108

6-receptors is increased by some 10-30 fold (depending on chain length), relative to the parent tetrapeptide, whereas activity at /z-receptors is relatively unchanged. Cor- respondingly, selectivity is lost and a small inversion in selectivity is seen with the higher members of the series (DTEn, n = 8, 10). However, DTE12 shows a 30-fold loss of activity at /z-receptors to give a 8//z selectivity ratio of 91. Thus, in the DTE~ series ~//z selectivity relative to the parent monomer DAPEA is lost and with DTE~2 a dramatic inversion in selectivity occurs. Relative to the monovalent ligand DTEx2 shows a 1 000-fold selectivity for the ~-receptor.

The data for the dimeric pentapeptide and tetrapeptide enkephalin series both suggest an arrangement of ~-mceptors, either pre-existing or induced, which can be bridged by bivalent ligands of appropriate geometry. /z-Receptors do not apparently share this cosy arrangement.

It is therefore of interest that similar con- clusions are apparent for entirely different opiate ligands, dimeric fl-naltrexamine derivatives s [ HI, n = 2; x = CH2CI-hOCt'hCH2OCH2CH2; x = (CI-hCH2OCH2CH2OCHzCH2) 20]. These ligands were assayed in guinea-pig ileum and mouse vas deferens against morphine, ethylketazocine and DADLE as/z, K and selective ligands respectively. Neither /~-naltrexamine dimer exhibited signifi- cantly enhanced activity against /J-responses. However, enhanced activity, relative to monomer, was observed against K- and ~-responses with dimers containing the short and long spanning groups respec- tively.

At least for these series of divalent ligands two heads are not only better than one but are also more selective. Extension of this principle to other receptors is of obvious interest.

T I P S - M a r c h 1 9 8 3

Reading list 1 De Lean, A., Munson, P. J. and Rodbard, D.

(1979) Mol. Pharmacol. 15, 60-70 2 Mmton, A. J (1981) Mol. Pharmacol. 19, 1-14 3 Straganian, R. P., Hook, W A and Levine, B. B.

(1975)Immunochem~stry 12, 149-158 4 Dembo, M. andGoldstem, B. (1978)J lmmunoL

121,345-353 5 Coy, D H., Kastm, A J., Walker, M. J.,

McGivern, R. F and Sandman, C. A. (1978) Btochem. Btophys Res Comm. 83,977-983

6 Shunohigashi, Y , Costa, T., Matsuura, S., Chen, H. C. and Rodbard, D. (1982) Mol Pharm. 21, 558-563

7 Shtmohigashi, Y., Costa, T., Chen, H. C. and Rodbard, D. (1982) Nature (London) 297, 333-335

8 Erez, M., Takemori, A. E. and Portoghese, P. S (1982)J. Med. Chem. 25,847~49

9 Hazum, E., Chang, K J. and Cuatrecasas, P (1980) Proc Natl Acad. Sct. U.S.A. 77, 3038-3041

D J. TR1GGLE

Department of Biochemtcal Pharmacology, State Unwersity of New York, Buffalo, NY 14260, USA.

Some persisting myths about fi-endorphin and related substances Despite vigorous research over the past few years, there are a few myths about the endorphins and enkephalins that still persist and are misleading. One is that they are very rapidly attacked by aminopeptidases in the blood or tissues, and are thus immediately converted into inactive des- TyP compounds. This statement is wrong on two counts. First, des-TyrO-Met ~- enkephalin 2,'' and the des-TyP-endorphins 7 are not inactive compounds; they do not act upon opiate receptors, but they do have strong behavioral effects. The effects of des-TyP-Met~-enkephalin may be ex- plained, like some of the naloxone- reversible effects of MeP- or Leu 5- enkephalin, morphine, ACTH or adrenaline, by a release of endogenous hypothalamic /3-endorphin 2. Secondly, Met~-enkephalin 3 and fl-endorphin 5. ~' circu- late in the blood; in the case of the latter, 50% of the radioactivity measured in the plasma of rats 45 min after the injection of tritiated human fi-endorphin arises from intact human fl-endorphin 5. It is true that the enkephalins and endorphins may read- ily lose their N-terminal tyrosine residue in contact with tissue homogenates, but this does not imply that aminopeptidases are normally running loose within intact tissues - which would certainly make life very dif- ficult for the tissues. Instead, it has been proposed that, in the case of fi-endorphin, the N-terminal cleavage may be the start of a rather selective metabolic route producing behaviorally active derivatives, such as des-Tyrl-3,-endorphin (see Ref. 7).

Another myth is that endorphins and enkephalins do not cross the blood-brain barrier. This myth is held both by adherents to the Oldendorf method of measuring blood-brain barrier restriction to the pas-

• sage of substances, and by people who take that method for granted without ever having studied it. The Oldendorf method is based on the bolus injection of a labeled substance into the common carotid artery and measur- ing radioactivity in the ipsilateral forebrain of animals decapitated 5 s after injection, as compared with the radioactivity resulting from simultaneously injected thiourea and EDTA*. Clearly, this method can only detect the permeability to substances that cross the barrier either by free diffusion or by very rapid transport, and will not detect the passage of such sub- stances as fl-endorphin, whose plasma dis- tribution time after i.v. injection is 2 min in the rat and 5 min in the rabbit, and whose plasma clearance is 1 to 8 h 5. In such cases, times considerably longer than 5 s should be considered. Some researchers in the opioid field should be reminded that by the Oldendorf method one is led to the conclu- sion that morphine does not enter the brain 4, a conclusion that most addicts would certainly dispute, It seems likely that such peptides as the enkephalins or endor- phins should cross the blood-brain barrier by saturable carrier systems similar to those that have been described in the gut (see Ref. 4), which is a process that takes a certain amount of time. Indeed, Houghten e t al. ~

carefully measured the penetration of

human fi-endorphin labeled with 3H on either Tyr 1 or Tyr 27, given i.v., into the CSF of rabbits. The substance penetrates rather slowly, reaching a peak at between 60 and 90 min; approximately 20% of the injected substance enters the CSF intact within 2 h of injection. Therefore, the numerous findings from several labora- tories on central effects of systemically injected/3-endorphin may certainly be attrib- uted to effects on the brain (see Refs 6, 7).

What happens to fl-endorphin after it enters into the brain is another matter. Within 2 h ofi.v, injection in which 20% of injected [~H]/3-endorphin enters the CSF, nearly all the radioactivity detectable in the brain hemispheres of rabbits or rats is attributable to free tyrosine residues 5. This suggests that fl-endorphin has a 'hit-and- run' effect on mostly periventricular struc- tures. The brain fl-endorphin system is in close proximity to the brain ventricles and many opiate-sensitive receptors also have a periventricular distributionL It is possible that fl-endorphin interacts with the recep- tors and is then immediately detyrosinated. It is also possible, however, that the method of detection of Houghten e t al. ~ was not suf- ficiently sensitive; they measured radio- activity in large samples of brain and there- fore probably diluted the more subtle changes occurring in restricted periven- tricular regions. The major inactive metab- olites of ~endorphin in the pituitary gland 8 and in some regions of the brain (hippo- campus, amygdala, colliculae, brain stem) 9 are a-N-acetylated compounds. It is not known whether des-tyrosinated/3-endorphin derivatives are major metabolites else- where, as the work of Houghten et al. 5 or of de Wied's group (see Ref. 7) may suggest.

I~l Elsevier Biomedical Press 1983 0165 - 6147/83/0000 - 0000/$01 00

TIPS - March 1 983

Reading list 1 Bloom, F. E and McGinty, J. F. (1981) in

Endogenous Pept~les and Learning and Mem- ory Processes (Martlnez, J. L Jr, Jensen, R. A., Messing, R B., Rigter. H. and McGaugh, J. L., eds), pp 199-230, Acadermc Press, New York

2 Carrasco, M A,DIas, R. D, Perry, M L. S , Wofchuk, S T , Souza, D. O. and Izqmerdo, I Psychoneuroendocrinology (in press)

3 Clement-Jones, U , Lowry, P J., Rees, L H. and Besser, G. M (1980) Nature (London) 283, 295-297

4 Corndorf, E M., Braun, L D , Crane, P. D and Oldendorf, W H (1978) Endocnnology 103, 1297-1303

5 Houghten, R A., Swarm, R. W and Ll, C H (1980) Proc. Natl Acad. Sci US.A 77, 4588--4591

6 lzqmerdo, I (1982) Trends Pharmacol Sct 3, 455--457

7 Kovhcs, G L and de Wled, D (1981) in Endogenous Pepades and Learning and Mem- ory Processes (Martmez, J L. Jr, Jensen, R A ,

Computer Club The world of pharmacology computing

109

Messmg, R B , Rlgter, H and McGaugh, J. L , eds), pp 231-247, Academic Press, New York

8 Weber, E,Evans, C J. andBarchas, J D (1981) Btochem Btophys. Res Commun. 103,982-989

9 Zakanan, S and Smyth, D. G (1982) Nature (London) 296,250-252

I V A N I Z Q U I E R D O

Departamento de Bioqufmica, lnstituto de Biocien- cias, UFRGS (centro), 90.000 Porto Alegre, RS, Brazd

Computer controlled tissue bath experiments: on-line control and data acquisition Programs have been written which permit the control of a number o f processes involved in the performance of experiments investigating the contractile responses of isolated portions o f rat vas deferens, and which also measure the twitch responses, and manipulate the 'size' of these responses to calculate certain parameters, such as the ratio of the size of successive responses.

memory usage. However, other sampling rates may be selected depending on the characteristics of the response being anal- ysed. Machine code routines are used to

Control A Biodata Microlink interface unit links

a CBM 8032 microcomputer with (1) a Grass $88 stimulator, (2) a motor-driven servomechanism which maintains a con- stant tension on the tissue except for the period of recording activity and (3) a motor driven syringe unit which delivers drug solu- tions to the tissue bath.

Data acquisition Twitch responses are monitored via a

strain gauge-amplifier and the analogue output is digitized through an 8-bit A-D converter which allows a resolution of 1:256 (approximately 0 .4%). At the same time, 'traditional' records may be kept via the chart recorder, or visualized via an oscil- loscope. The constant tension device gives the data acquisition system a stable base- line from which to operate as well as main- taining the tissue at a suitably constant ten- sion. The present system analyses both the magnitude of the developed tension as well as the time to reach the peak tension, but other parameters can also be measured. A sampling rate of 100 Hz has been found to be adequate for the analysis of the response of this particular muscle since it strikes a balance between resolution and economy of

capture data as well as to extract the desired parameters. Data is stored on disk, either from the start o f the experiment, or storage may commence at some predetermined time, for example when responses equili- brate (as calculated by sequential compari- son of responses until a preset level of simi- larity exists between consecutive responses). Hard copy is routinely obtained

I

i

i

T m ~

F~g. 1. Program logic flow diagram

Set uO pmc:~dutes

O~k T ~

S¢,o¢I S a m ~ ,-ate

[~tr, a Equt~tum

Number o! Select ~

El~v~*'t Blorae~..al Press 1983 0165 - 6147/83/~000 - 0(300/$01 00