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Leu-enkephalin binding to cultured human T lymphocytes
Cell Biology
international
Reports,
LEIJ-KNKEPHALIN BlNDINti Clara
Vol. 8, No. 2, February
1984
97
TO CULTURED HUMAN T LYMPHOCYTES.
M. Ausiello*
and L. tiiorgio
Roda
*
di Tipizzazione 'l'issutale e Problem1 CNR "lstituto "Laboratorio di Farmacologia, Italy; L'Aquila, di Tecnica b'isiologica, Ancona, Ancona, and Cattedra University, Via 0. Haimondi, 1 00173 Rome, Italy.
della Dialisi", University 01. 'ITor Vergata"
ABS'I'HACT Evidence is given for the existence of leu-enkephalin binding activity on the cell surface of a human lympnoid 'I' line. uur data structures which in all suggest that binding is due to receptor as is already known to be the case with likelyhood are lipoproteic, the receptors present in the mammalian brain. In spite OS these lymphocytes exhibit anomalous specificity as similarities, however, rar as the binding toward oploid alkaloids and their inhibitors is concerned.
'The concept o? the presence OS endogenous opioids has been advanced more than once since the late sixties (::lartin, 1967; Davis and LlJalsh , 197Uj. ln latter years the spec i TIC search for with considerable endo,gcnous opioids has been rewarded success identification, leading to the isolation structural and characterization of the smaller opioid peptides, leu and met-enkephalin (Hughes, 19,/b; 'I'erenius and :~;ahlstrom, lY'/5 ; Pasternak et al., 19/4j. Subsequently, quite a large - and perhaps even eccessively so - body oi endogenous substances related to them has been described. Furthermore, it has been possible to demonstrate the existence of binding activity of labellea opioids to brain homoqenates and to distinguish between speciric and non-speciIic binding on a stereospecificity basis. Mevertneless, tne guriiicatlon OI the membrane-bound receptors has proved to be quite aifficult, possibly on account OS the nature of the receptors itself (Lin and Simon, 19 /B; Pasternak and Snyder, 19'/4; Abood et 0309-I 651184/020097-10/$03.00/0
@ 1984 Academic
Press Inc. (London)
Ltd.
Cell Biology
98
International
Reports,
Vol. 8, No. 2, February
1984
800..
400-m
200..
I
1
I
2
3
cells
x to8
KIWJRE 1. tiinding ot‘ leu-enkephalin to Jurkat T cells. 6x10-4DP'M or' ('Hj leu-enkephalin were incubated (SO', 3'1°C) with the indicated number of cells. Solid squares indicate experimental points. The solid line indicates zhe least-squares interpolated curve.
al., 1977). Presently, a lipoproteic structure for the opioid receptors seems highly probable, and data consistent with this hypothesis have been reported by several groups (Lee and Smith, 1980; Eeites et al., 1979, Cherubini et al, 1903). To date opioid peptides have been shown to possess several different activities in addition to their hypotnesizcd role in pain Furthermore, it control. has been demonstrated that both enkephalins are p,,esent within the adrenal chromaffin medulla granule, and are thereby into released the blood stream (Schultzberg et al., 1970, Viveros et al, 1979). In addition, Clemente-Jones et al. (1930) demonstrated that met-enkephalin circulates in peripheral blood in quantities up to 50 pg/ml. On the basis of these latter data, it is not altogether surprising that in recent years evidence ilas been preacnted for the presence of opioid receptors on the surface of iympllocytes (liazum
Cell Biology
International
Reports,
Vol. 8, No. 2, February
a b M ?8 =: 23
1984
total mob
en&@mlin,
1.5
s : 5
1.0
a P
0.5
210
4.0
6.0
8.0
bound moles/ce(l x 10-14
FIGURE 2, panel a. Satxration binding curve OS leu-enkephalin to Jurkat T cells. 2,25x10 cells were incubated 130',35757uC) with she indicated amount of leu-enkephalin; to this 1x10 DPM or' ( H) labelled leu-enkephalin were added. Panel b: Scatchard plot from the data in panel a.
Cell Biology
100 et al., et al.,
1979; 1982).
!$ybran
et
International al.,
1979;
Reports,
Vol. 8, No. 2, February
plc Donough
et
al.,
1980;
1984 Gilman
show that The experiments hereunder reported erikephalin-binding structures are present even on the surface of Our evidence supports the cultured human T lymphoblastoid cells. with an external standard accessory (L'XB Produkter, Bromma, Sweden). The reagents used were of the following origin: proteinase ;< and phospholipase A * Calbiochem (San Diego CA., USA), tunicamycin: 2' Sigma (St. Louis, ilO, USA), naloxon: Endo Laboratories (Garden NY, USA), morphine: City, Angelini Medicinali (Ancona, Italy), leucine enkephalin: Serva (Heidelberg, DBR) and tyrosi1-3,5-31-:(N) 4 leu-enkephalin, specific activity 3x10 Ci/mole: The Radiochemical Centre (Amhersham, UK). All the other chemicals were of reagent grade and bought from local suppliers. RESULTS AND DISCUSSIOE To investigate the existence within the T lymphocyte menl'orane VJ of structures showing binding activity toward leu-enke halin, % 8 incubated (30' 37°C) a number 04f cells rangigg from 3x10 to 3x10 with a constant amount (6x10 DPX) of ( 1-I) leu-enkephalin in phosphate-saline buffer. Samples were analyzed as described and the resulting data interpolated between the experimental points with a least-square computer program. The resulting curve (Figure 1) shows that quite a good linear relationship (r=0.989) exists between cell number and bound enkephalin. In additi n, equilibrium experiments were performed. A fixed k3 number (2.25~10 ) of washed T cells - together $th an arnount of -9 2.5x10 leu-enkephalin ranging from moles - were 5x10 to lx105DPPI resuspenfqd in a 300 ul final volume of buffered saline. / (1.5x10 L moles) of labelled enkephalin were added to each tube and the mixture was incubated 30' at 3?cC. In figure 2, panel a, bound enkephalin is plotted against the total amount of enkephalin present in the incubation mixture. The plotted curve shows that the leu-enkephalin-T lymphocytes binding is saturable. To determine the kinetic parameters of the binding, the same data were analyzed and plotted according to Scatchard (1949), as shown in figure 2, panel b. From the plot in figure 2b it seems likely different binding sites - with that a population of different binding constants - exists on T cells surface, and this also appears to be the case with@-endorphin (Ilazum et al, 1979). However, from the linear portion of the Scatshard plot in figure 2b an apparent dissociation constant of 2.03x10 may be calculated on
Cell Biology
international
a per cell
basis.
Reports,
Vol. 8, No. 2, February
1984
101
demonstrate the existerice of stiructures IYXGUltS The &OVl: silowing receptor activity toward leu-en:cepllalin on tile surface of T ‘:Jiiether or no’; these structurc;s are CilN!iCaiiy cells. To ascertain ilypothesis L!mt both pilospholipids ant', protein are i:ecessary for 2e also deinoristrdx the existence of conspicuous binding activity. known oploid receptor. differences in regard to tile already
T cell iine (Artin et al., 1932) w~~s Jurkat human lymphoid Inc., maintained at 37OC in 5% CO in RPZU 1640 (Plow Laboratories with 10;: fetal call serum (710~) plus KcLean, Va, USA) su;lplementZcI 100 units/ml penicillin and 100 mg/r:il streptomycin. Binding assays were performed as follows: cultured cells, pfI 7.4, 0.9:1 :;aCl (phosphate-saline) washed in 15 rn:; Ila phosphate ih'ere counted by means of a StXliI3.I'd buffer, procedure a11d s resuspeildeci at a final concentratiorl of Z-1OxlC cells/ml. A volume Was then transferred in 250 PVC tubes. To ^,:I& "5 P1 ( F!)leu-enkephalin was added anti cells iilcu’dated were at 37‘C The incubation was (final volume 300 ul). stopped by the addition / of 3 ml of ice cold phosphate-saline buffer, the cells were pelletted (5' at 1000 x g) in a refrigerated centrifuge, resuspended in 3 ml of the same buffer alid centrifuged again. 21e pellet was then resuspended in 50G ul of 0.1 :j :;aOi-i and 450 ,ul of it were counted in a LXD Xini3et.a / scintiilation counter eq6Lpped similar to the ones described in the case of nervous tissue, we two of the opioid agonist inhibition experiments usin? performed for the and antagonist tha-t are known to compete with enkephalins - receptors. As shown in figure 3, naloxon same - or very similar -5 did not inhibit Leu-enkephalin bindinE up to 10 (solid line) a molar concentration of 3x10 . Similarly, inhibition except at a concentration OS ~~~~~~IZ" AiY' nZYc:uSJe tiowewer, the penultimate concentration moles/~X ul. 5x10 mof es of morphLne/YCC ul - caused approximately a assayea - 1x10 / lns$ad, no inhibition was caused by the next 5o:A Inhibition. 5x10 moles/ZOO ui. It is debatable however concentration, wnether the innihitory effect thu& determined can be considered meaniny,pl since itbis only detectable at a molar concentration or 3,3x10 , i.e. a 10 molar excess over enkephalin. me results of' these inhibition experiments seem to suggest that 'I' cell binding is due to structures which are not quite the same as those present in the nervous tissue. Since there is some
Cell Biology
102
International
Reports,
Vol. 8, No. 2, February
1984
knowledge - albeit poor - of' the chemical structure of the latter, we tried to partially characterize '1 lymphocyte receptors according to their chemical ‘I’0 obtain this information, we properties. treated the lymphocytes with different substances that are active on the protein primary structure, on the disulfide bonds of proteins, on phospholipids and on carbohydrates cell synthesis. binding
'Table 1 shows the by the following
lo+:
results of the action on the substances: mercaptoethanol,
lo-0
~8
10-7 total
leu-enkephalin tunicamycin,
lo-s mole8
FlGURE 3. lnhibition by morphine and leu-enkephalin-Jurkat cells inding. _152~olx,s, 11; yyeFabe;:%% !? incubated (30',37°C) with 1x10 DPM (7x10 leu-enkephalin plus the indicated amounts of both substances. Solid line and triangles indicate naloxon; dash-dotted line and circles indicate morphine. were incubated and proteinase i(. dasned cells phospho.lypase A under test in a 300 ul r'lnal volume (IL!,', :r';/"c) wUzh%the substance 01 phosphate-saline bui‘i'er, washed again and incub/ated (30' 37°C) volume with labelled leu-enkephalln. 'I'unicamycin in a 300 ul r'rnal / incuoation was .L’y nours under culture conciitlons. I( A and proteinase 1, both pnosphoiipase .4s silown in table of' the enkepnalln b;$ding to 1' cells. caused a market irthlbltlon even in the case of‘ 'L' lymphocytes, Mat, suggests TClj S result
-4 -s
-3
-2
-1
)
A2
l.36XlO-14
-13
5.o2x1o-14
1.17x10
1.44x10
0.8
0.08
1.12x10
1.36X10
bound
-13
-13
-14
-13
(moles)
Leu-enkephalin
PHOSPHOLYPASE
9.75x10-l4 1 .21X1o-13
7.94x10-l4
6.80X10-‘~
3.53X10
8
SO
(units/ml
concentration
Phospholypase
1x10 1x10
1x10
1x10
1x10
(moles/l)
)
K
in
A2
SIJBSTANCES
(moles)
Leu-enkephal
! mg/ml
K
DIFFERENT
PROTEISASE
OF
bound
i naso
Prote
EFFECT
Concentration
1:
I’AHLE
89
58 72
49
25
100
the
8
106
86
37
100
control
the
0.5
T-CELLS
-5
-3 -4
-2
-1
-13 -13
-13
-13
(moles)
1 .42X10
1
1.38X10
1.31x10
1.36x10
bound
-13
-13
-13
-13
(moles)
leu-enkephal
TUNICAMYCIN
~81x10-'~
1.93x10 1.95x10
1.43x10
1 .91x10-l4 3.63X10
bound
Leu-enkephalin
2 MERCAPTOETHANOL
BINDING
5
(ug/ml)
concentration
Tunicamycin
1x10
1x10 1x10
1x10
1x10
concentration
2-Mercaptoethanol
LEU-ENKEPHALIN
% of
control
% of
OS
in
95
101 102
7s
1
contra
102
104
96
100
the
the
% of
19
100
control
% of
2
2
G
6 b 2
.Q
$
8
B
Cell Biology
International
Reports,
Vol. 8, No. 2, February
1984
phospholipid are the protein and essential constituents of enkephalln receptor. On the contrary, no effect on binding activity has been detected after incubation with tunicamycin. This result suggesting no carbohydrate involvement in the receptor structure is also consistent with the data existing about opioid receptors in [Lee and Smith, nervous tissue 1980; Meites et al., 19'/9 ) . Finally, rather ambiguous results 'I' were given by incubating 2-mercaptoethanol. ln lymphocytes with this case, only -2 concentrations OS the reducing agent 01 10 1'4 and higher are in inhibiting binding activity. Since disulphide bonds effective to be relatively shielded, a nlgn in membrane proteins are likely concentration 01 reducing agent may actually be necessary to reduce the hand, 2-mercaptoethanol tnem effectivelly. On other indicated are likely to have a concentrations in the range non-specir.ic inhibitory effect. conclusion, tne results we obtained though only In the presence 01' structures with receptor preliminary - demonstrate activity toward leu-enkephalin on the surface of Jurkat '1 cells. 'The behavior OS these receptors is anomalous as far as opioid agonists and antagonists are concerned. Yet, these structures seem chemical characteristics known for brain to have the same gross and phospholipids - but no opioid receptors. 'I'hat is, both proteins carbohydrates - seem to be involved in the receptor structure. 'The existence of different kinds of receptors f‘or endogenous opioids in lymphoid cells has been so f'ar suggested by the results and by the present Of' Hazum et al (19'7Y) Wybran et al (19'7Y), seem to suggest the existence oi' a also study. 'I'hese r'indings closer and more direct connection between nervous and immune system Furthermore, this suggests that there than is commonly supposed. may exist new roles for opioid peptides, different rrom the ones that may be searched for. 'I'hese new roles may presently accepted, nervous activity on the mediators 01' the system's be like physiological iunctions of the immunological system, 1'or which a nervous control is not commonly recognized today.
out and partially supported by the work has been carried Fina1izzat.i "Chimica Fine e Secondaria" and "Controllo CYPI (Ital ian CouncJ.1 for Neoplastica" of the della Crescita Scl.entific HesearchJ. This
Progetti
Cell Biology
International
Reports,
Vol. 8, No. 2, February
105
1984
REFERENCES Salem, N., Macneil, Jr.M., Bloom, L. and Abood, 1) Abood, L.G., M.E. (1977) ENHANCEMENT OF OPIATE BlNDING BY VARIOUS MOLECULAR FORMS UF PHOSPHATIDYLSERINE AND INHIBITION BY OTHER UNSATURATED LIPID. Biochimica et Biophysics Acta, 468: 51-62
2) Cherubini, O., De Marco, V., Roscetti, ti., Possenti, R. and PEPTIDES WITH INTERACTION OF OPIOID VITRO Roda, L.G. IN FORMATION AND CHARACTERIZATION OF CUMPLEXES. PHOSPHCLIPIDS: International Journal of Peptides and Protein Research, in press September 1983 3) Clement-Jones, V., (1980) MET-ENKEPHALIN 295-297
Lowry, P.J., CIRCULATES
Rees, L.H., and Besser, IN HUMAN PLASMA. Nature
and Walsh, M.S. Davis, V.E. 4) A POSSlBLE BIOCHEMICAL ALKALOIDS: Science 167: 1005-1007.
G.M., 283:
(1970) ALCOHOL, AMINES, AND BASIS FOR ALCOHOL ADDICTION.
5) Gilman, S.C., Schwartz, J.M., Milner, R.J., Bloom, F.E., and -ENDORPHIN ENHANCES LYMPHUCYTE (1982) Feldman, J.D., b Academy of PROLIFERATIVE RESPONSES. Proceedings of the National Sciences. USA '/9: 4226-4230. and Cuatrecasas, Chang, K.S. 6) Hazum, E., NANOPIATE RECEPTOR FOH/3-ENDORPHINE. Science
P. (19*/g) SPECIFIC 205: 1033-1035
(1975) ISOLATION OF AN ENDOGENOUSCOMPOUNDFROM THE 7) Hughes, J., BHAlN WITH PHARMACULOGICAL PROPERTIES SIMILAR TO MORPHINE. Brain Research, 88: 295-308 8) Lee, N.M., and Smith, A.P., OPIATE RECEPTOR. Life Sciences,
(1980) A PROTEIN-LIPID 26: 1459-1464.
MODEL OF THE
\iY'/td) ~HtiSrkuLlPASE A 1NHIBITION OF 9J Lin, H.K. and Simon,E.S. OPlATE RECEPTOR BINDING CAN BE REVERSED BY ALBUMIN. Nature 271: 383-384 Giblett, E.H., Martin, P.J., and (1982) Hansen, J.A., 10) PHENOTYPlNG HUMAN LEUKEMlC 'I'-CELL LINES: ENZYME MARKERS, SUHE'ACE ANTltiENS, AND CYTOGENETICS. Immunogenetics 15: 385-398. Martin, W.R., 11) Reviews 19: 463-521 12) M.,
(1967)
OPIOID
McUonough, H.J., Madden, J.J., Gordon, D., kiokos, P., Kuehnle,
ANTAGONISTS. Falek, J-C.,
Pharmacological
A., Shafer, D.A., and Mendelson, J.,
Pline, (1980)
106
Cell Biology
International
Reports,
Vol. 8, No. 2, February
1984
ALTERATlON OF T AND NULL LYMPHUCYTE FHEUUENClES IN THE PEHlPHEHAL BLOOD OF HUMAN OPLATE ADDICTS: IN VlVU EVIDENCE FOR OPIATE RECEPTOR SITES UN T LYMPHUCYTES. Journal of Immunology, 125: 2539-2543 Bruni, J.F., Van Vugt, D.A., and Smith, A.Y., 13) Meites, J., (197Y) HELATlUN UF ENDOtiENUUS UPlUlD PEPTIDES ANU MUHPHINE TU NEUHUENUOCRINEFUNCTIONS. Life Sciences, 24: 1325-1336 141 Pasternak, G.W., and EFFECTS HiNDING: UF Pharmacology,lO: 183-193 lb)
Snyder, S.H., ENZYMATIC
Pasternak, G.W., Goodman, R., and Snyder, S.H., MOHPHINE-LIKE FACTOR IN MAMMALlAN BRAIN. Life
ENUOtitiNUUS 24:
(1974) UPIATE RECEPTOR TREATMENTS. Molecular (19'15) AN Sciences,
13251336
lb;) Scatchard, G. MOLECULES AND IONS.
(1949) Annals
THE ATTRACTION OF PROTElN FOR SMALL 01' the New York Academy of Sciences, 51:
660-672
1'7) Schultzberg, M., Lundberg, J.M., Hokfelt,T., Terenius, L., ENKEPHALINE-LIKE Brandt, J., Elde, R.P., and Goldstein, M., (lY'/8) 1MMUNOREACTIVITY IN GLAND CELLS AND NERVE TERMINAL OF THE ADRENAL MEDULLA. Neuroscience, 3: 1169-1186. L., and Wahlstrom, 181 Terenius, ENUOGENOUS LIGAND FOR THE OPIATE Scandinavica, 94: 74-81.
A., (19'/5) SEAHCH FOR AN RECEPTOR Acta Physiologica
O.H, Diliberto, E.J., Jr., Hazum, E. and Chang, K.,-J. 19) Viveros, OPIATE-LIKE MATERlAL IN THE ADRENAL MEDULLA: EVIDENCE FOR STORAGE AND SECRETION WITH CATHECOLAMINES (197Y)) Molecular Pharmacology 16: 1101-1108 20) Wybran, J., Appelboom, T., Famaey, J.P., and Govaerts, A., AND EVIDENCE RECEPTORS FUH MORPHINE SUGGESI’IVE (1979) FOR METHIUNINE-ENKEPHALINE ON NORMAL HUMAN BLOOD T LYMPHOCYTES. Journal of Immunology, 128: 1068-1070
Received:
13th
July
1983.
Revised version accepted: 14th November 1983
international
Reports,
LEIJ-KNKEPHALIN BlNDINti Clara
Vol. 8, No. 2, February
1984
97
TO CULTURED HUMAN T LYMPHOCYTES.
M. Ausiello*
and L. tiiorgio
Roda
*
di Tipizzazione 'l'issutale e Problem1 CNR "lstituto "Laboratorio di Farmacologia, Italy; L'Aquila, di Tecnica b'isiologica, Ancona, Ancona, and Cattedra University, Via 0. Haimondi, 1 00173 Rome, Italy.
della Dialisi", University 01. 'ITor Vergata"
ABS'I'HACT Evidence is given for the existence of leu-enkephalin binding activity on the cell surface of a human lympnoid 'I' line. uur data structures which in all suggest that binding is due to receptor as is already known to be the case with likelyhood are lipoproteic, the receptors present in the mammalian brain. In spite OS these lymphocytes exhibit anomalous specificity as similarities, however, rar as the binding toward oploid alkaloids and their inhibitors is concerned.
'The concept o? the presence OS endogenous opioids has been advanced more than once since the late sixties (::lartin, 1967; Davis and LlJalsh , 197Uj. ln latter years the spec i TIC search for with considerable endo,gcnous opioids has been rewarded success identification, leading to the isolation structural and characterization of the smaller opioid peptides, leu and met-enkephalin (Hughes, 19,/b; 'I'erenius and :~;ahlstrom, lY'/5 ; Pasternak et al., 19/4j. Subsequently, quite a large - and perhaps even eccessively so - body oi endogenous substances related to them has been described. Furthermore, it has been possible to demonstrate the existence of binding activity of labellea opioids to brain homoqenates and to distinguish between speciric and non-speciIic binding on a stereospecificity basis. Mevertneless, tne guriiicatlon OI the membrane-bound receptors has proved to be quite aifficult, possibly on account OS the nature of the receptors itself (Lin and Simon, 19 /B; Pasternak and Snyder, 19'/4; Abood et 0309-I 651184/020097-10/$03.00/0
@ 1984 Academic
Press Inc. (London)
Ltd.
Cell Biology
98
International
Reports,
Vol. 8, No. 2, February
1984
800..
400-m
200..
I
1
I
2
3
cells
x to8
KIWJRE 1. tiinding ot‘ leu-enkephalin to Jurkat T cells. 6x10-4DP'M or' ('Hj leu-enkephalin were incubated (SO', 3'1°C) with the indicated number of cells. Solid squares indicate experimental points. The solid line indicates zhe least-squares interpolated curve.
al., 1977). Presently, a lipoproteic structure for the opioid receptors seems highly probable, and data consistent with this hypothesis have been reported by several groups (Lee and Smith, 1980; Eeites et al., 1979, Cherubini et al, 1903). To date opioid peptides have been shown to possess several different activities in addition to their hypotnesizcd role in pain Furthermore, it control. has been demonstrated that both enkephalins are p,,esent within the adrenal chromaffin medulla granule, and are thereby into released the blood stream (Schultzberg et al., 1970, Viveros et al, 1979). In addition, Clemente-Jones et al. (1930) demonstrated that met-enkephalin circulates in peripheral blood in quantities up to 50 pg/ml. On the basis of these latter data, it is not altogether surprising that in recent years evidence ilas been preacnted for the presence of opioid receptors on the surface of iympllocytes (liazum
Cell Biology
International
Reports,
Vol. 8, No. 2, February
a b M ?8 =: 23
1984
total mob
en&@mlin,
1.5
s : 5
1.0
a P
0.5
210
4.0
6.0
8.0
bound moles/ce(l x 10-14
FIGURE 2, panel a. Satxration binding curve OS leu-enkephalin to Jurkat T cells. 2,25x10 cells were incubated 130',35757uC) with she indicated amount of leu-enkephalin; to this 1x10 DPM or' ( H) labelled leu-enkephalin were added. Panel b: Scatchard plot from the data in panel a.
Cell Biology
100 et al., et al.,
1979; 1982).
!$ybran
et
International al.,
1979;
Reports,
Vol. 8, No. 2, February
plc Donough
et
al.,
1980;
1984 Gilman
show that The experiments hereunder reported erikephalin-binding structures are present even on the surface of Our evidence supports the cultured human T lymphoblastoid cells. with an external standard accessory (L'XB Produkter, Bromma, Sweden). The reagents used were of the following origin: proteinase ;< and phospholipase A * Calbiochem (San Diego CA., USA), tunicamycin: 2' Sigma (St. Louis, ilO, USA), naloxon: Endo Laboratories (Garden NY, USA), morphine: City, Angelini Medicinali (Ancona, Italy), leucine enkephalin: Serva (Heidelberg, DBR) and tyrosi1-3,5-31-:(N) 4 leu-enkephalin, specific activity 3x10 Ci/mole: The Radiochemical Centre (Amhersham, UK). All the other chemicals were of reagent grade and bought from local suppliers. RESULTS AND DISCUSSIOE To investigate the existence within the T lymphocyte menl'orane VJ of structures showing binding activity toward leu-enke halin, % 8 incubated (30' 37°C) a number 04f cells rangigg from 3x10 to 3x10 with a constant amount (6x10 DPX) of ( 1-I) leu-enkephalin in phosphate-saline buffer. Samples were analyzed as described and the resulting data interpolated between the experimental points with a least-square computer program. The resulting curve (Figure 1) shows that quite a good linear relationship (r=0.989) exists between cell number and bound enkephalin. In additi n, equilibrium experiments were performed. A fixed k3 number (2.25~10 ) of washed T cells - together $th an arnount of -9 2.5x10 leu-enkephalin ranging from moles - were 5x10 to lx105DPPI resuspenfqd in a 300 ul final volume of buffered saline. / (1.5x10 L moles) of labelled enkephalin were added to each tube and the mixture was incubated 30' at 3?cC. In figure 2, panel a, bound enkephalin is plotted against the total amount of enkephalin present in the incubation mixture. The plotted curve shows that the leu-enkephalin-T lymphocytes binding is saturable. To determine the kinetic parameters of the binding, the same data were analyzed and plotted according to Scatchard (1949), as shown in figure 2, panel b. From the plot in figure 2b it seems likely different binding sites - with that a population of different binding constants - exists on T cells surface, and this also appears to be the case with@-endorphin (Ilazum et al, 1979). However, from the linear portion of the Scatshard plot in figure 2b an apparent dissociation constant of 2.03x10 may be calculated on
Cell Biology
international
a per cell
basis.
Reports,
Vol. 8, No. 2, February
1984
101
demonstrate the existerice of stiructures IYXGUltS The &OVl: silowing receptor activity toward leu-en:cepllalin on tile surface of T ‘:Jiiether or no’; these structurc;s are CilN!iCaiiy cells. To ascertain ilypothesis L!mt both pilospholipids ant', protein are i:ecessary for 2e also deinoristrdx the existence of conspicuous binding activity. known oploid receptor. differences in regard to tile already
T cell iine (Artin et al., 1932) w~~s Jurkat human lymphoid Inc., maintained at 37OC in 5% CO in RPZU 1640 (Plow Laboratories with 10;: fetal call serum (710~) plus KcLean, Va, USA) su;lplementZcI 100 units/ml penicillin and 100 mg/r:il streptomycin. Binding assays were performed as follows: cultured cells, pfI 7.4, 0.9:1 :;aCl (phosphate-saline) washed in 15 rn:; Ila phosphate ih'ere counted by means of a StXliI3.I'd buffer, procedure a11d s resuspeildeci at a final concentratiorl of Z-1OxlC cells/ml. A volume Was then transferred in 250 PVC tubes. To ^,:I& "5 P1 ( F!)leu-enkephalin was added anti cells iilcu’dated were at 37‘C The incubation was (final volume 300 ul). stopped by the addition / of 3 ml of ice cold phosphate-saline buffer, the cells were pelletted (5' at 1000 x g) in a refrigerated centrifuge, resuspended in 3 ml of the same buffer alid centrifuged again. 21e pellet was then resuspended in 50G ul of 0.1 :j :;aOi-i and 450 ,ul of it were counted in a LXD Xini3et.a / scintiilation counter eq6Lpped similar to the ones described in the case of nervous tissue, we two of the opioid agonist inhibition experiments usin? performed for the and antagonist tha-t are known to compete with enkephalins - receptors. As shown in figure 3, naloxon same - or very similar -5 did not inhibit Leu-enkephalin bindinE up to 10 (solid line) a molar concentration of 3x10 . Similarly, inhibition except at a concentration OS ~~~~~~IZ" AiY' nZYc:uSJe tiowewer, the penultimate concentration moles/~X ul. 5x10 mof es of morphLne/YCC ul - caused approximately a assayea - 1x10 / lns$ad, no inhibition was caused by the next 5o:A Inhibition. 5x10 moles/ZOO ui. It is debatable however concentration, wnether the innihitory effect thu& determined can be considered meaniny,pl since itbis only detectable at a molar concentration or 3,3x10 , i.e. a 10 molar excess over enkephalin. me results of' these inhibition experiments seem to suggest that 'I' cell binding is due to structures which are not quite the same as those present in the nervous tissue. Since there is some
Cell Biology
102
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Vol. 8, No. 2, February
1984
knowledge - albeit poor - of' the chemical structure of the latter, we tried to partially characterize '1 lymphocyte receptors according to their chemical ‘I’0 obtain this information, we properties. treated the lymphocytes with different substances that are active on the protein primary structure, on the disulfide bonds of proteins, on phospholipids and on carbohydrates cell synthesis. binding
'Table 1 shows the by the following
lo+:
results of the action on the substances: mercaptoethanol,
lo-0
~8
10-7 total
leu-enkephalin tunicamycin,
lo-s mole8
FlGURE 3. lnhibition by morphine and leu-enkephalin-Jurkat cells inding. _152~olx,s, 11; yyeFabe;:%% !? incubated (30',37°C) with 1x10 DPM (7x10 leu-enkephalin plus the indicated amounts of both substances. Solid line and triangles indicate naloxon; dash-dotted line and circles indicate morphine. were incubated and proteinase i(. dasned cells phospho.lypase A under test in a 300 ul r'lnal volume (IL!,', :r';/"c) wUzh%the substance 01 phosphate-saline bui‘i'er, washed again and incub/ated (30' 37°C) volume with labelled leu-enkephalln. 'I'unicamycin in a 300 ul r'rnal / incuoation was .L’y nours under culture conciitlons. I( A and proteinase 1, both pnosphoiipase .4s silown in table of' the enkepnalln b;$ding to 1' cells. caused a market irthlbltlon even in the case of‘ 'L' lymphocytes, Mat, suggests TClj S result
-4 -s
-3
-2
-1
)
A2
l.36XlO-14
-13
5.o2x1o-14
1.17x10
1.44x10
0.8
0.08
1.12x10
1.36X10
bound
-13
-13
-14
-13
(moles)
Leu-enkephalin
PHOSPHOLYPASE
9.75x10-l4 1 .21X1o-13
7.94x10-l4
6.80X10-‘~
3.53X10
8
SO
(units/ml
concentration
Phospholypase
1x10 1x10
1x10
1x10
1x10
(moles/l)
)
K
in
A2
SIJBSTANCES
(moles)
Leu-enkephal
! mg/ml
K
DIFFERENT
PROTEISASE
OF
bound
i naso
Prote
EFFECT
Concentration
1:
I’AHLE
89
58 72
49
25
100
the
8
106
86
37
100
control
the
0.5
T-CELLS
-5
-3 -4
-2
-1
-13 -13
-13
-13
(moles)
1 .42X10
1
1.38X10
1.31x10
1.36x10
bound
-13
-13
-13
-13
(moles)
leu-enkephal
TUNICAMYCIN
~81x10-'~
1.93x10 1.95x10
1.43x10
1 .91x10-l4 3.63X10
bound
Leu-enkephalin
2 MERCAPTOETHANOL
BINDING
5
(ug/ml)
concentration
Tunicamycin
1x10
1x10 1x10
1x10
1x10
concentration
2-Mercaptoethanol
LEU-ENKEPHALIN
% of
control
% of
OS
in
95
101 102
7s
1
contra
102
104
96
100
the
the
% of
19
100
control
% of
2
2
G
6 b 2
.Q
$
8
B
Cell Biology
International
Reports,
Vol. 8, No. 2, February
1984
phospholipid are the protein and essential constituents of enkephalln receptor. On the contrary, no effect on binding activity has been detected after incubation with tunicamycin. This result suggesting no carbohydrate involvement in the receptor structure is also consistent with the data existing about opioid receptors in [Lee and Smith, nervous tissue 1980; Meites et al., 19'/9 ) . Finally, rather ambiguous results 'I' were given by incubating 2-mercaptoethanol. ln lymphocytes with this case, only -2 concentrations OS the reducing agent 01 10 1'4 and higher are in inhibiting binding activity. Since disulphide bonds effective to be relatively shielded, a nlgn in membrane proteins are likely concentration 01 reducing agent may actually be necessary to reduce the hand, 2-mercaptoethanol tnem effectivelly. On other indicated are likely to have a concentrations in the range non-specir.ic inhibitory effect. conclusion, tne results we obtained though only In the presence 01' structures with receptor preliminary - demonstrate activity toward leu-enkephalin on the surface of Jurkat '1 cells. 'The behavior OS these receptors is anomalous as far as opioid agonists and antagonists are concerned. Yet, these structures seem chemical characteristics known for brain to have the same gross and phospholipids - but no opioid receptors. 'I'hat is, both proteins carbohydrates - seem to be involved in the receptor structure. 'The existence of different kinds of receptors f‘or endogenous opioids in lymphoid cells has been so f'ar suggested by the results and by the present Of' Hazum et al (19'7Y) Wybran et al (19'7Y), seem to suggest the existence oi' a also study. 'I'hese r'indings closer and more direct connection between nervous and immune system Furthermore, this suggests that there than is commonly supposed. may exist new roles for opioid peptides, different rrom the ones that may be searched for. 'I'hese new roles may presently accepted, nervous activity on the mediators 01' the system's be like physiological iunctions of the immunological system, 1'or which a nervous control is not commonly recognized today.
out and partially supported by the work has been carried Fina1izzat.i "Chimica Fine e Secondaria" and "Controllo CYPI (Ital ian CouncJ.1 for Neoplastica" of the della Crescita Scl.entific HesearchJ. This
Progetti
Cell Biology
International
Reports,
Vol. 8, No. 2, February
105
1984
REFERENCES Salem, N., Macneil, Jr.M., Bloom, L. and Abood, 1) Abood, L.G., M.E. (1977) ENHANCEMENT OF OPIATE BlNDING BY VARIOUS MOLECULAR FORMS UF PHOSPHATIDYLSERINE AND INHIBITION BY OTHER UNSATURATED LIPID. Biochimica et Biophysics Acta, 468: 51-62
2) Cherubini, O., De Marco, V., Roscetti, ti., Possenti, R. and PEPTIDES WITH INTERACTION OF OPIOID VITRO Roda, L.G. IN FORMATION AND CHARACTERIZATION OF CUMPLEXES. PHOSPHCLIPIDS: International Journal of Peptides and Protein Research, in press September 1983 3) Clement-Jones, V., (1980) MET-ENKEPHALIN 295-297
Lowry, P.J., CIRCULATES
Rees, L.H., and Besser, IN HUMAN PLASMA. Nature
and Walsh, M.S. Davis, V.E. 4) A POSSlBLE BIOCHEMICAL ALKALOIDS: Science 167: 1005-1007.
G.M., 283:
(1970) ALCOHOL, AMINES, AND BASIS FOR ALCOHOL ADDICTION.
5) Gilman, S.C., Schwartz, J.M., Milner, R.J., Bloom, F.E., and -ENDORPHIN ENHANCES LYMPHUCYTE (1982) Feldman, J.D., b Academy of PROLIFERATIVE RESPONSES. Proceedings of the National Sciences. USA '/9: 4226-4230. and Cuatrecasas, Chang, K.S. 6) Hazum, E., NANOPIATE RECEPTOR FOH/3-ENDORPHINE. Science
P. (19*/g) SPECIFIC 205: 1033-1035
(1975) ISOLATION OF AN ENDOGENOUSCOMPOUNDFROM THE 7) Hughes, J., BHAlN WITH PHARMACULOGICAL PROPERTIES SIMILAR TO MORPHINE. Brain Research, 88: 295-308 8) Lee, N.M., and Smith, A.P., OPIATE RECEPTOR. Life Sciences,
(1980) A PROTEIN-LIPID 26: 1459-1464.
MODEL OF THE
\iY'/td) ~HtiSrkuLlPASE A 1NHIBITION OF 9J Lin, H.K. and Simon,E.S. OPlATE RECEPTOR BINDING CAN BE REVERSED BY ALBUMIN. Nature 271: 383-384 Giblett, E.H., Martin, P.J., and (1982) Hansen, J.A., 10) PHENOTYPlNG HUMAN LEUKEMlC 'I'-CELL LINES: ENZYME MARKERS, SUHE'ACE ANTltiENS, AND CYTOGENETICS. Immunogenetics 15: 385-398. Martin, W.R., 11) Reviews 19: 463-521 12) M.,
(1967)
OPIOID
McUonough, H.J., Madden, J.J., Gordon, D., kiokos, P., Kuehnle,
ANTAGONISTS. Falek, J-C.,
Pharmacological
A., Shafer, D.A., and Mendelson, J.,
Pline, (1980)
106
Cell Biology
International
Reports,
Vol. 8, No. 2, February
1984
ALTERATlON OF T AND NULL LYMPHUCYTE FHEUUENClES IN THE PEHlPHEHAL BLOOD OF HUMAN OPLATE ADDICTS: IN VlVU EVIDENCE FOR OPIATE RECEPTOR SITES UN T LYMPHUCYTES. Journal of Immunology, 125: 2539-2543 Bruni, J.F., Van Vugt, D.A., and Smith, A.Y., 13) Meites, J., (197Y) HELATlUN UF ENDOtiENUUS UPlUlD PEPTIDES ANU MUHPHINE TU NEUHUENUOCRINEFUNCTIONS. Life Sciences, 24: 1325-1336 141 Pasternak, G.W., and EFFECTS HiNDING: UF Pharmacology,lO: 183-193 lb)
Snyder, S.H., ENZYMATIC
Pasternak, G.W., Goodman, R., and Snyder, S.H., MOHPHINE-LIKE FACTOR IN MAMMALlAN BRAIN. Life
ENUOtitiNUUS 24:
(1974) UPIATE RECEPTOR TREATMENTS. Molecular (19'15) AN Sciences,
13251336
lb;) Scatchard, G. MOLECULES AND IONS.
(1949) Annals
THE ATTRACTION OF PROTElN FOR SMALL 01' the New York Academy of Sciences, 51:
660-672
1'7) Schultzberg, M., Lundberg, J.M., Hokfelt,T., Terenius, L., ENKEPHALINE-LIKE Brandt, J., Elde, R.P., and Goldstein, M., (lY'/8) 1MMUNOREACTIVITY IN GLAND CELLS AND NERVE TERMINAL OF THE ADRENAL MEDULLA. Neuroscience, 3: 1169-1186. L., and Wahlstrom, 181 Terenius, ENUOGENOUS LIGAND FOR THE OPIATE Scandinavica, 94: 74-81.
A., (19'/5) SEAHCH FOR AN RECEPTOR Acta Physiologica
O.H, Diliberto, E.J., Jr., Hazum, E. and Chang, K.,-J. 19) Viveros, OPIATE-LIKE MATERlAL IN THE ADRENAL MEDULLA: EVIDENCE FOR STORAGE AND SECRETION WITH CATHECOLAMINES (197Y)) Molecular Pharmacology 16: 1101-1108 20) Wybran, J., Appelboom, T., Famaey, J.P., and Govaerts, A., AND EVIDENCE RECEPTORS FUH MORPHINE SUGGESI’IVE (1979) FOR METHIUNINE-ENKEPHALINE ON NORMAL HUMAN BLOOD T LYMPHOCYTES. Journal of Immunology, 128: 1068-1070
Received:
13th
July
1983.
Revised version accepted: 14th November 1983