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High molecular-weight renin and renin-binding protein
381
T I P S - S e p t e m b e r 1 983
Reading list 57-63 1 Sander, M. (1972)Lancet i, 618--619 16 Hall, D. W. (1968)./. R Coll. Gen. Pract. 15, 2 Welch, K M. A., Spira, P. J., Knowles, L. and 321-324 17 Ala-Hm'ula, V., Myllyla, V. V, H ~ n , E. Lance, J, M. (1974)Neurology 28, 705-710 and Tokola, O. (1981)Headache 2 I, 240-242 3 Varth, Y., Rahey, 1. M., St~eifler,M., Schwartz, 18 Hanington,E. (1978)Lancet n, 501-503 A., Lindner,H. R. and For, U. ( 1975) Neurology 26, 447--450 4 Horrobin,D. F. (1977) Headache 17, 113-117 5 Vapaatalo,H., Parantainen,J., Linden, I.-B. and Hakkarainen, H. (1977) m Headache, New Vis. tas (Sicuteri,F., ed.), pp. 287-300, Biochemical Press, Florence 6 Hakkammen,H., Vapaatalo,H., Gothoni,G and Parantamen,J. (1979) Lancet ii, 326-328 7 Peattield, R. C., Stelner, T J , Gawel, M. J. and Rose, C. F. (1981) in Progress m Migraine Research 1 (Rose, F. C and Zllkha, K J., eds), pp 124-129, PitmanBooks, London 8 Hakkarainen,H., Parantmnen,J., Gothom,G. and Vapaatalo,H. (1982)Cephalagia 2, 173-177 9 Parantamen, J and Vapaatalo, H. m Handbook of Biology and Cherntstry of Prostaglandins and Related Metabolites of Polyunsaturated Fatty Acids (Curtis-Prior,P. B., ed.), Churchill- Jouko Parantamen, M.A. ~ a pharmacologist in the Ltvingstone,Edinburghand New York (m press) Research Laboratories o[the Medica Pharmaceutt10 Bergstmm,S., Carlson,L. A, Ekelund,L. G. and cal Company Lid and is a vtsitmg scientist m the Oro, L. ( 1965)A cta Physiol. Scand. 64, 332-339 Section o[ Pharmacology, Department o[ Btometh11 Szezeklik, A , Gryglewski, R. J., Nizankowski, cal Sciences, Umversity of Tampere. H~s malor R., Skawinski, S., Gluszko, p. and Korbut, R. research interests are interacaons between the sym(1980) Thromb. Res. 19, 191-199 patheac nervous system and prostaglandms. 12 Barrie, M. and Jowett, A. (1967) Brain 90, 785-794 13 Anthony,M. (1976)Headache 16, 58--69 14 Amroyan, E. A. (1981) in Prostaglandins and Thromboxanes (F6rster, W, ed.), pp. 157-158, VEB GuslavFischer Veflag,Jena 15 Volans,G. N. (1975)Br.J. Clm. Pharmacol. 2,
19 Berde, B. and Fanchamps, A. (1975) in Kop#chmerz, Headache (Barolin, G. S., Saurugg, D. and Hemmer, W., eds), pp. 55-74, VerlagsgesellschaftOtto Spatz, Miinchen 20 Hilton, B. P. and Cummgs, J. N. (1972) J. Neurol. Neurosurg. Psychiatry 35,505--509
High molecular-weight renin and renin-binding protein
tigations of this protein were initiated by Boyd ~ who studied the pig kidney, and further evidence has been obtained in studies of dogs and rats.
Kenjiro Yamamoto and Fumihiko Ikemoto Department of Pharmacology, Osaka City University Medical School, 1-4-54, Asahtmachl, Abeno-ku, Osaka 545, Japan. R e n a l high mol, wt renin (mol. wt 6 0 000) is a c o m p l e x o f renin (mol. wt 4 0 00(9) a n d renin-binding protein. The renin-binding protein was thought to be present in renal conical cytosol fraction. Recently, however, this protein has been identified in renal cortical tubular cells a n d its physiological role in tubular f u n c t i o n has been elucidated.
Renin, a key enzyme in the renin-angiotensin-aldosterone cascade, is an aspartic protease which cleaves angiotensinogen to liberate angiotensin I which is, in turn, converted into a pressor octapeptide, angiotensin II. This enzyme exists in multiple forms in the plasma and kidney. Advances in the biochemistry of renin have led to a complete purification of renal renin from pigs,
rats, mice, dogs and humans, and mouse submaxillary gland renin o f extra-renal origin. The mol. wt of renin is approximately 40 000. A renin with a tool. wt o f approximately 60 000 has been detected in the kidney of pigs, dogs, rats and mice and this type o f renin is thought to be a complex of renin and a renal cortical protein which was termed 'renin-binding protein'. Inves-
Heikkt Vapaatalo, M.D. is Professor of Pharmacology in the Departmem of B~omedJcal Sciences of the University of Tampere. He received his training in pharmacology m the Department of pharmacology of the Umversity of Helsmki, Finland during 1963-1969 and m the Department of pharmacology of the Medical Highschool of Hanhover, F.R.G during 1970-1971. He waS AssocuUe Profesaor of Pharmacology in the University of Oalu, Finland#ore 1972-1974, a#er wluch he went to the University of Tampere. His main research interest is the role of prostaglandin$ in basic and clinicalsciences and m the mode ofaction of drugs. II
Properties o f renin-binding p r o t e i n Gel filtration is the procedure which has been most widely used to estimate the tool. wt of renin. In the dog kidney, renin with a tool. wt o f 40 000 (low mol. wt renin) is present in the renin granules, while renirv binding protein is present in the cytosol of the renal cortex. Funakawa et al. 2 isolated renin granules from the dog kidney using discontinuous sucrose density gradient centrifugation and found that these granules contained only the low mol. wt renin. In the renal cortical homogenate, the tool. wt of the renin was 40 000 but the low mol. wt renin disappeared and a renin of high mol. wt (60000) was identified after the sulfhydryl group oxidizing agent sodium tetrathionate was added to give a final concentration o f 5 mM. The authors concluded that the low mol. wt renin is the native form, and that this renin is converted into the high mol. wt (60 000) renin by sulfhydryl oxidation with sodium tetrathionate, probably by the interaction o f renin with renin-binding protein. This high mol. wt renin was experimentally produced in a
© ~983 Elsevier Soence Pl~hshers B V , ~
0165 - 6147/83/$01 00
382 mixture of renin from isolated renin granules and the soluble fraction of renal cortical homogenate in the presence of sodium tetrathionate, yet high tool. wt renin was not produced when the soluble fraction of renal medulla or liver was used. Thus, the presence of renin-binding protein in the renal cortical cytosol fraction was demonstrated 3. With gel filtration, this protein is eluted into the fractions corresponding to a mol. wt region of 100 000, although this value is not consistent with estimations expected from the mol. wt difference between high and low mol. wt renins. The mol. wt is not reduced by 4 M urea, therefore this protein is not an aggregated form of mol. wt 20 000 subunits. The renin-binding protein does not bind to concanavalin A 'Sepharose' beads. Chloroform and 1% 'Triton X- 100' have no influence on the ability of this protein to bind to renin. Therefore, the reninbinding protein is not a glycoprotein, and lipids are apparently not involved in the binding reaction4. Comparable properties of renin-binding protein were demonstrated in rats 5. A similar mOde of mol. wt conversion of renin was also noted in pig and mouse 6 kidneys. Intra-renal localization of renin-binding protein The renin-binding protein is present in the renal cortex but not in the renal medulla and liverL Thus, the physiological role of the high mol. wt renin can be considered in relation to the exact localization of the renin-binding protein. This protein may exist in juxtaglomerular cells for a specific role in the process of synthesis and/or storage of renin. If this is the case, then the high mol. wt renin produced in the renal cortical homogenate may be a re-constituted form of the intermediate renin/protein complex, through which renin is synthesized and stored. The enzyme was shown to be synthesized as a higher mol. wt pre-pro-form in the mouse submaxillary gland and kidney. However, if this protein exists in other tissues, such as tubular cells, the significance of high mol. wt renin in the kidney would be different. We have isolated glomeruli and tubular segments from the renal cortex of rats 7 and dogs s, and attempted to identify this binding protein. Glomeruli and tubular segments were isolated by microdissection. Great care was taken to prevent the detachment of afferent arterioles from the glomeruli and to avoid contamination of the glomeruli with tubular segments. The isolated glomeruli contained large quantities of renin, while in the isolated tubular segments renin activity was hardly detectable. Isolated glomeruli and tubular segments were separately pooled.
TIPS - September 1983
D
|
+ SH
SH
SH ~
Itlgh tool. ~ mnln Tetrathionate
if the renin-binding protein does exist in the proximal tubules, it may play a role in the process of reabsorption of the renin. Presumably, this protein is a carrier of renin and the reabsorbed renin is converted into a high mol. wt form to be further transported, or to be metabolized in the tubular cells.
MOI wt 40 000
Fig. 1. Schematw representation o f possible mode o f mol. wt conversion o f renin. Low tool. wt (40 000) renm binds to the renm-binding protein ( R B P) to form the high tool. wt (60 000) renin. The renin-bmding protein in renal cortwal tubular cells has a strong affinity for and ts bound to, renm A sulflzydryl-activated enzyme (SH-enzyme), however, cleaves the high tool. wt renin, thereby liberatmg the low moL wt renm. The bmdmg reaction proceeds in the absence or inacttvation o f this enzyme
Approximately 3 mg (wet weight) of the respective areas of the nephron were obtained from a single dissection. Since the method provided only minute quantities of the sample, the mol. wt of renin was estimated, using high performance liquid chromatography (HPLC) with 2 coupled TSK G3000 SW columns 7. Extracts of the glomeruli were examined for the presence of renin-binding protein. The extract did contain renin, therefore, if it also contained the renin-binding protein, the endogenous renin would be bound to form the high mol. wt renin. However, the extract of isolated glomeruli, in the presence of sodium tetrathionate, showed a single peak of renin activity on the chromatogram and the retention time of the peak position corresponded to that of the low tool. wt renin. This result suggested a lack of renin-binding protein in the glomeruli. In contrast, extracts of the isolated tubular segments were found to contain the renin-binding protein. When the low mol. wt renin was mixed with the extract of isolated tubular segments, in the presence of sodium tetrathionate, and the mixture was subjected to HPLC, there was a peak of renin activity at an earlier retention time than that of the low mol. wt renin. The mol. wt was estimated to be 60 0007'8. Reninbinding protein is thus considered to be present in the tubular, but not in the juxtaglomerular, cells. The question of the mode of action of renin-binding protein in the renal cortical tubules remains. Recent studies indicate that renin is filtered through glomerular capillaries and is reabsorbed by the proximal tubular cells. We have recently found that i.v. administered ~25I-labelled mouse submaxillary gland renin is predominantly concentrated in the mouse kidney. Microscopic autoradiography reveals that this labelled renin is incorporated into the proximal tubular cells, suggesting reabsorption 9. Therefore,
Possible enzymatic reaction involved in the mol. wt conversion of renin The mol. wt conversion of renin in the kidney involves oxidation of tissue sulfhydryl groups. In homogenates of the renal cortex, the high tool. wt renin can be formed in the presence of the sulfhydryl group oxidizing agent sodium tetrathionate, and can be re-converted into the low mol. wt renin by dithiothreitol2. Thus, the 2 types of renin seem to be inter-convertible by oxidation or reduction of the sulfhydryl groups. However, the sulfhydryl alkylating agent N-ethylmaleimide is also effective in the formation of the high mol. wt renin and the coupling of renin and renin-binding protein is not considered to be effected by disulfide bonds. Indeed, renin remains reactive with renin-binding protein even after it has been pre-treated with sulfhydryl group blocking agents, e.g. 5,5'-dithiobis(2-nitro benzoic acid) and N-ethylmaleimide. To assess the role of sulfhydryl groups in the conversion system, we examined, and demonstrated, the possible contribution of an enzyme-like substance which is sensitive to sulfhydryl oxidation in the conversion from the high mol. wt renin to the low mol. wt renin. The former was prepared by the addition of potassium tetrathionate to a renal cortical extract from dogs, followed by dialysis to remove the excess potassium tetrathionate. Two extracts from different sources were then prepared: one from renal cortex (renin was eliminated using pepstatin-aminohexylagarose affinity chromatography) and the other from the renal medulla. When the high mol. wt renin was mixed with the extract of renal cortex and incubated at 37°C for 15 min, this renin was converted into the low mol. wt form. However, there was no evidence of conversion in the extract from the renal medulla. When the extract from the renal cortex was pre-treated with perchloric acid or potassium tetrathionate, the mol. wt of the renin remained unchanged. All these findings taken together suggested that, in the kidney, the conversion from high mol. wt to low mol. wt renin may be catalysed by a sulfhydryl-activated enzyme-like substance 1°. The possible mode of mol. wt conversion of renin is shown in Fig. 1. Since the renin-binding protein is present in tubular cells, it may be that the conversion plays a role in the mechanism of reabsorption of
TIPS - September 1 983
383
renin from the proximal tubules. Whether the renin-binding protein and a 'molecular weight converting' enzyme exist in the same tubular cells where renin is reabsorbed, is the subject of continuing studies.
8 Takaofi, K., lwao, H , Ikemo~o, F. and Yamamoto, K (1982) CIm Exp. Hypertens. A4, 2097-2105 9 Iwao, H , Nakamura, N , Ikernoto, F. and
Yamamoto, K J. H~tochem Cytochem. (in press) 10 Ikemoto, F , Takaon, K , iwao, H and Yamamo~o, K (1982)C/in Sct. 62, 157-162
Kenliro Yamamoto M.D. has been Professor and Chairman of the Department of Pharmacology, Osaka City University Medtcal School since 1972. From 1960 he has concentrated on research of renm--angiotensin systems.
Fumlhdco lkemoto Ph D. graduated from the UniversUy of Kobe, Faculty of Sc4ence. Following research on biochemistry of lenttcular cells, he moved to Osaka Cay UniversUy Medical School in 1977 and has been studying b~ochemical pharmacology of renm and angiotensins. He ts currently Associate Professor
Acknowledgement We would like to thank M. Ohara for helping us prepare this article.
Reading list 1 Boyd, G. W. (1974) Ctrc. Res. 35,426--438 2 Funakawa, S., Funae, Y. and Yamamoto, K (1978) Biochem. J. 176, 977-981 3 Kawamura, M., lkemoto, F., Funakawa, S. and Yamamoto, K. (1979)Clin Sci. 57,345-350 4 Yamamoto, K., Ikemoto, F , Kawamura, M. and Takaon, K. (1980) Chn. Sct 59 (Suppl. 6),
25s---27s 5 Takaori, K., lkemoto, F. and Yamamoto, K. (1981)Clin. Exp. Hypertens.3,991-1000 6 lwao, H., Minarm,T., Ikemoto,F., Takaon, K., Nakamura, N. and Yamamoto, K. (1982) Btochem. Btophys. Res. Commun. 106, 933--939 7 lkemoto, F., Takaon, K., iwao, H. and Yamamoto,K. (1982)LifeSci. 31, 1011-1016 I
II
Pharmacological investigations in space medicine V. S. Shashkov Kosmicheskaya biologiya i A viakosraicheskaya Meditzina, Mezhdunarodnaya Kmga, Moscow G-200,
USSR. The exploration of the Universe, which started with the flight of Yu. A. Gagarin, has brought an awareness of the importance of assuring spacecrews' safety. The main aspect of this problem is the development of pharmacological methods of prevention and therapy o f the physiological changes occurring during weightlessness and the readaptation period. l'aarmacological supportof spoceflights Spaceflight influences a number of the physiological indices of the living organism. Functional changes in the cardiovascular system occur, together with a complex of symptoms similar to motion sickness, plus disturbances in fluid and electrolyte balance and the mineral saturation of bone tissue, anaemia, deterioration of immunological activity, reduction in muscle weight, etc. All these symptoms are reversible1. The effects of weightlessness are multiform and the pathogenic causes of some of them require further investiga-" tion and definition. The use of medication to prevent and cure the unwanted effects of spaceflight and subsequent re,adaptation has been the subject of wide discussion in the scientific
literature2"s'4. It should first be noted that drugs traditionally prescribed in response to various pathological changes cannot always prevent or cure the peculiar conditions occurring as a result of spaceflight. Spaceflight safety must be considered at several stages: the training period, launch and obtaining orbit, free spaceflight, prelanding and landing, and readaptation after return to the earth s . Each stage involves pharmacological support of a different kind. In the pre-flight period, the type and amount of medical substances which need to be carried are determined on the basis of likely functional disturbances and their prognosis, the number of the spacecrew members, etc. In this period idiosyncratic reactions to drugs which may produce toxic
effects are investigated. The prospective astronauts study drug administration, siteof-action, contra-indications, and drug interactions. Reactions of the body to medical substances under simulated spaceflight conditions, with particular reference to weightlessness are also studied. Modelling of most spaceflight factors (radiation, acceleration, altered gas environment, etc. ) at all levels and ranges of their effects can be accomplished in ground-based experiments. The main goal is the protection of the organism against potentially pathogenic agents. There is a danger, during long-term spaceflights, of the development of stable forms of micro-organisms, super-infection and reactions to drugs used for prevention and therapy (e.g. antibiotics). These dangets are increased by the decrease in general reactivity of the patient. Prophylactic measures are planned which take into account the individual characteristics of astronauts' microflora. Problems with the pharmacological support of launch and landing of spacecrews, particularly with respect to overload and vibration and including the biological background of gravitational physiology, are fully described by Smith ~, and by Vasilyev and Kotovskaya ~ in joint Soviet-American review articles. Physiological investigations in groundbased experiments and during flights on the orbital stations 'Salyut' and 'Skylab' have allowed us to obtain much information on man's reaction to long-term spaceflight and the post-flight period which has helped us to understand complexes of symptoms that will need prevention and therapy.
1983ElsevtefSctet~ctI~MIshersB V , Amsterdam 0165- 6147/83/$01t~O
T I P S - S e p t e m b e r 1 983
Reading list 57-63 1 Sander, M. (1972)Lancet i, 618--619 16 Hall, D. W. (1968)./. R Coll. Gen. Pract. 15, 2 Welch, K M. A., Spira, P. J., Knowles, L. and 321-324 17 Ala-Hm'ula, V., Myllyla, V. V, H ~ n , E. Lance, J, M. (1974)Neurology 28, 705-710 and Tokola, O. (1981)Headache 2 I, 240-242 3 Varth, Y., Rahey, 1. M., St~eifler,M., Schwartz, 18 Hanington,E. (1978)Lancet n, 501-503 A., Lindner,H. R. and For, U. ( 1975) Neurology 26, 447--450 4 Horrobin,D. F. (1977) Headache 17, 113-117 5 Vapaatalo,H., Parantainen,J., Linden, I.-B. and Hakkarainen, H. (1977) m Headache, New Vis. tas (Sicuteri,F., ed.), pp. 287-300, Biochemical Press, Florence 6 Hakkammen,H., Vapaatalo,H., Gothoni,G and Parantamen,J. (1979) Lancet ii, 326-328 7 Peattield, R. C., Stelner, T J , Gawel, M. J. and Rose, C. F. (1981) in Progress m Migraine Research 1 (Rose, F. C and Zllkha, K J., eds), pp 124-129, PitmanBooks, London 8 Hakkarainen,H., Parantmnen,J., Gothom,G. and Vapaatalo,H. (1982)Cephalagia 2, 173-177 9 Parantamen, J and Vapaatalo, H. m Handbook of Biology and Cherntstry of Prostaglandins and Related Metabolites of Polyunsaturated Fatty Acids (Curtis-Prior,P. B., ed.), Churchill- Jouko Parantamen, M.A. ~ a pharmacologist in the Ltvingstone,Edinburghand New York (m press) Research Laboratories o[the Medica Pharmaceutt10 Bergstmm,S., Carlson,L. A, Ekelund,L. G. and cal Company Lid and is a vtsitmg scientist m the Oro, L. ( 1965)A cta Physiol. Scand. 64, 332-339 Section o[ Pharmacology, Department o[ Btometh11 Szezeklik, A , Gryglewski, R. J., Nizankowski, cal Sciences, Umversity of Tampere. H~s malor R., Skawinski, S., Gluszko, p. and Korbut, R. research interests are interacaons between the sym(1980) Thromb. Res. 19, 191-199 patheac nervous system and prostaglandms. 12 Barrie, M. and Jowett, A. (1967) Brain 90, 785-794 13 Anthony,M. (1976)Headache 16, 58--69 14 Amroyan, E. A. (1981) in Prostaglandins and Thromboxanes (F6rster, W, ed.), pp. 157-158, VEB GuslavFischer Veflag,Jena 15 Volans,G. N. (1975)Br.J. Clm. Pharmacol. 2,
19 Berde, B. and Fanchamps, A. (1975) in Kop#chmerz, Headache (Barolin, G. S., Saurugg, D. and Hemmer, W., eds), pp. 55-74, VerlagsgesellschaftOtto Spatz, Miinchen 20 Hilton, B. P. and Cummgs, J. N. (1972) J. Neurol. Neurosurg. Psychiatry 35,505--509
High molecular-weight renin and renin-binding protein
tigations of this protein were initiated by Boyd ~ who studied the pig kidney, and further evidence has been obtained in studies of dogs and rats.
Kenjiro Yamamoto and Fumihiko Ikemoto Department of Pharmacology, Osaka City University Medical School, 1-4-54, Asahtmachl, Abeno-ku, Osaka 545, Japan. R e n a l high mol, wt renin (mol. wt 6 0 000) is a c o m p l e x o f renin (mol. wt 4 0 00(9) a n d renin-binding protein. The renin-binding protein was thought to be present in renal conical cytosol fraction. Recently, however, this protein has been identified in renal cortical tubular cells a n d its physiological role in tubular f u n c t i o n has been elucidated.
Renin, a key enzyme in the renin-angiotensin-aldosterone cascade, is an aspartic protease which cleaves angiotensinogen to liberate angiotensin I which is, in turn, converted into a pressor octapeptide, angiotensin II. This enzyme exists in multiple forms in the plasma and kidney. Advances in the biochemistry of renin have led to a complete purification of renal renin from pigs,
rats, mice, dogs and humans, and mouse submaxillary gland renin o f extra-renal origin. The mol. wt of renin is approximately 40 000. A renin with a tool. wt o f approximately 60 000 has been detected in the kidney of pigs, dogs, rats and mice and this type o f renin is thought to be a complex of renin and a renal cortical protein which was termed 'renin-binding protein'. Inves-
Heikkt Vapaatalo, M.D. is Professor of Pharmacology in the Departmem of B~omedJcal Sciences of the University of Tampere. He received his training in pharmacology m the Department of pharmacology of the Umversity of Helsmki, Finland during 1963-1969 and m the Department of pharmacology of the Medical Highschool of Hanhover, F.R.G during 1970-1971. He waS AssocuUe Profesaor of Pharmacology in the University of Oalu, Finland#ore 1972-1974, a#er wluch he went to the University of Tampere. His main research interest is the role of prostaglandin$ in basic and clinicalsciences and m the mode ofaction of drugs. II
Properties o f renin-binding p r o t e i n Gel filtration is the procedure which has been most widely used to estimate the tool. wt of renin. In the dog kidney, renin with a tool. wt o f 40 000 (low mol. wt renin) is present in the renin granules, while renirv binding protein is present in the cytosol of the renal cortex. Funakawa et al. 2 isolated renin granules from the dog kidney using discontinuous sucrose density gradient centrifugation and found that these granules contained only the low mol. wt renin. In the renal cortical homogenate, the tool. wt of the renin was 40 000 but the low mol. wt renin disappeared and a renin of high mol. wt (60000) was identified after the sulfhydryl group oxidizing agent sodium tetrathionate was added to give a final concentration o f 5 mM. The authors concluded that the low mol. wt renin is the native form, and that this renin is converted into the high mol. wt (60 000) renin by sulfhydryl oxidation with sodium tetrathionate, probably by the interaction o f renin with renin-binding protein. This high mol. wt renin was experimentally produced in a
© ~983 Elsevier Soence Pl~hshers B V , ~
0165 - 6147/83/$01 00
382 mixture of renin from isolated renin granules and the soluble fraction of renal cortical homogenate in the presence of sodium tetrathionate, yet high tool. wt renin was not produced when the soluble fraction of renal medulla or liver was used. Thus, the presence of renin-binding protein in the renal cortical cytosol fraction was demonstrated 3. With gel filtration, this protein is eluted into the fractions corresponding to a mol. wt region of 100 000, although this value is not consistent with estimations expected from the mol. wt difference between high and low mol. wt renins. The mol. wt is not reduced by 4 M urea, therefore this protein is not an aggregated form of mol. wt 20 000 subunits. The renin-binding protein does not bind to concanavalin A 'Sepharose' beads. Chloroform and 1% 'Triton X- 100' have no influence on the ability of this protein to bind to renin. Therefore, the reninbinding protein is not a glycoprotein, and lipids are apparently not involved in the binding reaction4. Comparable properties of renin-binding protein were demonstrated in rats 5. A similar mOde of mol. wt conversion of renin was also noted in pig and mouse 6 kidneys. Intra-renal localization of renin-binding protein The renin-binding protein is present in the renal cortex but not in the renal medulla and liverL Thus, the physiological role of the high mol. wt renin can be considered in relation to the exact localization of the renin-binding protein. This protein may exist in juxtaglomerular cells for a specific role in the process of synthesis and/or storage of renin. If this is the case, then the high mol. wt renin produced in the renal cortical homogenate may be a re-constituted form of the intermediate renin/protein complex, through which renin is synthesized and stored. The enzyme was shown to be synthesized as a higher mol. wt pre-pro-form in the mouse submaxillary gland and kidney. However, if this protein exists in other tissues, such as tubular cells, the significance of high mol. wt renin in the kidney would be different. We have isolated glomeruli and tubular segments from the renal cortex of rats 7 and dogs s, and attempted to identify this binding protein. Glomeruli and tubular segments were isolated by microdissection. Great care was taken to prevent the detachment of afferent arterioles from the glomeruli and to avoid contamination of the glomeruli with tubular segments. The isolated glomeruli contained large quantities of renin, while in the isolated tubular segments renin activity was hardly detectable. Isolated glomeruli and tubular segments were separately pooled.
TIPS - September 1983
D
|
+ SH
SH
SH ~
Itlgh tool. ~ mnln Tetrathionate
if the renin-binding protein does exist in the proximal tubules, it may play a role in the process of reabsorption of the renin. Presumably, this protein is a carrier of renin and the reabsorbed renin is converted into a high mol. wt form to be further transported, or to be metabolized in the tubular cells.
MOI wt 40 000
Fig. 1. Schematw representation o f possible mode o f mol. wt conversion o f renin. Low tool. wt (40 000) renm binds to the renm-binding protein ( R B P) to form the high tool. wt (60 000) renin. The renin-bmding protein in renal cortwal tubular cells has a strong affinity for and ts bound to, renm A sulflzydryl-activated enzyme (SH-enzyme), however, cleaves the high tool. wt renin, thereby liberatmg the low moL wt renm. The bmdmg reaction proceeds in the absence or inacttvation o f this enzyme
Approximately 3 mg (wet weight) of the respective areas of the nephron were obtained from a single dissection. Since the method provided only minute quantities of the sample, the mol. wt of renin was estimated, using high performance liquid chromatography (HPLC) with 2 coupled TSK G3000 SW columns 7. Extracts of the glomeruli were examined for the presence of renin-binding protein. The extract did contain renin, therefore, if it also contained the renin-binding protein, the endogenous renin would be bound to form the high mol. wt renin. However, the extract of isolated glomeruli, in the presence of sodium tetrathionate, showed a single peak of renin activity on the chromatogram and the retention time of the peak position corresponded to that of the low tool. wt renin. This result suggested a lack of renin-binding protein in the glomeruli. In contrast, extracts of the isolated tubular segments were found to contain the renin-binding protein. When the low mol. wt renin was mixed with the extract of isolated tubular segments, in the presence of sodium tetrathionate, and the mixture was subjected to HPLC, there was a peak of renin activity at an earlier retention time than that of the low mol. wt renin. The mol. wt was estimated to be 60 0007'8. Reninbinding protein is thus considered to be present in the tubular, but not in the juxtaglomerular, cells. The question of the mode of action of renin-binding protein in the renal cortical tubules remains. Recent studies indicate that renin is filtered through glomerular capillaries and is reabsorbed by the proximal tubular cells. We have recently found that i.v. administered ~25I-labelled mouse submaxillary gland renin is predominantly concentrated in the mouse kidney. Microscopic autoradiography reveals that this labelled renin is incorporated into the proximal tubular cells, suggesting reabsorption 9. Therefore,
Possible enzymatic reaction involved in the mol. wt conversion of renin The mol. wt conversion of renin in the kidney involves oxidation of tissue sulfhydryl groups. In homogenates of the renal cortex, the high tool. wt renin can be formed in the presence of the sulfhydryl group oxidizing agent sodium tetrathionate, and can be re-converted into the low mol. wt renin by dithiothreitol2. Thus, the 2 types of renin seem to be inter-convertible by oxidation or reduction of the sulfhydryl groups. However, the sulfhydryl alkylating agent N-ethylmaleimide is also effective in the formation of the high mol. wt renin and the coupling of renin and renin-binding protein is not considered to be effected by disulfide bonds. Indeed, renin remains reactive with renin-binding protein even after it has been pre-treated with sulfhydryl group blocking agents, e.g. 5,5'-dithiobis(2-nitro benzoic acid) and N-ethylmaleimide. To assess the role of sulfhydryl groups in the conversion system, we examined, and demonstrated, the possible contribution of an enzyme-like substance which is sensitive to sulfhydryl oxidation in the conversion from the high mol. wt renin to the low mol. wt renin. The former was prepared by the addition of potassium tetrathionate to a renal cortical extract from dogs, followed by dialysis to remove the excess potassium tetrathionate. Two extracts from different sources were then prepared: one from renal cortex (renin was eliminated using pepstatin-aminohexylagarose affinity chromatography) and the other from the renal medulla. When the high mol. wt renin was mixed with the extract of renal cortex and incubated at 37°C for 15 min, this renin was converted into the low mol. wt form. However, there was no evidence of conversion in the extract from the renal medulla. When the extract from the renal cortex was pre-treated with perchloric acid or potassium tetrathionate, the mol. wt of the renin remained unchanged. All these findings taken together suggested that, in the kidney, the conversion from high mol. wt to low mol. wt renin may be catalysed by a sulfhydryl-activated enzyme-like substance 1°. The possible mode of mol. wt conversion of renin is shown in Fig. 1. Since the renin-binding protein is present in tubular cells, it may be that the conversion plays a role in the mechanism of reabsorption of
TIPS - September 1 983
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renin from the proximal tubules. Whether the renin-binding protein and a 'molecular weight converting' enzyme exist in the same tubular cells where renin is reabsorbed, is the subject of continuing studies.
8 Takaofi, K., lwao, H , Ikemo~o, F. and Yamamoto, K (1982) CIm Exp. Hypertens. A4, 2097-2105 9 Iwao, H , Nakamura, N , Ikernoto, F. and
Yamamoto, K J. H~tochem Cytochem. (in press) 10 Ikemoto, F , Takaon, K , iwao, H and Yamamo~o, K (1982)C/in Sct. 62, 157-162
Kenliro Yamamoto M.D. has been Professor and Chairman of the Department of Pharmacology, Osaka City University Medtcal School since 1972. From 1960 he has concentrated on research of renm--angiotensin systems.
Fumlhdco lkemoto Ph D. graduated from the UniversUy of Kobe, Faculty of Sc4ence. Following research on biochemistry of lenttcular cells, he moved to Osaka Cay UniversUy Medical School in 1977 and has been studying b~ochemical pharmacology of renm and angiotensins. He ts currently Associate Professor
Acknowledgement We would like to thank M. Ohara for helping us prepare this article.
Reading list 1 Boyd, G. W. (1974) Ctrc. Res. 35,426--438 2 Funakawa, S., Funae, Y. and Yamamoto, K (1978) Biochem. J. 176, 977-981 3 Kawamura, M., lkemoto, F., Funakawa, S. and Yamamoto, K. (1979)Clin Sci. 57,345-350 4 Yamamoto, K., Ikemoto, F , Kawamura, M. and Takaon, K. (1980) Chn. Sct 59 (Suppl. 6),
25s---27s 5 Takaori, K., lkemoto, F. and Yamamoto, K. (1981)Clin. Exp. Hypertens.3,991-1000 6 lwao, H., Minarm,T., Ikemoto,F., Takaon, K., Nakamura, N. and Yamamoto, K. (1982) Btochem. Btophys. Res. Commun. 106, 933--939 7 lkemoto, F., Takaon, K., iwao, H. and Yamamoto,K. (1982)LifeSci. 31, 1011-1016 I
II
Pharmacological investigations in space medicine V. S. Shashkov Kosmicheskaya biologiya i A viakosraicheskaya Meditzina, Mezhdunarodnaya Kmga, Moscow G-200,
USSR. The exploration of the Universe, which started with the flight of Yu. A. Gagarin, has brought an awareness of the importance of assuring spacecrews' safety. The main aspect of this problem is the development of pharmacological methods of prevention and therapy o f the physiological changes occurring during weightlessness and the readaptation period. l'aarmacological supportof spoceflights Spaceflight influences a number of the physiological indices of the living organism. Functional changes in the cardiovascular system occur, together with a complex of symptoms similar to motion sickness, plus disturbances in fluid and electrolyte balance and the mineral saturation of bone tissue, anaemia, deterioration of immunological activity, reduction in muscle weight, etc. All these symptoms are reversible1. The effects of weightlessness are multiform and the pathogenic causes of some of them require further investiga-" tion and definition. The use of medication to prevent and cure the unwanted effects of spaceflight and subsequent re,adaptation has been the subject of wide discussion in the scientific
literature2"s'4. It should first be noted that drugs traditionally prescribed in response to various pathological changes cannot always prevent or cure the peculiar conditions occurring as a result of spaceflight. Spaceflight safety must be considered at several stages: the training period, launch and obtaining orbit, free spaceflight, prelanding and landing, and readaptation after return to the earth s . Each stage involves pharmacological support of a different kind. In the pre-flight period, the type and amount of medical substances which need to be carried are determined on the basis of likely functional disturbances and their prognosis, the number of the spacecrew members, etc. In this period idiosyncratic reactions to drugs which may produce toxic
effects are investigated. The prospective astronauts study drug administration, siteof-action, contra-indications, and drug interactions. Reactions of the body to medical substances under simulated spaceflight conditions, with particular reference to weightlessness are also studied. Modelling of most spaceflight factors (radiation, acceleration, altered gas environment, etc. ) at all levels and ranges of their effects can be accomplished in ground-based experiments. The main goal is the protection of the organism against potentially pathogenic agents. There is a danger, during long-term spaceflights, of the development of stable forms of micro-organisms, super-infection and reactions to drugs used for prevention and therapy (e.g. antibiotics). These dangets are increased by the decrease in general reactivity of the patient. Prophylactic measures are planned which take into account the individual characteristics of astronauts' microflora. Problems with the pharmacological support of launch and landing of spacecrews, particularly with respect to overload and vibration and including the biological background of gravitational physiology, are fully described by Smith ~, and by Vasilyev and Kotovskaya ~ in joint Soviet-American review articles. Physiological investigations in groundbased experiments and during flights on the orbital stations 'Salyut' and 'Skylab' have allowed us to obtain much information on man's reaction to long-term spaceflight and the post-flight period which has helped us to understand complexes of symptoms that will need prevention and therapy.
1983ElsevtefSctet~ctI~MIshersB V , Amsterdam 0165- 6147/83/$01t~O