Methods Rabbit aortic strips were suspended in a 5-mL organ bath
NOTES
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in a modified Krebs solution (0.50 m M Ca2+instead of 2.48 m M ) according to Needleman et ( I / . (1972). The pH of the Krebs solution was adjusted immediately prior to addition to the tissue? with 1 AT NaOH or 1 N HC1. The tissues were equilibrated with the lowest pH (6.8), pure oxygen was bubbled through the bath, and no carbon dioxide was added. Dose-response curves were performed after 90 min of equilibration. Then the tissues were mlashed and equilibrated for 90 rnin with the next higher pH solution. Every I S min the solution was replaced by fresh Krehs buffer of the same pH. The peptides tested were described by Escher, Nguyen, Robert ct a/. (1978). General techniques such as tissue preparation and recording of responses were described by St-1,ouis eb QI. (1977). The results were easily reproduced if pH 9.0 was not exceeded. Inversion of the pH seqrlcnce did not significantly influence the results. At pH 9.5 and higher. the tissues were very weakly responsive and often contracted continiiously without addition of agonists. The intrinsic activity ( Ariens 1964) a,, was determined for each pH, with ctE at pH 7.4 = 1 as internal standard. The half-mm~aximaldoses (ED;,!) are the calculated concentrations of an agonist which produced half-maximal contraction at a given pH.
319
FIG.2- Relationship betwecn pH and the ratio between the half-maximal doses of [Sar1, Tyr4]AT and [Sar1, Amp4]AT (ED,,,(Tyr4)/ED,,(AImpq)).Each point) . 4 is the mean k SEhI of at least seven experiments and the straight line is a calculated linear regression. A indicates the phenolic deprotonation of Tvr in percentage as a function of pH according to Nakaie et ul. (1977).
The dose differential does not show any dependence on the pH within the given limits; within the error, Results and nissussion the dose differential is always equal. This supports the initial hypothesis that [Sarl]AT and [Sar1, At pH 6.8, 7.4, $.8, 8.5, and 9.0 dose-response curves of [Sarl]AT and [SarL,Arnp4]AT were meas- Arnp4]AT have the same mode of action on the vascular smooth muscle receptor of AT. The higher ured on rabbit aortic strips and their ED,,, and values calculated. It is seen that with higher pH, the activity of [Sar1, Tyri]AT has therefore to be exintrinsic activity and the affinity decrease. The results plained in a different way because the electronegativity of the Anlp side chain is lowel- than for are summarized in Table 1. tyrosine. The ratio of ED,,,(Tyr" over ED,o(Amp4) The observed affjnity of [Sarl]AT was higher, as values was recorded as a furaction of pH (Fig. 2 ) . If any activity increase had occurred due to tyrosine expected from the electronegativity relation. This deprotonation or to a different mode of action, the difference has to come from some additional binding ratio would increase with rising pH. Conversely, an energy of the hormone-receptor interaction. Again, activity decrease would have resulted in a fall in the reason must be in the nlolecular structure, speratio with rising pH. Neither change was observed cifically in some bonding that a phenolic hydroxyl and we therefore conclude that the deprotonated can undergo and an aromatic amine cannot. One form of Tyr4 in A T is not the most or even only possibility could be a weak hydrogen bond, with active principle of A T on smooth muscle, as sug- the hydroxyl acting as hydrogen acceptor through gested by earlier investigations (Methot et nl. 1964). its free sp2 orbital on the oxygen. The amine has already a hydrogen in this position which would result rather in steric hindrance. This hydrogen bond TABI.L 1. Effect of pH on the relative biological activities of could be intramolecularly within the peptide itself [Sar1, Tyr I]AT and [Sar1, Amp 4]AT or intermolecularly with the receptor. These possi[Sar1, Tyr "AT [Sar l . Amp 4]AT bilities are actually under investigation with spectroscopic methods. pH
6.8 7.4 8.0 8.5 9.0
En:,o, ng,'rnl,
1.5f0.9 3.111.7 5.2k2.7 7.5-F-2.6 10.5k2.9
CVE
1.02f0.36
!-:.
0.8310.20 0.67fQ.12 0.31k0.14
EDSO, ng,'mL
aF:
6.7f2.4 1.04+0.14 1 3 . 8 k 3 . 1 1.00 23.3k8.9 0.85f0.13 30.7k14.9 0.68k0.18 4 9 . 7 5 15.4 0 . 1 8 k 0 . 1 2
NOTE: E D x is the dose of agonist \vhicIl gives the half-maxirnal contmction at this pH. All values are the mean o f at least seven estimations. aE is the intrinsic activity (Ariens 1964) anti is related to the libility of a drug to produce the maximal contraction at a given pH. At pH 9.4, aE is 1.00 for both compounds.
Acknowledgments Thanks are due to Dr. Domenico Regoli and Dr. Francis Rioux for helpful advice and discussions. AKIENS,E. J. 1964. Molecular Pharmacology. Vols. I and 11. Academic Press Tnc., London. ESCFIER, E., NGUYEN, T. M. I)., and WEC;OLI, Ll. 1978. Photoaffinity labeling of the angiotensin I I receptor; pharmacology of the labeling peptides in the dark. Can. J. Physiol. Pharmacol. 56, 956-962.
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3 20
CAN. 9. PHYSIBL. PHARMACBL. VOL. 57, 1979
NEEDLEMAN, P., FREER? R. J., and MARSHALL, @. R. 1972. ESC~IER, E., NOUYFN,r. M. D., RORFRT, H . , ST-PIFRRE, S. A.. Angiotensin-receptor Interaction: Influence of pH on and REOOLI,D. 1978. Photoaffinity labeling of the angioangiotensin inhibition by Phe4-TyrY-angiotensin11 in rat tensin II-receptor. I. Synthesis of the photolabeling peptides. uterine smooth muscle. Arch. Int. Pharmacodyn. Ther. 280, J. Med. Chern. 21, 280-284. METHOT, A. L., MEYER. P., BIRON,P., LORAIN, M. I-., LAORUE, 118-1 25. I)., PARK,W. K., and Rrsux, F'. 1974. [Pharmacology G., and MILIEZ, P. 1964. Hepatic inactivation of angiotensin. REGOLI, Nature (London), 203, 531-532. of angiotensin. Pharmacol. Rev. 26, 69-123. NAKAIE, C . R., OSIIIRO, M. E. M., ~ O I S S I G., S , and PAIVA. ST-LOUIS,J., REUOLI:D., BARARE, J., and PARK,W. K. 19'77. Myotropic actions of ailgiotensin and noradrenaline in A. C. M. 19'77. ElTect of tyrosine ionization upon biological strips of rabbit aortae. Can. J. Physiol. Pharmacol. 55, activities of angiotensin I1 and two new peptide analogues. Biochirn. Biophys. Acta, 495, 151--158. 1056-1069.
Altered effect of insulin and catecholamines in brown adipose tissue of cold-acclinlatedrats NICOLE B~GIN-HEICK, IRISNQLAND, MARTHE D A L PAND ~ , H. M. C. HEICK Department o f Biochemistry, University o f Ottawa, Ottawa, Ont., Canada Eleanor M . Patterson Department of Laboratoo Medicine, Chiklren's Hospital o f Ea.~ternOnrcirio, Ottawcl, ddnt., Canada Received July 14, 1978 B~GIN-HEICK, N., NOLAND, I., D A L P ~M., , and HEICK,H. M. C. 1979. Altered effect of insulin and catecholamines in brown adipose tissue of cold-acclimated rats. Can. J. Physiol. Pharmacol. 57, 320-324. Data are presented indicating that in brown adipose tissue (BAT) of cold-acclimated ( C A ) , but not cold-exposed ( C E ) rats, there was an alteration in the relative response to catecholamines and insulin as evidenced by increased binding of alprenolol and decreased binding of insulin to plasma membrane enriched fractions. In addition, the stirnulatory effect of insulin on glucose incorporation into glycogen and its inhibitory action on adenylate cyciase activity were both blunted in the CA tissues. It is proposed that shifts in the capacity of BAT to respond to catecholamines and insulin may be involved in the mechanism of cold acclimation. B~GIN-HEICK, N., NOLAND, I., D A L P ~M., , et HEICK,H. hf. C. 1979. Altered effect of insulin and catecholamines in brown adipose tissue s f cold-acclimated rats. Can. J. Physiol. Pharmacol. 57, 320-324. On presente des rCsultats indiquant que le tissu adipeux brun (BAT) de rats asclirnat6s au froid (CA) est le sikge d9unemodification de la reponse relative aux catCsholamines et h l'insuline qui n9estpas prksente chez les rats qui ont CtC uniquernent exposes au froid (CE). Cette modification est mise eta Cvide~acepar une augmentation de la liaison de l'alprenolol et une diminution de la liaison de l'insuline h des fractions subcellulaires enrishies en membranes plasmatiques. De plus, i'efret stinlulant de l'insuline sur l'incorporation du glucose dans le glycogkne ainsi que son action inhibitrice sur i'activite de l'adenylate cyclase sont diniinuks dans le tissu adipeux brun des rats du groupe CA. On propose que ces changements de la capacitk du BAT h repondre aux catCcholarnines et ? L'insuline i pourraient 2tre impliquks dans le mechanisme de l'acclimation au froid.
Introduction or by drug treatment (LeBlanc et al. 1972; Heick et It is well documented that rats rendered cold re- al. i973 )'increase their oxygen consumption in reof catecholamines. In sistant by cold acclimation ( H ~ ~ 197s) ~ ~ sponse - Hto an ~ acute ~ ~injection ~ the case of CA rats. the catecholamine-stimulated ABBREV~ATIONS: CA. cold acclimated: CE, cold exposed; illcrease in consumption was not due to an inNST, nonshivering therrnogenesis; BAT, brown adipose tissi,e: KRI,, Krebs-Rinper-.,hosphate; DHA, dihydroal- crease in sensitivity but rather to an increased capacity to respond (Himms-Hagen B 975 ) . prenolol ; JNA, isoproterenol. 0008-4212/79 /030320-05$01.OO/O
F) 1979 National Research Council of Canada/Conseil national de recherches du Canada
NOTES
activity. It is probable, therefore that the effect of hypotonicity is due to directIy induced changes in ion gradients rather than to ATPase-induced changes. ,4lthough a reduction of 10%) in the [K+] of the bathing solution did not affect the response to oxytocin, a reduction in tonicity of the bathing solution and consequent entry of water into cells with their much higher [K'] could temporarily have a more marked effect on the K+ gradient and therefore on membrane polarity. Results of studies of the involvement of cyclic nucleotides in the response of neurohypophysia1 hornlone target tissues have been equivocal. Oxytocin has been found to be without efiect on adenyIate cyclase levels in mammary gland (Bousa 1977). From the presently described experiments, it is d e a r that the hypotonicity-induced enhancement of the response to oxytocin is not due to increased levels of CAMP or cGMP. It may be of some significance that co-administration of oxytocin with the cyclic nucleotides consistently resulted in diminished responses. It was considered possible that the effect of hypotonicity could be caused by increased levels of prostaglandins in the mammary gland, perhaps as a result of release of arachidonic acid from the membranes of the swollen cells. The administration of indomethacin together with oxytocin, or for up to 45-min peric~dsbefore oxytocin, was without effect on the hypotonicity response, or on the standard response. suggesting that prostaglandin synthesis is not a necessary component of either. Bentley and McEwen ( 1961 ) reported that cell swelling did not correlate with uterine sensitivity to oxytoci~~.
317
In a11 conditions studied, the responses sf the strip of mammary gland to acetylcholine and to oxytocin were altered similarly. This further supports the idea that the influence of the changed conditions is at a stage beyond hormone-receptor interaction. The use of hypotonic Tprode soIution for routine biological assay of oxytocin enhances the sensitivity of the isolated strip of lactating mouse mammary gland, as claimed (Roca rt cil. 1972). It does not increase the assay's seIectivity. Acknowledgment This work was supported by the Medical Research Council of Canada. BENTLEY, P. J . , and MCEWFN,E. 1961. The effects of ion changes in the rat uterus stimulated by oxytocin. Br. J. Pharmacol. 16,45-49. DANIEL, E. E. 1964. The iilterconnection between active tramport and contracture in uterine tissues. Can. J. Phy4iol. Pharmacol. 42, 453-495. Dousa, T. P. 1977. Cyclic nucleotides in the cellular action of neurcohypophywal hormones. Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, 1867-1871. FIFI~ITI, C., ROCA,R., MATTEI,A., MFLANDER, S.. GAROFALO, E., GIOIADE COCH.M. N.. and C o c t ~J., A. 1970. A sensitive and specific bioa\say for oxytocin. l'roc. Soc. Exp. Biol. Med. 133, I 155-1 157. MARKLL, H.V.,~ V A KJK. ,, and BRANDA. L. A. 1978. Oxytocin binding site4 in lactating rabbit rnamrriary gland. Can. J. Biochern. 56, 968-976. ROCA, R. A.. G ~ R O F A L E, O , G., GIOIADE COCH, M. N., AROCINA, hl., and C o c ~ r .J. A. 1972. Tmprovemcnt in the bioassay of oxytocin by the mou\e mammary glai-md iiz vitro. Proc. Soc. Exp. Biol. Med. 139, 1010-1012. SOLOFF,M. S., SCHROEDEII, B. T., CHAKRABOIITY, J., and P E A R L ~ I U ' I ~A.L RF. , 1977. Characterization of oxytocin reccptors in the uterus and nlamtnary gland. Fetf. Proc. Fed. Am. Soc. Exp. Biol. 36. 1861-1866.
Study on the influence of tyrosine deprotonation on the myotropic action of angiotensin 111 E. ESCIIER~ AND G. GUII,I,EMETTE~ Department ofPhysiology and I'lmarnzacology, Unir*ersityofSherbrookc Medical ,School, Slmerbrooke, P.Q.,Carlada J l H 5N4 Received June 2, 1978 ESCHER,E., and GUILLEMETTE, C;. 1979. Study on the influence of tyrosine deprotonation on the myotropic action of angiotensin 11. Can. J. Physiol. Pharmacol. 57, 317-320. The influence of phenolic tyrosine ionization in angiotensin 11 on the myotropis action of this peptide has been investigated in vitro on rabbit aortic strips. [Sar1, Tyr'langiotensi~~ ABBREVIA? IONS:AT, angiotensin 11; Amp. L-4'-aminophenylalaninc. 'This work was supported by the grant DG154 from the Medical Kesearch Council of Canada and an installation grant from the Conseil de la Recherche en Sante du Quebec, No. 770150. "uthor to whom correspondence should be addressed. Tart of M.Sc. thesis of G.G.
0008-4212/79/030317-04$01.00/0 @) 1979 National Research Couilcil of Canada/Conseil national de recherches du Canada
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CAN. J . PHYSIOE. PHARMACOL. VOE. 57, 1979
I1 and [Sar1, (4'-amino) Phe4]angi~tensinI1 (as a reference which cannot undergo the same ionization) were tested over a pH range from 6.8 to 9.0 and their activities compared. The results clezlrly indicate that angiotensin 11 with a deprotonated phenolic hydroxyl group on Tyr in position 4 is not the most active o r only active form ~f angiotensin 11.
ESCHER,E., et GL;ILI.IF,~IETTE, G. 1979. Study on the influence of tyrosine deprotonation on the myotropic action of angiotensin 11. Can. 5 . Physiol. Pharrnacol. 57, 3 17-320. L'influence d'ionisation phknolique de tyrosine en angiotensine I1 fut investiganke pour B'actiom myotropique in \*it/.ode ce peptide SLIP des bandelettcs d'norte de lapila. T.es analogues [Sar', Tyr4]angiotensine11et [Sar1, (4'-amino)Phe4]angistensine I1 (cornme rkfkrence qui ne pcut pas subir la m6me ionisation) furent testees ii p H variant entre 6.8 ct 9.0 et leurs actia~itescomparies. kes rbsultats indiq~aentclairernent que l'ungiotensine 11 dbprotonee sur- I'hydroxyl phholique de Tyr en position 4 n'est pas la seule forme active ou plus active de l'angiotensine 11.
Introduction The high activity of the octapeptide hormone A T (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) is strongly reduced orn any modification in the Tyri residue. We recently investigated the myotropic action of [Sari]AT analogues, modified in position 4 with several aromatic amino acids, on rabbit aortic strips. Our results suggested a dependence of the agonistic properties on the electronegativity of the substituted aromatic nucleus of position 4 (Escher, Nguyen, and Regoli 1978 ) . High electronegativity gave low affinity for the receptors on aortic smooth muscle and vice versa, only the electronegativity of Tyr did not fit the relation well. The deprotonated form of Tyr is leqs electronegative than Tyr itself and would fit the relation much bctter. Furthermore earlier experiments with A T at lzigher pH gave higher activities (Paladini pt al. 1963; Methot et (11. 1964); therefore, we suspected that the ionized form of Tyr might be the active species. However, Nakaie et al. (1977) recently reported experiments with monochlorinated [Tyri]AT and concluded that deprotonation of the phenolic hydroxyl of Tyr is even unfavorable. Unfortunately the introduction of a chlorine raises the electro~~egativity of the aromatic nezclells of Tyr4 and explains the reduced poteracy of this analogue. The experiments of Nakaie et al. were performed ~virh[Aspl]AT analogues on rat uterus and guinea pig ileum; our earlier expcriments were performed with [Sarl]AT analogues on rabbit aorta. Although the bioassays and the peptides were not the same, the values can be co~nparedwith some reserve. We therefore undertook experiments at a range of different pH values with the two peptides [Sar1, Tyrt]AT and the Amp-containing analogue [Sar1, Amp4]A% (Escher, Nguyen. Robert et al. 1978) (Fig. 1 ) . The phenolic function of Tyr has a dissociation constan; pK,, of 10.0 and the aromatic amino func-
FIG.I . Structure of the t w investigated ~ AT analogues.
tion of Amp a pKB of >> 14. This means that Amp does not deprotonatc at all in aqueous solutio~~s Since both compounds are identical with the exception of the -OH or the -NH2 group, any difference in conformation or biological activity would result from this molecular difference. Position 4 is not directly linked to the myotropic response release. This is mainly the function of position 8 (Regoli ct cal. 1 974). Because of this, i.e., the specificity of their action and great similarity of these two peptides, it is reasonable to assume that both have the same mode of action. If, as suspected, the deprotonated [Sar1, TyrYAT were the most or only active form, the activity would increase with higher deprotonation. Extreme pH values affect the tissues quite severely; therefore any change in AT activity could be seen only if a nonionizable AT analogue like [Sar1, Amp4]A% acts as internal reference. At a given pH the half-maximal doses (EDr,,,)of each peptide give a dose differential ( = E19,,(Tyr4)/ ED5,,(Amp4)). If any increase were to occur due to deprstonation, this dose differential should develop a pH dependence similar to that of Tyr deprotonation (Fig. 2).
Materials and Methods Rabbit aortic strips were suspended in a 5-mL organ bath
NOTES
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in a modified Krebs solution (0.50 m M Ca2+instead of 2.48 m M ) according to Needleman et ( I / . (1972). The pH of the Krebs solution was adjusted immediately prior to addition to the tissue? with 1 AT NaOH or 1 N HC1. The tissues were equilibrated with the lowest pH (6.8), pure oxygen was bubbled through the bath, and no carbon dioxide was added. Dose-response curves were performed after 90 min of equilibration. Then the tissues were mlashed and equilibrated for 90 rnin with the next higher pH solution. Every I S min the solution was replaced by fresh Krehs buffer of the same pH. The peptides tested were described by Escher, Nguyen, Robert ct a/. (1978). General techniques such as tissue preparation and recording of responses were described by St-1,ouis eb QI. (1977). The results were easily reproduced if pH 9.0 was not exceeded. Inversion of the pH seqrlcnce did not significantly influence the results. At pH 9.5 and higher. the tissues were very weakly responsive and often contracted continiiously without addition of agonists. The intrinsic activity ( Ariens 1964) a,, was determined for each pH, with ctE at pH 7.4 = 1 as internal standard. The half-mm~aximaldoses (ED;,!) are the calculated concentrations of an agonist which produced half-maximal contraction at a given pH.
319
FIG.2- Relationship betwecn pH and the ratio between the half-maximal doses of [Sar1, Tyr4]AT and [Sar1, Amp4]AT (ED,,,(Tyr4)/ED,,(AImpq)).Each point) . 4 is the mean k SEhI of at least seven experiments and the straight line is a calculated linear regression. A indicates the phenolic deprotonation of Tvr in percentage as a function of pH according to Nakaie et ul. (1977).
The dose differential does not show any dependence on the pH within the given limits; within the error, Results and nissussion the dose differential is always equal. This supports the initial hypothesis that [Sarl]AT and [Sar1, At pH 6.8, 7.4, $.8, 8.5, and 9.0 dose-response curves of [Sarl]AT and [SarL,Arnp4]AT were meas- Arnp4]AT have the same mode of action on the vascular smooth muscle receptor of AT. The higher ured on rabbit aortic strips and their ED,,, and values calculated. It is seen that with higher pH, the activity of [Sar1, Tyri]AT has therefore to be exintrinsic activity and the affinity decrease. The results plained in a different way because the electronegativity of the Anlp side chain is lowel- than for are summarized in Table 1. tyrosine. The ratio of ED,,,(Tyr" over ED,o(Amp4) The observed affjnity of [Sarl]AT was higher, as values was recorded as a furaction of pH (Fig. 2 ) . If any activity increase had occurred due to tyrosine expected from the electronegativity relation. This deprotonation or to a different mode of action, the difference has to come from some additional binding ratio would increase with rising pH. Conversely, an energy of the hormone-receptor interaction. Again, activity decrease would have resulted in a fall in the reason must be in the nlolecular structure, speratio with rising pH. Neither change was observed cifically in some bonding that a phenolic hydroxyl and we therefore conclude that the deprotonated can undergo and an aromatic amine cannot. One form of Tyr4 in A T is not the most or even only possibility could be a weak hydrogen bond, with active principle of A T on smooth muscle, as sug- the hydroxyl acting as hydrogen acceptor through gested by earlier investigations (Methot et nl. 1964). its free sp2 orbital on the oxygen. The amine has already a hydrogen in this position which would result rather in steric hindrance. This hydrogen bond TABI.L 1. Effect of pH on the relative biological activities of could be intramolecularly within the peptide itself [Sar1, Tyr I]AT and [Sar1, Amp 4]AT or intermolecularly with the receptor. These possi[Sar1, Tyr "AT [Sar l . Amp 4]AT bilities are actually under investigation with spectroscopic methods. pH
6.8 7.4 8.0 8.5 9.0
En:,o, ng,'rnl,
1.5f0.9 3.111.7 5.2k2.7 7.5-F-2.6 10.5k2.9
CVE
1.02f0.36
!-:.
0.8310.20 0.67fQ.12 0.31k0.14
EDSO, ng,'mL
aF:
6.7f2.4 1.04+0.14 1 3 . 8 k 3 . 1 1.00 23.3k8.9 0.85f0.13 30.7k14.9 0.68k0.18 4 9 . 7 5 15.4 0 . 1 8 k 0 . 1 2
NOTE: E D x is the dose of agonist \vhicIl gives the half-maxirnal contmction at this pH. All values are the mean o f at least seven estimations. aE is the intrinsic activity (Ariens 1964) anti is related to the libility of a drug to produce the maximal contraction at a given pH. At pH 9.4, aE is 1.00 for both compounds.
Acknowledgments Thanks are due to Dr. Domenico Regoli and Dr. Francis Rioux for helpful advice and discussions. AKIENS,E. J. 1964. Molecular Pharmacology. Vols. I and 11. Academic Press Tnc., London. ESCFIER, E., NGUYEN, T. M. I)., and WEC;OLI, Ll. 1978. Photoaffinity labeling of the angiotensin I I receptor; pharmacology of the labeling peptides in the dark. Can. J. Physiol. Pharmacol. 56, 956-962.
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/30/14 For personal use only.
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CAN. 9. PHYSIBL. PHARMACBL. VOL. 57, 1979
NEEDLEMAN, P., FREER? R. J., and MARSHALL, @. R. 1972. ESC~IER, E., NOUYFN,r. M. D., RORFRT, H . , ST-PIFRRE, S. A.. Angiotensin-receptor Interaction: Influence of pH on and REOOLI,D. 1978. Photoaffinity labeling of the angioangiotensin inhibition by Phe4-TyrY-angiotensin11 in rat tensin II-receptor. I. Synthesis of the photolabeling peptides. uterine smooth muscle. Arch. Int. Pharmacodyn. Ther. 280, J. Med. Chern. 21, 280-284. METHOT, A. L., MEYER. P., BIRON,P., LORAIN, M. I-., LAORUE, 118-1 25. I)., PARK,W. K., and Rrsux, F'. 1974. [Pharmacology G., and MILIEZ, P. 1964. Hepatic inactivation of angiotensin. REGOLI, Nature (London), 203, 531-532. of angiotensin. Pharmacol. Rev. 26, 69-123. NAKAIE, C . R., OSIIIRO, M. E. M., ~ O I S S I G., S , and PAIVA. ST-LOUIS,J., REUOLI:D., BARARE, J., and PARK,W. K. 19'77. Myotropic actions of ailgiotensin and noradrenaline in A. C. M. 19'77. ElTect of tyrosine ionization upon biological strips of rabbit aortae. Can. J. Physiol. Pharmacol. 55, activities of angiotensin I1 and two new peptide analogues. Biochirn. Biophys. Acta, 495, 151--158. 1056-1069.
Altered effect of insulin and catecholamines in brown adipose tissue of cold-acclinlatedrats NICOLE B~GIN-HEICK, IRISNQLAND, MARTHE D A L PAND ~ , H. M. C. HEICK Department o f Biochemistry, University o f Ottawa, Ottawa, Ont., Canada Eleanor M . Patterson Department of Laboratoo Medicine, Chiklren's Hospital o f Ea.~ternOnrcirio, Ottawcl, ddnt., Canada Received July 14, 1978 B~GIN-HEICK, N., NOLAND, I., D A L P ~M., , and HEICK,H. M. C. 1979. Altered effect of insulin and catecholamines in brown adipose tissue of cold-acclimated rats. Can. J. Physiol. Pharmacol. 57, 320-324. Data are presented indicating that in brown adipose tissue (BAT) of cold-acclimated ( C A ) , but not cold-exposed ( C E ) rats, there was an alteration in the relative response to catecholamines and insulin as evidenced by increased binding of alprenolol and decreased binding of insulin to plasma membrane enriched fractions. In addition, the stirnulatory effect of insulin on glucose incorporation into glycogen and its inhibitory action on adenylate cyciase activity were both blunted in the CA tissues. It is proposed that shifts in the capacity of BAT to respond to catecholamines and insulin may be involved in the mechanism of cold acclimation. B~GIN-HEICK, N., NOLAND, I., D A L P ~M., , et HEICK,H. hf. C. 1979. Altered effect of insulin and catecholamines in brown adipose tissue s f cold-acclimated rats. Can. J. Physiol. Pharmacol. 57, 320-324. On presente des rCsultats indiquant que le tissu adipeux brun (BAT) de rats asclirnat6s au froid (CA) est le sikge d9unemodification de la reponse relative aux catCsholamines et h l'insuline qui n9estpas prksente chez les rats qui ont CtC uniquernent exposes au froid (CE). Cette modification est mise eta Cvide~acepar une augmentation de la liaison de l'alprenolol et une diminution de la liaison de l'insuline h des fractions subcellulaires enrishies en membranes plasmatiques. De plus, i'efret stinlulant de l'insuline sur l'incorporation du glucose dans le glycogkne ainsi que son action inhibitrice sur i'activite de l'adenylate cyclase sont diniinuks dans le tissu adipeux brun des rats du groupe CA. On propose que ces changements de la capacitk du BAT h repondre aux catCcholarnines et ? L'insuline i pourraient 2tre impliquks dans le mechanisme de l'acclimation au froid.
Introduction or by drug treatment (LeBlanc et al. 1972; Heick et It is well documented that rats rendered cold re- al. i973 )'increase their oxygen consumption in reof catecholamines. In sistant by cold acclimation ( H ~ ~ 197s) ~ ~ sponse - Hto an ~ acute ~ ~injection ~ the case of CA rats. the catecholamine-stimulated ABBREV~ATIONS: CA. cold acclimated: CE, cold exposed; illcrease in consumption was not due to an inNST, nonshivering therrnogenesis; BAT, brown adipose tissi,e: KRI,, Krebs-Rinper-.,hosphate; DHA, dihydroal- crease in sensitivity but rather to an increased capacity to respond (Himms-Hagen B 975 ) . prenolol ; JNA, isoproterenol. 0008-4212/79 /030320-05$01.OO/O
F) 1979 National Research Council of Canada/Conseil national de recherches du Canada
