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Platelet vasopressin receptors in bipolar affective illness
f’~~~~hiczrr.~~ Reseawh.
7, 83-86
( 1982)
83
Elsevier Biomedical Press
Platelet
Vasopressin
Receptors
in Bipolar Affective
Illness
Wade H. Berrettini, John I. Nurnberger, Jr., E. Kathyrn Worthington, Susan Simmons-Ailing, and Elliot S. Gershon Received
December
28, 1981; accepted
April
19, 1982.
Abstract. Determinations of the affinity constants (KD) and number of binding sites (Bmax) for platelet vasopressin receptors were done in euthymic bipolar patients and normal volunteers. No significant differences were found between these groups. In addition, lithium treatment did not alter these receptor parameters in the bipolar group. Key Words.
Platelet
vasopressin
receptor,
lithium,
bipolar
affective
illness.
Vasopressin is a nonapeptide, synthesized in the hypothalamus and released from posterior pituitary, which regulates free water clearance in the kidney (Kamm et al., 1928). Additionally, vasopressin has been demonstrated to play important roles in memory and learning, as described by de Wied (1980). More recently, vasopressin has been found in high concentrations in various brain regions, including locus ceruleus, hypothalamus, and substantia nigra (Rossor et al., 1981). Iontophoretically applied vasopressin produces an excitatory response in the locus ceruleus (Olpe and Baltzer, 1981). There is evidence to suggest that vasopressin may be a neurotransmitter or a neuromodulator (Versteeg et al., 1979). Recent studies of vasopressin in affective illness may indicate a dysfunction in the physiology of this neuropeptide. These include a report of lower cerebrospinal fluid (CSF) vasopressin in nonpsychotic depression (Gold et al., 1978) and a study demonstrating cognitive improvement of some depressed patients in response to administration of a vasopressin analogue (Gold et al., in press). Finally, lithium’s antagonism of vasopressin function in the kidney (Forrest, 1979) might reflect a pharmacologically important action in the central nervous system (CNS). These considerations led us to investigate vasopressin function in bipolar affective illness, using a recently described assay for platelet vasopressin receptors (Berrettini et al., 1982).
Methods The patients in this study attended a clinic for bipolar affective illness in the Section on Psychogenetics, Biological Psychiatry Branch, National Institute of Mental Health. All patients satisfied DSM-I/I(American Psychiatric Association, 1980) criteria for bipolar affective illness. All were carefully screened, using general, psychiatric, and neurological examinations. Additionally, all patients received electrocardiogram, electroencephalogram, and skull and chest
Wade El. Berrettini. M.D., Ph.D., John I. Nurnberger, Gershon. M.D.. Psychobiology, (Reprint requests Rock>llle Pike. 0165-1781
M.D., Susan Simmons-Ailing, M.S.N., and Elliot S. are in the Section on Psychogenetics, and E. Kathyrn Worthington is in the Section on Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, MD. to Dr. W.H. Berrettini at Section on Psychogenetics, NIMH. Bldg. 10, Rm. 3N218,9000 Bethesda, MD 20205, U.S.A.)
‘X2:0000-0000:$02.75
0 Elsevier Biomedical
Press
84 films, as well as multiple blood tests to rule out coexisting major illness. All patients either were taking lithium alone or were medication free for 2 weeks before the study began. If patients were on lithium, the most recent blood level was between 0.5 and 1.5 mEq/l. All patients were euthymic for at least 2 weeks before study, by history and clinical observation. Normal volunteers were selected from persons responding to advertisements. They were carefully screened as outlined above. All normal volunteers had been drug free for 2 weeks before study. Approximately 60 ml of venous blood was drawn into four 20 ml evacuated glass tubes, each containing 1.5 ml of a 10% sodium citrate solution. The blood was transferred to two 50 ml polypropylene centrifuge tubes containing 2.5 ml of a 5% disodium ethylenediaminetetraacetic acid solution. Platelets were separated using the following technique. Blood was centrifuged at 275g for 30 minutes at room temperature. The platelet-rich plasma was carefully removed and spun at 4” C for 15 minutes at 4950g. The supernatant plasma was discarded. The pellet was washed twice in cold assay buffer (see below), and homogenized in 10 ml of that buffer. All preparations were used fresh, as a 10% decline in binding was noted after freezing. The binding parameters were determined as described previously (Berrettini et al., 1982), with some modification. Briefly, I251-arginine vasopressin (New England Nuclear) was diluted to a specific activity of 2 X lo4 Ci/ mole using arginine vasopressin (Sigma Chemical Co.) in the assay buffer (100 mM Tris, IO mM MgCI,, pH 8.0 at 4°C). Aliquots of this ligand (yielding seven final concentrations ranging from 0.6 nM to 4.0 nM) were added to glass test tubes containing 25 ~1 of 4y0 bovine serum albumin, 300 ~1 of platelet preparation and assay buffer. The final volume was 500 ~1. Nonspecific binding was defined as the ligand bound in the presence of 10 PM arginine vasopressin, and ranged from 15 to 40% of total binding. Incubations were for 90 minutes on ice. Assay mixtures were diluted rapidly with 4 ml of cold assay buffer and filtered through Whatman GFjC filters (presoaked for 2 hours in 1% bovine serum albumin). Filters were counted in a gamma counter at a 70% efficiency. Equilibrium constant (KD) and maximal binding (B mu) values were calculated from at least six different concentrations of labeled ligand. Scatchard analysis using linear regression was
used. If the correlation coefficient for the line was less than -0.85 for any six of seven points, the experiment was rejected. Approximately 1 of IO experiments was rejected in this fashion. Protein measurements were made using the method of Lowry et al. (I 95 I). Results As shown
in Table 1, there were no significant differences among the controls, the medication-free bipolar patients and the lithium-treated bipolar patients. There were no significant changes in binding parameters in the same patients during lithium treatment and during the medication-free period.
Discussion These results indicate that this parameter of vasopressin function does not differentiate controls from euthymic bipolar patients regardless of lithium treatment. In
Table 1.12%Arg-vasopressin binding to platelets of euthymic bipolar patients and normal volunteers (mean L SD) M/F
Group
Mean we
Mean
Mean
KD nM
Bmaxl
Lithium-treated
bipolar
416
35f
13
2.2 + 1.4
101 i 53
Medication-free
bipolar
417
36?
12
2.1 4 0.8
100 k 37
7/9
34k
16
2.0 k 1.0
94 i 42
Normal volunteers 1.
Bmax
in fmoles/mg protein.
85 addition, these results suggest that lithium treatment does not affect the binding parameters for the platelet vasopressin receptor. The platelet vasopressin receptor seems to be quite similar, in kinetic and pharmacologic profiles (Berrettini et al., 1982), to the kidney medulla receptor described previously (Bockaert et al., 1973). Previous studies of the lithium effect on renal free-water clearance have suggested that this ion may act to inhibit the vasopressinstimulated adenyl cyclase in the collecting duct (Forrest, 1979). This inhibition may occur in the absence of changes in KD or Bmax. These results, although negative, do not in any way exclude alterations in vasopressin function among bipolar patients. First, alterations may be state dependent, occurring in depression or mania (or both states); these changes may not be detected in a study of euthymic patients. Second, the function of the platelet receptor most probably involves hemostasis. It has been shown that vasopressin, in high concentrations, can cause platelets to release serotonin and to aggregate (Pearce et al., 1978). Alterations in vasopressin physiology among bipolar patients may be limited to CNS sites. However, vasopressin receptors in brain have not, as yet, been described. The relationship of platelet receptors to CNS receptors remains to be determined. Although the literature suggests that alterations in vasopressin physiology may occur in affective illness. no changes in the platelet receptor were detectable in the euthymic state. Acknowledgment. determinations.
The authors
wish to thank
N. Susan
Nadi,
Ph.D.,
for protein
References American Psychiatric Association. DSM-III: Diagnostic and Statistical Manual of Mental Disorders. 3rd ed. APA, Washington, DC (1980). Berrettini, W.H., Post, R.M., Worthington, E.K., and Casper, J.B. Human platelet vasopressin receptors. Life Sciences, 30, 425 (1982). Bockaert, J., Christian, R., Rajerison, R., and Jord, S. Specific binding of 3H-Lys-vasopressin to pig kidney plasma membranes. Journal of Biological Chemistry, 248, 5922 (1973). de Wied, D. Behavioral action of neurohypophyseal peptides. Proceedings of the Ro_~al Society, (B), 210, 183 (1980). Forrest, J.N. Lithium-induced polyuria: Cellular mechanisms and response to diuretics. In: Cooper, T.B., Gershon, S., Kline, N.S., and Schou, M., eds. Lithium: Controversies and Unresolved Issues. Excerpta Medica, Amsterdam (1979). Gold, P.W. Ballenger, J., Robertson, G.L., Post, R.M., and Goodwin, F.K. Vasopressin in affective illness: Direct measurement, clinical trials, and response to hypertonic saline. In: Post, R.M., and Ballenger, J., eds. The Neurobiology of Mood Disorders. Williams & Wilkins Company, Baltimore (in press). Gold, P.W., Goodwin, F.K., and Reus, V.I. Vasopressin in affective illness. Lancet, 1(8076), 1233 (1978). Kamm, O., Aldrich, T.B., Grote, I.W., Rowe, L.W., and Bugbee, E.P. The active principles of the posterior lobe of the pituitary gland. Journal of the American Chemical Societ,v. 50,573 (1928). Lowry, O.H., Rosebrough, N.J.. Farr, A.L., and Randall, R.J. Protein measurements with the Folin phenol reagent. Journal of Biological Chemistry. 193, 265 (1951). Olpe, H.-R., and Baltzer, V. Vasopressin activates noradrenergic neurons in the rat locus coeruleus: A microiontophoretic investigation. European Journal of Pharmacology. 73, 377 (1981).
86
Pearce, P.H., Wright, J.M., Egan, C.M., and Scrutton, M.C. Interaction of human blood platelets with the 2’,3’-dialdehyde and 2’,3’-dialcohol derivatives of adenosine 5’-diphosphate and adenosine S-triphosphate. European Journal of Biochemistry’, 88, 543 (1978). Rossor, M.V., Iversen, L.L., Hawthorn, J., Ang, V.T.Y., and Jenkins, J.S. Extrahypothalamic vasopressin in human brain. Brain Research, 214, 349 (198 1). Versteeg, D.H.G., de Kloet, E.R., van Wimersma Greidanos, T., and de Wied, D. Vasopressin modulates the activity of catecholamine containing neurons in specific brain regions. Neuroscience Letters, 11, 69 (1979).
7, 83-86
( 1982)
83
Elsevier Biomedical Press
Platelet
Vasopressin
Receptors
in Bipolar Affective
Illness
Wade H. Berrettini, John I. Nurnberger, Jr., E. Kathyrn Worthington, Susan Simmons-Ailing, and Elliot S. Gershon Received
December
28, 1981; accepted
April
19, 1982.
Abstract. Determinations of the affinity constants (KD) and number of binding sites (Bmax) for platelet vasopressin receptors were done in euthymic bipolar patients and normal volunteers. No significant differences were found between these groups. In addition, lithium treatment did not alter these receptor parameters in the bipolar group. Key Words.
Platelet
vasopressin
receptor,
lithium,
bipolar
affective
illness.
Vasopressin is a nonapeptide, synthesized in the hypothalamus and released from posterior pituitary, which regulates free water clearance in the kidney (Kamm et al., 1928). Additionally, vasopressin has been demonstrated to play important roles in memory and learning, as described by de Wied (1980). More recently, vasopressin has been found in high concentrations in various brain regions, including locus ceruleus, hypothalamus, and substantia nigra (Rossor et al., 1981). Iontophoretically applied vasopressin produces an excitatory response in the locus ceruleus (Olpe and Baltzer, 1981). There is evidence to suggest that vasopressin may be a neurotransmitter or a neuromodulator (Versteeg et al., 1979). Recent studies of vasopressin in affective illness may indicate a dysfunction in the physiology of this neuropeptide. These include a report of lower cerebrospinal fluid (CSF) vasopressin in nonpsychotic depression (Gold et al., 1978) and a study demonstrating cognitive improvement of some depressed patients in response to administration of a vasopressin analogue (Gold et al., in press). Finally, lithium’s antagonism of vasopressin function in the kidney (Forrest, 1979) might reflect a pharmacologically important action in the central nervous system (CNS). These considerations led us to investigate vasopressin function in bipolar affective illness, using a recently described assay for platelet vasopressin receptors (Berrettini et al., 1982).
Methods The patients in this study attended a clinic for bipolar affective illness in the Section on Psychogenetics, Biological Psychiatry Branch, National Institute of Mental Health. All patients satisfied DSM-I/I(American Psychiatric Association, 1980) criteria for bipolar affective illness. All were carefully screened, using general, psychiatric, and neurological examinations. Additionally, all patients received electrocardiogram, electroencephalogram, and skull and chest
Wade El. Berrettini. M.D., Ph.D., John I. Nurnberger, Gershon. M.D.. Psychobiology, (Reprint requests Rock>llle Pike. 0165-1781
M.D., Susan Simmons-Ailing, M.S.N., and Elliot S. are in the Section on Psychogenetics, and E. Kathyrn Worthington is in the Section on Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, MD. to Dr. W.H. Berrettini at Section on Psychogenetics, NIMH. Bldg. 10, Rm. 3N218,9000 Bethesda, MD 20205, U.S.A.)
‘X2:0000-0000:$02.75
0 Elsevier Biomedical
Press
84 films, as well as multiple blood tests to rule out coexisting major illness. All patients either were taking lithium alone or were medication free for 2 weeks before the study began. If patients were on lithium, the most recent blood level was between 0.5 and 1.5 mEq/l. All patients were euthymic for at least 2 weeks before study, by history and clinical observation. Normal volunteers were selected from persons responding to advertisements. They were carefully screened as outlined above. All normal volunteers had been drug free for 2 weeks before study. Approximately 60 ml of venous blood was drawn into four 20 ml evacuated glass tubes, each containing 1.5 ml of a 10% sodium citrate solution. The blood was transferred to two 50 ml polypropylene centrifuge tubes containing 2.5 ml of a 5% disodium ethylenediaminetetraacetic acid solution. Platelets were separated using the following technique. Blood was centrifuged at 275g for 30 minutes at room temperature. The platelet-rich plasma was carefully removed and spun at 4” C for 15 minutes at 4950g. The supernatant plasma was discarded. The pellet was washed twice in cold assay buffer (see below), and homogenized in 10 ml of that buffer. All preparations were used fresh, as a 10% decline in binding was noted after freezing. The binding parameters were determined as described previously (Berrettini et al., 1982), with some modification. Briefly, I251-arginine vasopressin (New England Nuclear) was diluted to a specific activity of 2 X lo4 Ci/ mole using arginine vasopressin (Sigma Chemical Co.) in the assay buffer (100 mM Tris, IO mM MgCI,, pH 8.0 at 4°C). Aliquots of this ligand (yielding seven final concentrations ranging from 0.6 nM to 4.0 nM) were added to glass test tubes containing 25 ~1 of 4y0 bovine serum albumin, 300 ~1 of platelet preparation and assay buffer. The final volume was 500 ~1. Nonspecific binding was defined as the ligand bound in the presence of 10 PM arginine vasopressin, and ranged from 15 to 40% of total binding. Incubations were for 90 minutes on ice. Assay mixtures were diluted rapidly with 4 ml of cold assay buffer and filtered through Whatman GFjC filters (presoaked for 2 hours in 1% bovine serum albumin). Filters were counted in a gamma counter at a 70% efficiency. Equilibrium constant (KD) and maximal binding (B mu) values were calculated from at least six different concentrations of labeled ligand. Scatchard analysis using linear regression was
used. If the correlation coefficient for the line was less than -0.85 for any six of seven points, the experiment was rejected. Approximately 1 of IO experiments was rejected in this fashion. Protein measurements were made using the method of Lowry et al. (I 95 I). Results As shown
in Table 1, there were no significant differences among the controls, the medication-free bipolar patients and the lithium-treated bipolar patients. There were no significant changes in binding parameters in the same patients during lithium treatment and during the medication-free period.
Discussion These results indicate that this parameter of vasopressin function does not differentiate controls from euthymic bipolar patients regardless of lithium treatment. In
Table 1.12%Arg-vasopressin binding to platelets of euthymic bipolar patients and normal volunteers (mean L SD) M/F
Group
Mean we
Mean
Mean
KD nM
Bmaxl
Lithium-treated
bipolar
416
35f
13
2.2 + 1.4
101 i 53
Medication-free
bipolar
417
36?
12
2.1 4 0.8
100 k 37
7/9
34k
16
2.0 k 1.0
94 i 42
Normal volunteers 1.
Bmax
in fmoles/mg protein.
85 addition, these results suggest that lithium treatment does not affect the binding parameters for the platelet vasopressin receptor. The platelet vasopressin receptor seems to be quite similar, in kinetic and pharmacologic profiles (Berrettini et al., 1982), to the kidney medulla receptor described previously (Bockaert et al., 1973). Previous studies of the lithium effect on renal free-water clearance have suggested that this ion may act to inhibit the vasopressinstimulated adenyl cyclase in the collecting duct (Forrest, 1979). This inhibition may occur in the absence of changes in KD or Bmax. These results, although negative, do not in any way exclude alterations in vasopressin function among bipolar patients. First, alterations may be state dependent, occurring in depression or mania (or both states); these changes may not be detected in a study of euthymic patients. Second, the function of the platelet receptor most probably involves hemostasis. It has been shown that vasopressin, in high concentrations, can cause platelets to release serotonin and to aggregate (Pearce et al., 1978). Alterations in vasopressin physiology among bipolar patients may be limited to CNS sites. However, vasopressin receptors in brain have not, as yet, been described. The relationship of platelet receptors to CNS receptors remains to be determined. Although the literature suggests that alterations in vasopressin physiology may occur in affective illness. no changes in the platelet receptor were detectable in the euthymic state. Acknowledgment. determinations.
The authors
wish to thank
N. Susan
Nadi,
Ph.D.,
for protein
References American Psychiatric Association. DSM-III: Diagnostic and Statistical Manual of Mental Disorders. 3rd ed. APA, Washington, DC (1980). Berrettini, W.H., Post, R.M., Worthington, E.K., and Casper, J.B. Human platelet vasopressin receptors. Life Sciences, 30, 425 (1982). Bockaert, J., Christian, R., Rajerison, R., and Jord, S. Specific binding of 3H-Lys-vasopressin to pig kidney plasma membranes. Journal of Biological Chemistry, 248, 5922 (1973). de Wied, D. Behavioral action of neurohypophyseal peptides. Proceedings of the Ro_~al Society, (B), 210, 183 (1980). Forrest, J.N. Lithium-induced polyuria: Cellular mechanisms and response to diuretics. In: Cooper, T.B., Gershon, S., Kline, N.S., and Schou, M., eds. Lithium: Controversies and Unresolved Issues. Excerpta Medica, Amsterdam (1979). Gold, P.W. Ballenger, J., Robertson, G.L., Post, R.M., and Goodwin, F.K. Vasopressin in affective illness: Direct measurement, clinical trials, and response to hypertonic saline. In: Post, R.M., and Ballenger, J., eds. The Neurobiology of Mood Disorders. Williams & Wilkins Company, Baltimore (in press). Gold, P.W., Goodwin, F.K., and Reus, V.I. Vasopressin in affective illness. Lancet, 1(8076), 1233 (1978). Kamm, O., Aldrich, T.B., Grote, I.W., Rowe, L.W., and Bugbee, E.P. The active principles of the posterior lobe of the pituitary gland. Journal of the American Chemical Societ,v. 50,573 (1928). Lowry, O.H., Rosebrough, N.J.. Farr, A.L., and Randall, R.J. Protein measurements with the Folin phenol reagent. Journal of Biological Chemistry. 193, 265 (1951). Olpe, H.-R., and Baltzer, V. Vasopressin activates noradrenergic neurons in the rat locus coeruleus: A microiontophoretic investigation. European Journal of Pharmacology. 73, 377 (1981).
86
Pearce, P.H., Wright, J.M., Egan, C.M., and Scrutton, M.C. Interaction of human blood platelets with the 2’,3’-dialdehyde and 2’,3’-dialcohol derivatives of adenosine 5’-diphosphate and adenosine S-triphosphate. European Journal of Biochemistry’, 88, 543 (1978). Rossor, M.V., Iversen, L.L., Hawthorn, J., Ang, V.T.Y., and Jenkins, J.S. Extrahypothalamic vasopressin in human brain. Brain Research, 214, 349 (198 1). Versteeg, D.H.G., de Kloet, E.R., van Wimersma Greidanos, T., and de Wied, D. Vasopressin modulates the activity of catecholamine containing neurons in specific brain regions. Neuroscience Letters, 11, 69 (1979).