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Prolactin binding sites in human erythrocytes and lymphocytes
Life Sciences, Vol. 41, pp. 951-959 Printed in the U.S.A.
PROLACTIN
BINDING
SITES IN HUMAN LYMPHOCYTES
Pergamon Journals
ERYTHROCYTES
AND
1 I i Giuseppina Bellussi , Giampiero Muccioli , Corrado Gh~ and Raffaele Di Carlo 2. IInstitute of Pharmacology, University of Turin, Corso Raffaello 30, 10125 Turin, Italy. ~Department of Experimental Pharmacology, University of Naples, via Rodin5 22, 80138 Naples, Italy.
(Received in final form June 9, 1987) Summary Specific binding sites for prolactin (PRL) have been studied in human peripheral lymphocytes and erythrocytes of normal adult volunteers and of term cord bloods. In erythrocytes from healthy adult subjects of both sexes a very low specific binding of 1251-human PRL was found (0.24%),whereas a higher binding was found in term cord blood (I.I %). The binding was hormone specific, the binding capacity was 2.6 fmol/4xl09 cells and the Kd was 3.4x10-10M. In lymphocytes of both adults and term cord bloods an evident specific binding was observed (male adults: 1.6%; female adults: 1.7%; cord blood: 1.8%). The binding was specific for lactogenic hormones and the binding capacity was 3.7 fmol/2xl06 cells and the Kd was 3.gx10-10M. The presence of specific binding sites for PRL on human erythrocytes and lymphocytes could be used to study PRL binding on blood cells of patients in different physiological or pathological situations. Specific binding sites for lactogenic hormones (PRL and human growth hormone) have been described in a variety of tissues of different animal species (1,2,3). On the other hand few data are available in literature on the binding of lactogenic hormones to peripheral human blood cells. Positive studies, using human growth hormone as ligand, have generally been performed on special lines of cultured human lymphocytes (4,5), whereas controversial results are reported concerning
Presented in part to the XXII Congresso della Societ~ Italiana di Farmacologia, Bologna, October 10-13, 1984. 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
952
Specific PRL Binding Sites
Vol. 41, No. 8, 1987
human circulating lymphocytes (6,7,8). Using rat PRL as ligand recently Russel et al. (9,10) have identified PRL receptors on human peripheral blood lymphocytes suggesting that these receptors may be involved in regulating lymphocyte function. Concerning erythrocytes no data are to our knowledge, available on the presence of binding sites on their surface, even if on the basis of the role exerted by PRL on the regulation of human red cell sodium transport (ii), it could be suggested that erythrocytes may bind prolactin. In our research we have examined in more details PRL binding to human peripheral lymphocytes and for the first time studied the PRL binding to human erythrocytes. Materials
and Methods
Erythrocytes and lymphocytes from 15 male and female volunteers (20 to 40 years of age) and 15 normal term cord blood were studied. Patients were studied after fasting overnight. Blood was collected into heparinized tubes; after centrifugation (i0 min at 400 x g, 4°C) the plasma was aspirated. The cell pellet was mixed with one part of physiological saline and then layered on Hypaque (33.9%)-Ficoli (9%) mixture in a glass tube according to the method of BSyum (12) to separate erythrocytes and lymphocytes. Erythrocytes were resuspended with buffer G, as described by Gambhir et al. (13). Lymphocytes were washed in Dulbecco's Medium containing 0.1% bovine serum albumin (BSA) and then resuspended with the same medium. More than 95% of cells were viable, as determined by the trypan blue dye exclusion technique (14). PRL and insulin binding to erythrocytes was made according to the method used for insulin by Gambhir et al. (13). In the binding assays about 50,000 cpm of 1251-human prolactin (1251-hPRL, 43-49 ~Ci/ug, New England Nuclear) or 1251-porcine insulin (1251-pINS, 82-87 uCi/~g, New England Nuclear) were added to each tube containing 2x109 cells in a final volume of 0.5 ml assay buffer G. After 3.5 h of incubation at 15°C, 100 ~i of the incubated suspension was aliquoted into microfuge tubes containing 200 ul buffer G and i00 ~i of dibutylphthalate. These tubes were then centrifuged in a Beckman Microfuge for 2 min at 4°C. The buffer and dibutylphthalate layers were aspirated and the cell pellet was counted in a Packard auto-gamma counter. For PRL binding studies to lymphocytes about 50,000 cpm of 125I-hPRL were added to each tube containing 2xl06 cells in a final volume of 0.5 ml Dulbecco's Medium. After 6 h of incubation at 37°C, cells were centrifuged for 1 min in a Beckman Microfuge, were washed once in 1 ml of Hank's balanced salt solution plus 0.1% BSA and were re-centrifuged again for 1 min. The buffer was aspirated and the cell pellet counted in a Packard auto-gamma counter. Dissociation of endogenously bound performed by exposing lymphocytes to
PRL high
from the receptor was concentrations of MgCI2,
Vol. 41, No. 8, 1987
Specific PRL Binding Sites
953
essentially as described by Russell et al. (I0). Lymphocytes (2x106 cells) were treated with 0.2 ml of 4 M MgCI 2 on ice. After 5 min, I ml of cold Dulbecco's medium was added and was centrifuged in a Beckman Microfuge. The supernatant was removed and the pellet was washed further by suspension in I ml Dulbecco's medium and was recentrifuged as before. Binding of 1251-hPRL was assayed as described above. The specific binding to erythrocytes or lymphocytes was evaluated as the difference between the binding in the absence and the presence of excess unlabeled hormone (2 ug/ml) expressed as per cent of the total counts present in i00 ~i of the incubated cell suspension (for erythrocytes) or present in the incubation medium (for lymphocytes). Scatchard (15) analysis of the PRL binding to erythrocytes or lymphocytes was performed by transformation of binding data obtained from the competition studies with increasing concentrations of unlabeled human PRL mixed with a fixed amount of tracer. The dissociation constant (Kd) and binding capacity were determined for unlabeled hormone concentration between 0.8 and i00 ng/ml. The specificity of 1251-hPRL binding was tested with the following unlabeled hormones: human prolactin (hPRL, NIADDK-I-6), ovine prolactin (oPRL, NIADDK-oPRL-I-I), human growth hormone (hGH, NIADDK-RP-I), ovine growth hormone (oGH, NIH-GH-SII), rat follicle
0
z © H
H H Z
--
20
--
40
-
60
_
80
-
O
rFSH pINS
•
oPRL
PRL
H
7// t 10 0
''""J, 10 t
UNLABELED
I
i
10 2
10 3
HO~ONE
(ng/ml)
FIG. 1 Gompetitive inhibition of the specific binding of 125I-hPRL to erythrocytes of term cord blood by increasing concentrations of unlabeled hPRL and by a single concentration of oPRL, rFSH and pINS. Inhibition in the presence of 2x104 ng/ml hPRL was considered to be 100%. Non specific binding was 0.5%.
954
Specific PRL Binding Sites
Vol. 41, No. 8, 1987
stimulating hormone (rFSH, NIADDK-RP-I) and porcine insulin (pINS, Schwarz/Mann). All determinations were performed in triplicate and the results expressed as mean values. The deviation from the mean was always less than 15% of the mean value. Plasma PRL concentrations were determined by radioimmunoassay using the kit supplied by Sorin (Saluggia, Italy) and the results expressed as ng/ml. Results In erythrocytes from healthy adult subjects of both sexes a very low specific binding of 1251-hPRL was found : 120 ± 95 cpm/4xl09 cells (0.24 ± 0.19%; n=5), whereas a higher binding was found in term cord blood: 530 ± 270 cpm/4xl09 cells (i.i ± 0.5%; n=9). Non-specific binding was between 200 and 300 cpm (0.4-0.6%). The binding specificity of 1251-hPRI to erythrocytes of term cord blood is reported in Fig. I. The binding was inhibited by unlabeled hPRL or oPRL, but not by rFSH or pINS. The binding specific capacity calculated by Scatchard analysis was 2.6 fmol/4xl09 cells and the Kd was 3.4x10-10M (Fig. 2). As binding
a of
comparison we have also studied in the same blood samples the 125I-pINS. With this hormone the binding both to adults
0.016
0.012
0.008 Z D 0
0.004
I
[
I hPRL
]
2
3
BOUND
(fmol/4x109
cells)
FIG. 2 Scatchard analysis of binding data from the competition studies 1251-hPRL and unlabeled hPRL using erythrocytes of term cord blood.
between
Vol. 41, No. 8, 1987
Specific PRL Binding Sites
955
and term cord blood samples was about one order of magnitude higher (5.4% ± 1.0 and 8.5% ± 1.9 respectively). The results are in agreement with data from Kappy & Plotnick (16).
TABLE I Specific Binding of 1251-hPRL to Lymphocytes Obtained from Blood Samples of Adults and Term Cord and Corresponding Plasma PRL Concentrations. Specific binding/2xl06cells PRL plasma level(ng/ml)
Subject cpm
Cord blood (n=5) Adult male (n=6) Adult female(n=4)
% total counts
920 ± 480 795 ± 230 845 ± 90
1.8 ± 1.0 1.6 ± 0.5 1.7 ± 0.2
408 9.3 16.8
± ± ±
127.9 2.5 3.1
Values are means ± S.D. Non specific binding was between 250 and 400 cpm (0.5 - 0.8%).
2.0
16
D
B
A
o 1.5
37°C
~o~_ 12
m
O .Q
~C 1.0
21
8
I
H u~ c~
'~
0.5
4
,'N ,e--
I I I 2 4 6 INCUBATION T I M E (h)
I 8
I I I I 0.5 I .0 I .5 2.0 1 2 5 I - h P R L (ng added)
FIG. 3 Specific binding of 1251-hPRL to adult human lymphocytes as a function of incubation time and temperature (A) and as a function of increasing concentrations of labeled hormone added (B). Non specific binding was 0.6%.
956
Specific PRL Binding Sites
Vol. 41, No. 8, 1987
Table I reports the specific binding of 1251-hPRL to adult human lymphocytes of both sexes and term cord bloods and the corresponding PRL plasma levels. An evident specific binding was observed in all samples examined without significant difference noted between the mean values of adults and term cord blood samples and between male and female adult volunteers. As reported in literature, very high PRL plasma levels were found in all term cord blood samples. Also the levels in male and female adult volunteers were in the normal range. After in vitro desaturation only slight increase in specific binding was found (+19.3 ± 7.6%) in lymphocytes from adult subjects of both sexes, whereas in lymphocytes from term cord blood a much more pronounced increase was observed (+54.3 ± 22.7%). The specific binding of 1251-hPRL to adult human lymphocytes was timeand temperature-dependent (Fig. 3A). Binding was maximal at 37°C reaching equilibrium after 6 h, whereas at 40C lower levels of binding were attained even after 8 h. When increasing amounts of 1251-hPRL were incubated with a fixed number of cells, specific binding was detected over the whole range of concentration studied (0.5-1.8 ng) and appeared to reach saturation by approximately 1.8 ng (Fig. 3B). The
binding
specificity
of
125I-hPRL
to adult
human
lymphocytes
0--
20-Z O ~40H H Z60-H
80--
--// I
' ''"'"I
10 0
, b
,
101
UNLADI~LED H O R M O N E
I
r,
10 2
,
1
10 3
(ng/ml)
FIG. 4 Competitive inhibition of the specific binding of 1251-hPRL to adult human lymphocytes by increasing concentration of the indicated unlabeled hormones. Inhibition in the presence of 2x104 ng/ml hPRL was considered to be 100%. Non specific binding was 0.5%.
Vol. 41, No. 8, 1987
Specific PRL Binding Sites
957
is reported in Fig.4. Addition of increasing amounts of unlabeled hPRL resulted in a substantial decrease in binding of labeled tracer, a simil~r inhibition was observed for oPRL. Of other peptide hormones tested, only hGH, a hormone structurally and functionally similar to hPRL, was able to compete for the 125I-hPRL binding, oGH was much less effective, whereas no competition was displayed by the other hormones used. The binding specific capacity calculated by Scatchard analysis was 3.7 fmol/2xl06 cells and the Kd was 3.9xlO-lOM (Fig. 5).
0.020~
0"0151~
c~ Z o
°°°V 0"0051
~ 2I hPRL
4I
6I
BOUND
( f m o l / 2 x 1 0 6 cells) FIG. 5 Scatchard analysis of binding data from the competition studies 125I-hPRL and unlabeled hPRL using adult human lymphocytes.
between
Discussion Our results show for the first time the presence of PRL receptor on erythrocytes both of adults and term cord blood. The binding is quite low, especially in the adult cells, whereas in samples from newborn blood it is much higher and also specific for lactogenic hormones. On the same cells the binding values of insulin, a hormone which also binds to membrane receptors, resulted very close to the values reported in literature, confirming the validity of our technique. The significance of finding specific PRL receptors on human red blood cells remains not clearly defined. The physiologic role of these binding sites on erythrocytes membranes could be related to the influence exerted by PRL on the regulation of human red cell sodium transport. As reported by Gopalakrishnan et al. (II) incubation of red cells with higher concentrations of PRL in vitro enhanced the cellular sodium level and produced a significant reduction in erythrocyte membrane adenosine triphosphatase activity, through an inhibition of the active sodium pump. On the other hand Parke and Horrobin (17) have observed a significant decrease in plasma levels of PRL when the same blood sample
958
Specific PRL Binding Sites
Vol. 41, No. 8, 1987
was estimated immediately after veinpuncture and 6 hours later. This finding suggested the possibility that erythrocytes may bind PRL, exerting a sort of buffering action. Cord blood erythrocytes showed a binding to PRL higher than did those from adults. This finding could suggest that may be PRL has a special role in intrauterine growth and development. We have found also on lymphocytes from adults and term cord blood the presence of PRL receptors. The binding was specific for lactogenic hormones (hPRL and hGH), temperature-dependent, showed high affinity for hPRL and increased after exposition of lymphocytes to high concentrations of MgCI 2. The more pronounced effect exerted by desaturation on the binding of the lymphocytes from term cord blood seems to be related to the very high PRL plasma levels which could occupate a larger number of binding sites. Our results generally are in agreement with the data on lymphocytes reported by Russel et al. (9); however our binding values are slightly higher than those obtained by these authors. This could be ascribed to the different ligand we used which was human labeled PRL instead of rat labeled PRL, obviously less specific for human cells. As far as the possible role of PRL on lymphocyte membranes is concerned, a large body of evidence exists on the effect of lactogenic hormones, and of PRL in particular, on the human lymphocytes both peripheral and in culture. Mercola et al. (18) have studied the effect of PRL and growth hormone on human T-lymphocyte proliferation in vitro and have observed a direct and species-specific stimulation of growth on normal and some neoplastic human T-cells. The role of PRL in the regulation of humoral and cell-mediated immunoresponses also appears important. Nagy et al. (19) demonstrated that antibody formation to sheep red blood cells and the development of contact dermatitis in response to dinitrochlorobenzene are impaired in hypophysectomized rats. PRL, growth hormone and placental lactogen restoled the immunocompetence of hypophysectomized animals. On the other hand, bromocriptine depressed PRL serum levels and suppressed both antibody formation processes and skin reactivity to dinitrochlorobenzene (20). The ability to restore immunocompetence in hypophysectomized animals by administration of PRL suggests the PRL receptors may be important in the regulation of lymphocyte-implicated immunoresponses. Russel et al. (9) further demonstrated that PRL receptors on human peripheral lymphocytes can be enhanced in number or blocked by cyclosporine, a potent immunosuppressive agent used in human organ transplant patients. Investigations on possible variations of the PRL binding capacity of red blood cells and lymphocytes in different physiological and pathological conditions are in progress in our laboratories.
Vol. 41, No. 8, 1987
Specific PRL Binding Sites
959
Acknowledgments We thank the NIADDK Pituitary Hormone supplying polypeptide hormones and Mrs. A. assistance.
Distribution Program for Biavati for secretarial
References I. B.I.POSNER, P.A. KELLY, R.P.C. SHIU and H.G. FRIESEN, Endocrinology 95 521-531 (1974) 2. R.P.C. SHIU and H.G. FRIESEN, Biochem. J. 140 301-311 (1974). 3. R. DI CARLO and G. MUCCIOLI, in Advances in Biochemical Psychopharmacology, vol. 37; CNS-Receptors-From Molecular PharmacoloK-~ to Behavior ed. by P.Mandel and F.V. De Feudis, Raven Press, New York (1983) pp. 429-439. 4. M.A. LESNIAK, J. HOT}{, P. GORDEN and J.R. III GAVIN, Nature New Biology 241 20-22 (1973). 5. M.A. LESNIAK, P. GORDEN, J. ROT}{ and J.R. III GAVIN, J. Biol. Chem. 249 1661-1667 (1974). 6. R. ESHET, S. MANHEIMER, P. CHOBSIENG and Z. LARON, Horm. Metab. Res.7 352-353 (1975). 7. W. HINTERBERGER, K. KLETTER and W. WALDHAUSL, Horm. Metab. Res. 8 492-493 (1975). 8. C. STEWART, S. CLEJAN, L. FUGLER, T. CHERUVANKY and P.J. COLLIPP, Arch. Biochem. Biophys. 220 309-313 (1983). 9. D.H. RUSSEL, L. MATRISIAN, R.KIBLER, D.F. LARSON, B. POULOS and B.E. MAGUN, Biochem. Biophys. Res. Commun. 121 899-906 (1984). 10. D.H. RUSSEL, R. KIBLER, L. MATRISIAN, D.F. LARSON, B. POULOS and B.E. MAGUN, J. Immunol. 134 3027-3031 (1985). II. V. GOPALAKRISHNAN, S. RAMASWAMY, N. PADMANABHAPILLAI, S. R A N G A N A T ~ and M.N. GHOSH, Experientia 36 1423-1425 (1980). 12. A. BOYUM, Scand. J. Clin. Invest. 21 (suppl. 97) 77-89 (1968). 13. K.K. GAMBHIR, J.A. ARCHER and C.J. BRADLEY, Diabetes 27 701-708 (1978). 14. G.E. MOORE, E. ITO, K. ULRICH and A.A. SANDERBERGER, Cancer 19 713-723 (1966). 15. G.SCATCHARD, Ann. N. Y. Acad. Sci. 51 660-672 (1949). 16. M.S. KAPPY and L. PLOTNICK, Diabetes 28 1001-1005 (1979). 17. L. PARKE and D.F. HOP/ROBIN, Brit. Med. J. 1 262-263 (1976). 18. K.E. MERCOLA, M.J. CLINE and D.W. GOLDE, Blood 58 337-340 (1981). 19. E.NAGY, I.BERCZI and H.G.FRIESEN, Aeta Endocrinol. 102 351-357 (1983a). 20. E. NAGY,I. BERCZI, G.E. WREN, S.L. ASA and K. KOVACS, I~nunopharmacol. 6 231-243 (1983b).
PROLACTIN
BINDING
SITES IN HUMAN LYMPHOCYTES
Pergamon Journals
ERYTHROCYTES
AND
1 I i Giuseppina Bellussi , Giampiero Muccioli , Corrado Gh~ and Raffaele Di Carlo 2. IInstitute of Pharmacology, University of Turin, Corso Raffaello 30, 10125 Turin, Italy. ~Department of Experimental Pharmacology, University of Naples, via Rodin5 22, 80138 Naples, Italy.
(Received in final form June 9, 1987) Summary Specific binding sites for prolactin (PRL) have been studied in human peripheral lymphocytes and erythrocytes of normal adult volunteers and of term cord bloods. In erythrocytes from healthy adult subjects of both sexes a very low specific binding of 1251-human PRL was found (0.24%),whereas a higher binding was found in term cord blood (I.I %). The binding was hormone specific, the binding capacity was 2.6 fmol/4xl09 cells and the Kd was 3.4x10-10M. In lymphocytes of both adults and term cord bloods an evident specific binding was observed (male adults: 1.6%; female adults: 1.7%; cord blood: 1.8%). The binding was specific for lactogenic hormones and the binding capacity was 3.7 fmol/2xl06 cells and the Kd was 3.gx10-10M. The presence of specific binding sites for PRL on human erythrocytes and lymphocytes could be used to study PRL binding on blood cells of patients in different physiological or pathological situations. Specific binding sites for lactogenic hormones (PRL and human growth hormone) have been described in a variety of tissues of different animal species (1,2,3). On the other hand few data are available in literature on the binding of lactogenic hormones to peripheral human blood cells. Positive studies, using human growth hormone as ligand, have generally been performed on special lines of cultured human lymphocytes (4,5), whereas controversial results are reported concerning
Presented in part to the XXII Congresso della Societ~ Italiana di Farmacologia, Bologna, October 10-13, 1984. 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
952
Specific PRL Binding Sites
Vol. 41, No. 8, 1987
human circulating lymphocytes (6,7,8). Using rat PRL as ligand recently Russel et al. (9,10) have identified PRL receptors on human peripheral blood lymphocytes suggesting that these receptors may be involved in regulating lymphocyte function. Concerning erythrocytes no data are to our knowledge, available on the presence of binding sites on their surface, even if on the basis of the role exerted by PRL on the regulation of human red cell sodium transport (ii), it could be suggested that erythrocytes may bind prolactin. In our research we have examined in more details PRL binding to human peripheral lymphocytes and for the first time studied the PRL binding to human erythrocytes. Materials
and Methods
Erythrocytes and lymphocytes from 15 male and female volunteers (20 to 40 years of age) and 15 normal term cord blood were studied. Patients were studied after fasting overnight. Blood was collected into heparinized tubes; after centrifugation (i0 min at 400 x g, 4°C) the plasma was aspirated. The cell pellet was mixed with one part of physiological saline and then layered on Hypaque (33.9%)-Ficoli (9%) mixture in a glass tube according to the method of BSyum (12) to separate erythrocytes and lymphocytes. Erythrocytes were resuspended with buffer G, as described by Gambhir et al. (13). Lymphocytes were washed in Dulbecco's Medium containing 0.1% bovine serum albumin (BSA) and then resuspended with the same medium. More than 95% of cells were viable, as determined by the trypan blue dye exclusion technique (14). PRL and insulin binding to erythrocytes was made according to the method used for insulin by Gambhir et al. (13). In the binding assays about 50,000 cpm of 1251-human prolactin (1251-hPRL, 43-49 ~Ci/ug, New England Nuclear) or 1251-porcine insulin (1251-pINS, 82-87 uCi/~g, New England Nuclear) were added to each tube containing 2x109 cells in a final volume of 0.5 ml assay buffer G. After 3.5 h of incubation at 15°C, 100 ~i of the incubated suspension was aliquoted into microfuge tubes containing 200 ul buffer G and i00 ~i of dibutylphthalate. These tubes were then centrifuged in a Beckman Microfuge for 2 min at 4°C. The buffer and dibutylphthalate layers were aspirated and the cell pellet was counted in a Packard auto-gamma counter. For PRL binding studies to lymphocytes about 50,000 cpm of 125I-hPRL were added to each tube containing 2xl06 cells in a final volume of 0.5 ml Dulbecco's Medium. After 6 h of incubation at 37°C, cells were centrifuged for 1 min in a Beckman Microfuge, were washed once in 1 ml of Hank's balanced salt solution plus 0.1% BSA and were re-centrifuged again for 1 min. The buffer was aspirated and the cell pellet counted in a Packard auto-gamma counter. Dissociation of endogenously bound performed by exposing lymphocytes to
PRL high
from the receptor was concentrations of MgCI2,
Vol. 41, No. 8, 1987
Specific PRL Binding Sites
953
essentially as described by Russell et al. (I0). Lymphocytes (2x106 cells) were treated with 0.2 ml of 4 M MgCI 2 on ice. After 5 min, I ml of cold Dulbecco's medium was added and was centrifuged in a Beckman Microfuge. The supernatant was removed and the pellet was washed further by suspension in I ml Dulbecco's medium and was recentrifuged as before. Binding of 1251-hPRL was assayed as described above. The specific binding to erythrocytes or lymphocytes was evaluated as the difference between the binding in the absence and the presence of excess unlabeled hormone (2 ug/ml) expressed as per cent of the total counts present in i00 ~i of the incubated cell suspension (for erythrocytes) or present in the incubation medium (for lymphocytes). Scatchard (15) analysis of the PRL binding to erythrocytes or lymphocytes was performed by transformation of binding data obtained from the competition studies with increasing concentrations of unlabeled human PRL mixed with a fixed amount of tracer. The dissociation constant (Kd) and binding capacity were determined for unlabeled hormone concentration between 0.8 and i00 ng/ml. The specificity of 1251-hPRL binding was tested with the following unlabeled hormones: human prolactin (hPRL, NIADDK-I-6), ovine prolactin (oPRL, NIADDK-oPRL-I-I), human growth hormone (hGH, NIADDK-RP-I), ovine growth hormone (oGH, NIH-GH-SII), rat follicle
0
z © H
H H Z
--
20
--
40
-
60
_
80
-
O
rFSH pINS
•
oPRL
PRL
H
7// t 10 0
''""J, 10 t
UNLABELED
I
i
10 2
10 3
HO~ONE
(ng/ml)
FIG. 1 Gompetitive inhibition of the specific binding of 125I-hPRL to erythrocytes of term cord blood by increasing concentrations of unlabeled hPRL and by a single concentration of oPRL, rFSH and pINS. Inhibition in the presence of 2x104 ng/ml hPRL was considered to be 100%. Non specific binding was 0.5%.
954
Specific PRL Binding Sites
Vol. 41, No. 8, 1987
stimulating hormone (rFSH, NIADDK-RP-I) and porcine insulin (pINS, Schwarz/Mann). All determinations were performed in triplicate and the results expressed as mean values. The deviation from the mean was always less than 15% of the mean value. Plasma PRL concentrations were determined by radioimmunoassay using the kit supplied by Sorin (Saluggia, Italy) and the results expressed as ng/ml. Results In erythrocytes from healthy adult subjects of both sexes a very low specific binding of 1251-hPRL was found : 120 ± 95 cpm/4xl09 cells (0.24 ± 0.19%; n=5), whereas a higher binding was found in term cord blood: 530 ± 270 cpm/4xl09 cells (i.i ± 0.5%; n=9). Non-specific binding was between 200 and 300 cpm (0.4-0.6%). The binding specificity of 1251-hPRI to erythrocytes of term cord blood is reported in Fig. I. The binding was inhibited by unlabeled hPRL or oPRL, but not by rFSH or pINS. The binding specific capacity calculated by Scatchard analysis was 2.6 fmol/4xl09 cells and the Kd was 3.4x10-10M (Fig. 2). As binding
a of
comparison we have also studied in the same blood samples the 125I-pINS. With this hormone the binding both to adults
0.016
0.012
0.008 Z D 0
0.004
I
[
I hPRL
]
2
3
BOUND
(fmol/4x109
cells)
FIG. 2 Scatchard analysis of binding data from the competition studies 1251-hPRL and unlabeled hPRL using erythrocytes of term cord blood.
between
Vol. 41, No. 8, 1987
Specific PRL Binding Sites
955
and term cord blood samples was about one order of magnitude higher (5.4% ± 1.0 and 8.5% ± 1.9 respectively). The results are in agreement with data from Kappy & Plotnick (16).
TABLE I Specific Binding of 1251-hPRL to Lymphocytes Obtained from Blood Samples of Adults and Term Cord and Corresponding Plasma PRL Concentrations. Specific binding/2xl06cells PRL plasma level(ng/ml)
Subject cpm
Cord blood (n=5) Adult male (n=6) Adult female(n=4)
% total counts
920 ± 480 795 ± 230 845 ± 90
1.8 ± 1.0 1.6 ± 0.5 1.7 ± 0.2
408 9.3 16.8
± ± ±
127.9 2.5 3.1
Values are means ± S.D. Non specific binding was between 250 and 400 cpm (0.5 - 0.8%).
2.0
16
D
B
A
o 1.5
37°C
~o~_ 12
m
O .Q
~C 1.0
21
8
I
H u~ c~
'~
0.5
4
,'N ,e--
I I I 2 4 6 INCUBATION T I M E (h)
I 8
I I I I 0.5 I .0 I .5 2.0 1 2 5 I - h P R L (ng added)
FIG. 3 Specific binding of 1251-hPRL to adult human lymphocytes as a function of incubation time and temperature (A) and as a function of increasing concentrations of labeled hormone added (B). Non specific binding was 0.6%.
956
Specific PRL Binding Sites
Vol. 41, No. 8, 1987
Table I reports the specific binding of 1251-hPRL to adult human lymphocytes of both sexes and term cord bloods and the corresponding PRL plasma levels. An evident specific binding was observed in all samples examined without significant difference noted between the mean values of adults and term cord blood samples and between male and female adult volunteers. As reported in literature, very high PRL plasma levels were found in all term cord blood samples. Also the levels in male and female adult volunteers were in the normal range. After in vitro desaturation only slight increase in specific binding was found (+19.3 ± 7.6%) in lymphocytes from adult subjects of both sexes, whereas in lymphocytes from term cord blood a much more pronounced increase was observed (+54.3 ± 22.7%). The specific binding of 1251-hPRL to adult human lymphocytes was timeand temperature-dependent (Fig. 3A). Binding was maximal at 37°C reaching equilibrium after 6 h, whereas at 40C lower levels of binding were attained even after 8 h. When increasing amounts of 1251-hPRL were incubated with a fixed number of cells, specific binding was detected over the whole range of concentration studied (0.5-1.8 ng) and appeared to reach saturation by approximately 1.8 ng (Fig. 3B). The
binding
specificity
of
125I-hPRL
to adult
human
lymphocytes
0--
20-Z O ~40H H Z60-H
80--
--// I
' ''"'"I
10 0
, b
,
101
UNLADI~LED H O R M O N E
I
r,
10 2
,
1
10 3
(ng/ml)
FIG. 4 Competitive inhibition of the specific binding of 1251-hPRL to adult human lymphocytes by increasing concentration of the indicated unlabeled hormones. Inhibition in the presence of 2x104 ng/ml hPRL was considered to be 100%. Non specific binding was 0.5%.
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is reported in Fig.4. Addition of increasing amounts of unlabeled hPRL resulted in a substantial decrease in binding of labeled tracer, a simil~r inhibition was observed for oPRL. Of other peptide hormones tested, only hGH, a hormone structurally and functionally similar to hPRL, was able to compete for the 125I-hPRL binding, oGH was much less effective, whereas no competition was displayed by the other hormones used. The binding specific capacity calculated by Scatchard analysis was 3.7 fmol/2xl06 cells and the Kd was 3.9xlO-lOM (Fig. 5).
0.020~
0"0151~
c~ Z o
°°°V 0"0051
~ 2I hPRL
4I
6I
BOUND
( f m o l / 2 x 1 0 6 cells) FIG. 5 Scatchard analysis of binding data from the competition studies 125I-hPRL and unlabeled hPRL using adult human lymphocytes.
between
Discussion Our results show for the first time the presence of PRL receptor on erythrocytes both of adults and term cord blood. The binding is quite low, especially in the adult cells, whereas in samples from newborn blood it is much higher and also specific for lactogenic hormones. On the same cells the binding values of insulin, a hormone which also binds to membrane receptors, resulted very close to the values reported in literature, confirming the validity of our technique. The significance of finding specific PRL receptors on human red blood cells remains not clearly defined. The physiologic role of these binding sites on erythrocytes membranes could be related to the influence exerted by PRL on the regulation of human red cell sodium transport. As reported by Gopalakrishnan et al. (II) incubation of red cells with higher concentrations of PRL in vitro enhanced the cellular sodium level and produced a significant reduction in erythrocyte membrane adenosine triphosphatase activity, through an inhibition of the active sodium pump. On the other hand Parke and Horrobin (17) have observed a significant decrease in plasma levels of PRL when the same blood sample
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Specific PRL Binding Sites
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was estimated immediately after veinpuncture and 6 hours later. This finding suggested the possibility that erythrocytes may bind PRL, exerting a sort of buffering action. Cord blood erythrocytes showed a binding to PRL higher than did those from adults. This finding could suggest that may be PRL has a special role in intrauterine growth and development. We have found also on lymphocytes from adults and term cord blood the presence of PRL receptors. The binding was specific for lactogenic hormones (hPRL and hGH), temperature-dependent, showed high affinity for hPRL and increased after exposition of lymphocytes to high concentrations of MgCI 2. The more pronounced effect exerted by desaturation on the binding of the lymphocytes from term cord blood seems to be related to the very high PRL plasma levels which could occupate a larger number of binding sites. Our results generally are in agreement with the data on lymphocytes reported by Russel et al. (9); however our binding values are slightly higher than those obtained by these authors. This could be ascribed to the different ligand we used which was human labeled PRL instead of rat labeled PRL, obviously less specific for human cells. As far as the possible role of PRL on lymphocyte membranes is concerned, a large body of evidence exists on the effect of lactogenic hormones, and of PRL in particular, on the human lymphocytes both peripheral and in culture. Mercola et al. (18) have studied the effect of PRL and growth hormone on human T-lymphocyte proliferation in vitro and have observed a direct and species-specific stimulation of growth on normal and some neoplastic human T-cells. The role of PRL in the regulation of humoral and cell-mediated immunoresponses also appears important. Nagy et al. (19) demonstrated that antibody formation to sheep red blood cells and the development of contact dermatitis in response to dinitrochlorobenzene are impaired in hypophysectomized rats. PRL, growth hormone and placental lactogen restoled the immunocompetence of hypophysectomized animals. On the other hand, bromocriptine depressed PRL serum levels and suppressed both antibody formation processes and skin reactivity to dinitrochlorobenzene (20). The ability to restore immunocompetence in hypophysectomized animals by administration of PRL suggests the PRL receptors may be important in the regulation of lymphocyte-implicated immunoresponses. Russel et al. (9) further demonstrated that PRL receptors on human peripheral lymphocytes can be enhanced in number or blocked by cyclosporine, a potent immunosuppressive agent used in human organ transplant patients. Investigations on possible variations of the PRL binding capacity of red blood cells and lymphocytes in different physiological and pathological conditions are in progress in our laboratories.
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Acknowledgments We thank the NIADDK Pituitary Hormone supplying polypeptide hormones and Mrs. A. assistance.
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