400
TiPS-
proton NMR studies of biological samples. In all such s t u d i e s selfshielding gradient coils are n e e d e d in order that data ~ccumulation can be o b t a i n e d in short t i m e p e r i o d s (allowing short delay times a n d spin echo experiments). These s h i e l d e d g r a d i e n t coils avoid the effect of the g r a d i e n t s w i t c h i n g on the m a g n e t itself, w h i c h produces artifactual e d d y currents. With such g r a d i e n t coils, effective biological data can be o b t a i n e d w i t h proton NMR.
[]
[]
[]
Cost is often a major cons,~deration in d e t e r m i n i n g the availability of e x p e n s i v e i n s t r u m e n t s . Pharmaceutical compan:'es m a y soon see NMR as a cost-effective t e c h n i q u e , because it reduces the n u m b e r s of animals that n e e d to be s t u d i e d to d e t e r m i n e the metabolic effects of a drug. The ability to study cells g r o w n in culture in a ' m o d e l turnoff could also h a v e significant effects on the e x p e n s e s r e q u i r e d for d r u g research. Fig. 3. Specially built wide-bore probe for studying tumor metabolism by NMR. The subcutaneous tumor is placed inside the solenoidal coil.
ance imaging. The powerful combination of i m a g i n g and spectroscopy is currently an area of active research, notably in the development of localized proton spectroscopy m e t h o d s using shielded gradient coils 15. However, these in v i v o i n s t r u m e n t s are q u i t e different from the high-resolution, narrov,--bore i n s t r u m e n t s used for chemical analysis; they may have radiofrequencies of 200 MHz, compared w i t h 500 MHz for the high-resolution machines. There is an important niche b e t w e e n these two extremes (say 400 MHz with an i n t e r m e d i a t e - b o r e magnet) that is optimal for pharmaceutical applications. The a d v e n t of m i c r o - i m a g i n g accessories on vertical magnets of high-resolution NMR systems should give an i m p e t u s for the more general application of these methods. Firstly, the i m a g i n g should allow the region b e i n g studied spectroscopically to be viewed; secondly, the presence of the gradient coils should i m p r o v e both spectroscopic localization and resolution I", and allow effective water signal suppression 17 in
OFER KAPLANAND JACK S. COHEN Pharmacology Department, Georgetown University Medical Center, Washington DC 20007, USA.
. . .L. . . . E
T r.......... T E~ R
$......} 1
O c t o b e r 1990 [Vol. 11]
References I Cohen, J. S., Lyon, R. C. and Daly, P. F. (1988) Methods Enzymol. 48, 435-438 2 Cohen, J. S. et aL (1986) Cancer Res. 46, 4087-4090 3 Lyon, R. C., Cohen, J. S., Faustino, P. J., Megnin, F. and Myers, C. E. (1988) Cancer Res. 48, 870--877 4 Kaplan, O. et al. (1990) Cancer Res. 50, 544-551 5 Jaroszweski, J. W., Kaplan, O. and Cohen, I. S. Cancer Res. (in press) 6 Neeman, M. and Degani, H. (1986) Proc. Natl Acad. Sci. USA 86, 5585--5589 7 Kaplan, O. et al. ]. Biol. Chem. (in press) 8 Ugurbil, K. et al. (1981) Proc. Nail Acad. Sci. USA 78, 4843--4847 9 Shankar, N. K., Moress, E. A., Chatham, J. C. and Barker, P. B. (1990) NMR Biomed. 3, 23-26 10 Daly, P. F. et al. (1990) Cancer Res. 50, 552-557 11 Ugurbil, K. et al. (1987) Ann. NY Acad. Sci. 508, 265-286 i2 Ng, T. C., Evanochko, W. T. and G!ickson, J. D. (1982)]. Magn. Resort. 49, 526-529 13 Lyon, R. '~--,, c- Tschudin, R. G., Dalv, P. F. and Cohen, J.S. (1988) Magn. Resort. Med. 6, 1-14 14 Navon, G., Lyon, R. C., Kaplan, O. and Cohen, J. S. (1989) FEBS Lett. 247, 86-90 15 van Zijl, P. C. M., Mooned, C. W. T., Alger, J. R., Cohen, J. S. and Chesnick, S. A. (1989) Magn. Reso,. Med. 10, 256-265 16 Moonen, C. W. T., von Kienlin, M., van Zijl, P. C. M., Daly, P. and Wolf, G. (1989) NMR Biomed. 2, 201-208 17 van Zijl, P. C. M. and Moonen, C. W. T. (1990) ]. Magn. Resort. 87, 18-25
~. . . . -. . . ~......}........~..........4....... 1 ~.........~i...........i....... !cZ~ 1I.
I............. 4 !............;.....
Receptor-independent action of bradykinin
Direct activation of G proteins In a recent article ( T I P S , September 1990) 1, Mousli et al. o u t l i n e d the e v i d e n c e that substance P a n d o t h e r basic p e p t i d e s i n c l u d i n g b r a d y k i n i n 2-5 stimulate release of h i s t a m i n e from mast cells by interacting directiy w i t h G proteins. We w o u l d like to p o i n t out that b r a d y k i n i n has at least o n e m o r e effect that appears to be receptor i n d e p e n d e n t . Contrac-
tion of isolated g u i n e a - p i g trachea by b r a d y k i n i n is m e d i a t e d in part by m e t a b o l i t e s of the a r a c h i d o n i c acid cascade. In this case, involvem e n t of B2 rcceptors can be ruled out b e c a u s e B2 receptor antagonists not only fail to block the r e s p o n s e , but also contract guineapig trachea ° . Mousli et al. p r o p o s e that the basic character of substance P a n d
TABLE I. Partial amino acid sequences of third cytoplasmic loop of membrane receptors Receptor Sequence Ref. ~2-Adrenoceptor -Val- Ala -Lys-Arg-Thr -Thr -Lys-Asn-Leu10 1~2-Adrenoceptor -Gly- Leu-Arg-Ar§-Ser -Ser -Lys-Phe-Cys9 NK1 -Ala- Lys -Arg-Lys-Val -Val -Lys-Met-Met11 NK2 -Ala- Lys -Lys-Lys-Phe-Val -Lys-Ala -Met12 NK3 -Ala- Lys -Arg-Lys-Val -Val -Lys-Met-Mett3 5-HT1A -Arg-Glu -Arg-Lys-Thr -Val -Lys-Thr -Leu14 TSH -Ala- Met-Arg-Ly¢-Ala -lie -Lys-Thr -Asp15
TiPS - October 1990 [Vol. 11]
401 other peptides allows them to penetrate deeply into the plasma membrane, as predicted by Schwyzer's theory of insertion of a m p h i p h i l i c peptides into membranes 7,8. Is it also their basic character that allows these peptides to interact directly with G proteins? Comparisons of receptor amino acid sequences (Table I) suggest that this may indeed be the case. According to Lefkowitz et al. 9, "Interaction of the receptor molecule with G protein appears to be mediated by cytoplasmic regions of the receptor protein and e~pecially those functions of the third cytoplasmic loop which lie in close apposition to the plasma membrane'. In addition to the receptors shown in Table I, four h u m a n muscarinic receptors have been found to have
a cluster of three or four basic a m i n o acids in the third cytoplasmic loop 16. DOMENICO REGOLI A N D FRANCOIS NANTEL
Departnlent of Pharmacology, University of Sherbrooke, Sherbrooke [1H 5N4, Q1wbec, Canada.
References 1 Mousli, M., Bueb, J-L., Bronner, C., Rouot, B. and Landry, Y. (1990) Trends Pharnlacol. Sci. 11, 358-362 2 Devillier, P., Renoux, M., Giroud, J-P. and Regoli, D. (1985) Eur. J. Pharnlacoi. 117, 89-96 3 Devillier, P., Renoux, M., Drapeau, G. and Regoli, D. (1988) Eur. J. Pharnlacol. 149, 137-140 4 DeviUier, P., Drapeau, G., Renoux, M. and Regoli, D. (1989) Eur. J. Pharntacoi. 168, 53--60 5 Bueb, J-L., Mousli, M., Landry, Y. and
Bronner, C. (1990) Agents Actiot:s 30, 98-101 6 Regoli, D., Rhaleb, N. E., Dion, S. and Drapeau, G. (1990) Trends Pharmaco!. Sci. 11, 156-161 7 Schwyzer, R. (1986) Biochenzistry 25, 4281-4296 8 Schwyzer, R. (1987) EMBO J. 6, 2255-2259 9 Lefkowitz, R. J., Kobilka, B. K. and Caron, M. G. (1989) Biochem. Pharnlacoi. 38, 2941-2948 10 Kobilka, B. et al. (1987) Science 238, 650--656 11 Yokota, v. ef el (~989) I. Biol. Chem. 264, 17649-17653 12 Masu, Y. et al. (1987) Nature 329, 836-838 13 Shigemoto, R., Yokota, Y., Tsuchida, K. and Nakanishi, S. (1989) J. Biol. Chenz. 262, 623-628 14 Fargin, A. e t a l . (1988) Nature 335, 358--360 15 Libert, F. et ai. (1989) Biochem. Biophys. Res. Conlmun. 165, 1250-1255 16 Peraita, E. G. et al. (1988) Trends Pharmacol. Sci. 9 (Suppl.), 6-11
This and That: drug tolerance and great expectations IT IS S O M E T I M E S V A L U A B L E to see t h e w o r l d f r o m a n extraterrestrial p e r s p e c t i v e . The fiat g r o u n d w e w a l k o n is s e e n f r o m the m o o n to b e a globe, g e o m e t r i c a l l y m o r e c o m p l e x t h a n t h e fiate a r t h e r s u s p e c t e d . The w a y b e h a v i o r a l p s y c h o l o g i s t s v i e w d r u g tolerance can make us pharthe special cases of receptor downm a c o l o g i s t s see s u c h a p h e n regulation or depletion of eno m e n o n as h a v i n g as n o v e l a dogenous ligands. We do feel, howgeometry. ever, that despite our ignorance To pharmacologists, tolerance is tolerance is a strictly pharmaa displacement to the right of the cological p h e n o m e n o n . But supdose-response curve for a drug. pose the dose-response curve for Thus, a dose of 100-200 l-ng mora drug moved to the left or right phine to people not previously d e p e n d i n g on whether the test exposed to narcotics is likeiy to animal 'knew' it was being given cause respiratory depression and a drug? death. Morphine addicts, howPavlov's salivating dogs have ever, can take up to 4 g of the drug become icons of the 20th century, without suffering severe respirone of those milestones marking atory depression, because of the h u m a n progress in understanding tolerance they have acquired. In the i~-~terface of m i n d and matter. this case, there has been a rightPavlov considered that the giving ward shift of the dose-response of a drug comprises a conditioncurve of the order of 1.3 log units, ing trial, the circumstances in impressive to the layman but perwhich the drug is given constituthaps r:ot so impressive to the ing a conditioned stimulus that pharmacologist familiar with, for becomes associated with the example, the 20000-fold increase pharmacological effects of the in potency of isoprenaline in drug, which comprise the unconh u m a n s previously exposed to ditioned stimulus 2. His dogs salicorticosteroids (a shift of 4.3 log vated w h e n they heard a bell they units) l. To name a p h e n o m e n o n is had been conditioned to associate not necessarily to understand it, with food. Similarly, a dog was and it is doubtful if m a n y pharmaconditioned by being given a subcologists would claim to undercutaneous injection of apomorstand the mecbanism underlying p h i n e followed by the sounding of tolerance once they had exhausted a note of a definite pitch.
While the note was still sounding the drug began to take effect upon the dog: the animal grew restless, began to nloisten its lips with its tongue, secreted saliva and showed some disposition to vonlit. After the experimenter had reinforced the tone with aponlorphine several times, it was found that the SOltnd of the note alone sufficed to pro.tuce all the active symptoms of the drug. 2
In other words, in a conditioned animal, the presence of the circumstances associated with the giving of a drug is sufficient to elicit a "drug effect'. However, behavioral psychologists want both to keep their cake and eat it. It is now postulated that although the effect that Pavlov observed can occur, the reverse effect may also occur. In a conditioned animal, an anticipatory response can be produced that antagonizes the eff?cts of the drug; for example, hyperalgesia in response to opiate-induced analgesia. This anticipatory response is held to constitute tolerance 3,4. If all this sounds a little airy, the very attic or garret of a theory, through which the wind whistles ignorant of receptors and endogenous opiates, it is disturbing to find a row of great, dirty chimneys blocking the skyline.
(~1Iq~0,ElsevierSciencePublishersLid.(UK) 01o5- bl47190/$02.00
TiPS-
proton NMR studies of biological samples. In all such s t u d i e s selfshielding gradient coils are n e e d e d in order that data ~ccumulation can be o b t a i n e d in short t i m e p e r i o d s (allowing short delay times a n d spin echo experiments). These s h i e l d e d g r a d i e n t coils avoid the effect of the g r a d i e n t s w i t c h i n g on the m a g n e t itself, w h i c h produces artifactual e d d y currents. With such g r a d i e n t coils, effective biological data can be o b t a i n e d w i t h proton NMR.
[]
[]
[]
Cost is often a major cons,~deration in d e t e r m i n i n g the availability of e x p e n s i v e i n s t r u m e n t s . Pharmaceutical compan:'es m a y soon see NMR as a cost-effective t e c h n i q u e , because it reduces the n u m b e r s of animals that n e e d to be s t u d i e d to d e t e r m i n e the metabolic effects of a drug. The ability to study cells g r o w n in culture in a ' m o d e l turnoff could also h a v e significant effects on the e x p e n s e s r e q u i r e d for d r u g research. Fig. 3. Specially built wide-bore probe for studying tumor metabolism by NMR. The subcutaneous tumor is placed inside the solenoidal coil.
ance imaging. The powerful combination of i m a g i n g and spectroscopy is currently an area of active research, notably in the development of localized proton spectroscopy m e t h o d s using shielded gradient coils 15. However, these in v i v o i n s t r u m e n t s are q u i t e different from the high-resolution, narrov,--bore i n s t r u m e n t s used for chemical analysis; they may have radiofrequencies of 200 MHz, compared w i t h 500 MHz for the high-resolution machines. There is an important niche b e t w e e n these two extremes (say 400 MHz with an i n t e r m e d i a t e - b o r e magnet) that is optimal for pharmaceutical applications. The a d v e n t of m i c r o - i m a g i n g accessories on vertical magnets of high-resolution NMR systems should give an i m p e t u s for the more general application of these methods. Firstly, the i m a g i n g should allow the region b e i n g studied spectroscopically to be viewed; secondly, the presence of the gradient coils should i m p r o v e both spectroscopic localization and resolution I", and allow effective water signal suppression 17 in
OFER KAPLANAND JACK S. COHEN Pharmacology Department, Georgetown University Medical Center, Washington DC 20007, USA.
. . .L. . . . E
T r.......... T E~ R
$......} 1
O c t o b e r 1990 [Vol. 11]
References I Cohen, J. S., Lyon, R. C. and Daly, P. F. (1988) Methods Enzymol. 48, 435-438 2 Cohen, J. S. et aL (1986) Cancer Res. 46, 4087-4090 3 Lyon, R. C., Cohen, J. S., Faustino, P. J., Megnin, F. and Myers, C. E. (1988) Cancer Res. 48, 870--877 4 Kaplan, O. et al. (1990) Cancer Res. 50, 544-551 5 Jaroszweski, J. W., Kaplan, O. and Cohen, I. S. Cancer Res. (in press) 6 Neeman, M. and Degani, H. (1986) Proc. Natl Acad. Sci. USA 86, 5585--5589 7 Kaplan, O. et al. ]. Biol. Chem. (in press) 8 Ugurbil, K. et al. (1981) Proc. Nail Acad. Sci. USA 78, 4843--4847 9 Shankar, N. K., Moress, E. A., Chatham, J. C. and Barker, P. B. (1990) NMR Biomed. 3, 23-26 10 Daly, P. F. et al. (1990) Cancer Res. 50, 552-557 11 Ugurbil, K. et al. (1987) Ann. NY Acad. Sci. 508, 265-286 i2 Ng, T. C., Evanochko, W. T. and G!ickson, J. D. (1982)]. Magn. Resort. 49, 526-529 13 Lyon, R. '~--,, c- Tschudin, R. G., Dalv, P. F. and Cohen, J.S. (1988) Magn. Resort. Med. 6, 1-14 14 Navon, G., Lyon, R. C., Kaplan, O. and Cohen, J. S. (1989) FEBS Lett. 247, 86-90 15 van Zijl, P. C. M., Mooned, C. W. T., Alger, J. R., Cohen, J. S. and Chesnick, S. A. (1989) Magn. Reso,. Med. 10, 256-265 16 Moonen, C. W. T., von Kienlin, M., van Zijl, P. C. M., Daly, P. and Wolf, G. (1989) NMR Biomed. 2, 201-208 17 van Zijl, P. C. M. and Moonen, C. W. T. (1990) ]. Magn. Resort. 87, 18-25
~. . . . -. . . ~......}........~..........4....... 1 ~.........~i...........i....... !cZ~ 1I.
I............. 4 !............;.....
Receptor-independent action of bradykinin
Direct activation of G proteins In a recent article ( T I P S , September 1990) 1, Mousli et al. o u t l i n e d the e v i d e n c e that substance P a n d o t h e r basic p e p t i d e s i n c l u d i n g b r a d y k i n i n 2-5 stimulate release of h i s t a m i n e from mast cells by interacting directiy w i t h G proteins. We w o u l d like to p o i n t out that b r a d y k i n i n has at least o n e m o r e effect that appears to be receptor i n d e p e n d e n t . Contrac-
tion of isolated g u i n e a - p i g trachea by b r a d y k i n i n is m e d i a t e d in part by m e t a b o l i t e s of the a r a c h i d o n i c acid cascade. In this case, involvem e n t of B2 rcceptors can be ruled out b e c a u s e B2 receptor antagonists not only fail to block the r e s p o n s e , but also contract guineapig trachea ° . Mousli et al. p r o p o s e that the basic character of substance P a n d
TABLE I. Partial amino acid sequences of third cytoplasmic loop of membrane receptors Receptor Sequence Ref. ~2-Adrenoceptor -Val- Ala -Lys-Arg-Thr -Thr -Lys-Asn-Leu10 1~2-Adrenoceptor -Gly- Leu-Arg-Ar§-Ser -Ser -Lys-Phe-Cys9 NK1 -Ala- Lys -Arg-Lys-Val -Val -Lys-Met-Met11 NK2 -Ala- Lys -Lys-Lys-Phe-Val -Lys-Ala -Met12 NK3 -Ala- Lys -Arg-Lys-Val -Val -Lys-Met-Mett3 5-HT1A -Arg-Glu -Arg-Lys-Thr -Val -Lys-Thr -Leu14 TSH -Ala- Met-Arg-Ly¢-Ala -lie -Lys-Thr -Asp15
TiPS - October 1990 [Vol. 11]
401 other peptides allows them to penetrate deeply into the plasma membrane, as predicted by Schwyzer's theory of insertion of a m p h i p h i l i c peptides into membranes 7,8. Is it also their basic character that allows these peptides to interact directly with G proteins? Comparisons of receptor amino acid sequences (Table I) suggest that this may indeed be the case. According to Lefkowitz et al. 9, "Interaction of the receptor molecule with G protein appears to be mediated by cytoplasmic regions of the receptor protein and e~pecially those functions of the third cytoplasmic loop which lie in close apposition to the plasma membrane'. In addition to the receptors shown in Table I, four h u m a n muscarinic receptors have been found to have
a cluster of three or four basic a m i n o acids in the third cytoplasmic loop 16. DOMENICO REGOLI A N D FRANCOIS NANTEL
Departnlent of Pharmacology, University of Sherbrooke, Sherbrooke [1H 5N4, Q1wbec, Canada.
References 1 Mousli, M., Bueb, J-L., Bronner, C., Rouot, B. and Landry, Y. (1990) Trends Pharnlacol. Sci. 11, 358-362 2 Devillier, P., Renoux, M., Giroud, J-P. and Regoli, D. (1985) Eur. J. Pharnlacoi. 117, 89-96 3 Devillier, P., Renoux, M., Drapeau, G. and Regoli, D. (1988) Eur. J. Pharnlacol. 149, 137-140 4 DeviUier, P., Drapeau, G., Renoux, M. and Regoli, D. (1989) Eur. J. Pharntacoi. 168, 53--60 5 Bueb, J-L., Mousli, M., Landry, Y. and
Bronner, C. (1990) Agents Actiot:s 30, 98-101 6 Regoli, D., Rhaleb, N. E., Dion, S. and Drapeau, G. (1990) Trends Pharmaco!. Sci. 11, 156-161 7 Schwyzer, R. (1986) Biochenzistry 25, 4281-4296 8 Schwyzer, R. (1987) EMBO J. 6, 2255-2259 9 Lefkowitz, R. J., Kobilka, B. K. and Caron, M. G. (1989) Biochem. Pharnlacoi. 38, 2941-2948 10 Kobilka, B. et al. (1987) Science 238, 650--656 11 Yokota, v. ef el (~989) I. Biol. Chem. 264, 17649-17653 12 Masu, Y. et al. (1987) Nature 329, 836-838 13 Shigemoto, R., Yokota, Y., Tsuchida, K. and Nakanishi, S. (1989) J. Biol. Chenz. 262, 623-628 14 Fargin, A. e t a l . (1988) Nature 335, 358--360 15 Libert, F. et ai. (1989) Biochem. Biophys. Res. Conlmun. 165, 1250-1255 16 Peraita, E. G. et al. (1988) Trends Pharmacol. Sci. 9 (Suppl.), 6-11
This and That: drug tolerance and great expectations IT IS S O M E T I M E S V A L U A B L E to see t h e w o r l d f r o m a n extraterrestrial p e r s p e c t i v e . The fiat g r o u n d w e w a l k o n is s e e n f r o m the m o o n to b e a globe, g e o m e t r i c a l l y m o r e c o m p l e x t h a n t h e fiate a r t h e r s u s p e c t e d . The w a y b e h a v i o r a l p s y c h o l o g i s t s v i e w d r u g tolerance can make us pharthe special cases of receptor downm a c o l o g i s t s see s u c h a p h e n regulation or depletion of eno m e n o n as h a v i n g as n o v e l a dogenous ligands. We do feel, howgeometry. ever, that despite our ignorance To pharmacologists, tolerance is tolerance is a strictly pharmaa displacement to the right of the cological p h e n o m e n o n . But supdose-response curve for a drug. pose the dose-response curve for Thus, a dose of 100-200 l-ng mora drug moved to the left or right phine to people not previously d e p e n d i n g on whether the test exposed to narcotics is likeiy to animal 'knew' it was being given cause respiratory depression and a drug? death. Morphine addicts, howPavlov's salivating dogs have ever, can take up to 4 g of the drug become icons of the 20th century, without suffering severe respirone of those milestones marking atory depression, because of the h u m a n progress in understanding tolerance they have acquired. In the i~-~terface of m i n d and matter. this case, there has been a rightPavlov considered that the giving ward shift of the dose-response of a drug comprises a conditioncurve of the order of 1.3 log units, ing trial, the circumstances in impressive to the layman but perwhich the drug is given constituthaps r:ot so impressive to the ing a conditioned stimulus that pharmacologist familiar with, for becomes associated with the example, the 20000-fold increase pharmacological effects of the in potency of isoprenaline in drug, which comprise the unconh u m a n s previously exposed to ditioned stimulus 2. His dogs salicorticosteroids (a shift of 4.3 log vated w h e n they heard a bell they units) l. To name a p h e n o m e n o n is had been conditioned to associate not necessarily to understand it, with food. Similarly, a dog was and it is doubtful if m a n y pharmaconditioned by being given a subcologists would claim to undercutaneous injection of apomorstand the mecbanism underlying p h i n e followed by the sounding of tolerance once they had exhausted a note of a definite pitch.
While the note was still sounding the drug began to take effect upon the dog: the animal grew restless, began to nloisten its lips with its tongue, secreted saliva and showed some disposition to vonlit. After the experimenter had reinforced the tone with aponlorphine several times, it was found that the SOltnd of the note alone sufficed to pro.tuce all the active symptoms of the drug. 2
In other words, in a conditioned animal, the presence of the circumstances associated with the giving of a drug is sufficient to elicit a "drug effect'. However, behavioral psychologists want both to keep their cake and eat it. It is now postulated that although the effect that Pavlov observed can occur, the reverse effect may also occur. In a conditioned animal, an anticipatory response can be produced that antagonizes the eff?cts of the drug; for example, hyperalgesia in response to opiate-induced analgesia. This anticipatory response is held to constitute tolerance 3,4. If all this sounds a little airy, the very attic or garret of a theory, through which the wind whistles ignorant of receptors and endogenous opiates, it is disturbing to find a row of great, dirty chimneys blocking the skyline.
(~1Iq~0,ElsevierSciencePublishersLid.(UK) 01o5- bl47190/$02.00
