STIMULATION OF ADENYLATE CYCLASE ACTIVITY IN DIFFERENT AREAS OF HUMAN BRAIN BY SUBSTANCE P M. 3. DUFFY”, J. WONG and D. POWELL Department of Biochemistry, Trinity College, Dublin 2, Eire (Accepted 2YJanuary
1975)
Summary-Substance P and adenylate cyclase activity were measured in different areas of human brain. Highest levels of both substance P and basal adenyiate cycla.seactivity were found in the hypothalmus, pineal gland and substantia nigra. Substance P was found to stimulate adenylate cyclase activity in all brain areas but not in liver tissue.
Substance P is an undecapeptide found in high concentrations in brain and intestine. The peptide has been purified from bovine hypothalamus (CHANG and LEEMAN, 1970), its amino acid sequence determined (CHANG, LEEMAN and NIALL, 1971) and synthesised by the solid phase procedure (TREGEAR, NIALL, POTTS, LEEMAN and CHANG, 1971). The synthetic product was found to possess equipotent activity in multiple bioassays and in immunoas~ys when compared with natural bovine substance P (TREGBAR ef at., 1971; POWELL, LEEHAN, TREGEAR, NIALL and POTTS, 1973}, suggesting identity between the synthetic and natural substance. A sensitive and specific radioimmunoassay for measuring the peptide was developed (POWELL et al., 1973) and this has shown immunochemical similarity between substance P in bovine, rat and human brains. The peptide causes a wide variety of effects both in vitro and in viva, which include excitation of neurones (KRNJEVI~: and MORRIS, 1974) stimulation of gut contraction and salivation (CHANG and LEEMAN, 1970) antagonism to morphine analgesia (STERN and HADZOVIC, 1973) potentiation of LSD-induced tremor (STERN, 1973) and stimulation of Na’ excretion in the kidney (MILLS, MACFARLANE and WARD, 1974). The physiological function of substance P is unknown. Its high concentration in brain and its uneven distribution in this tissue as determined by bioassay and by immunoassay suggests that the peptide might be a neurotransmitter (LEMBECK, 1953; PowELL et al., 1973). Since cyclic AMP is also thought to influence neurotransmission (DRUMMOND,1973) we decided to investigate the effect of substance P on cyclic AMP formation in human brain. We also decided to compare the distribution of substance P in brain with the distribution of basal adenylate cyclase activity and the extent of activation of adenylate cyclase activity by substance P in different brain regions. MATERIALS
AND METHODS
Drugs
Synthetic substance P and tyrosyl-8 substance P were kindly provided by Drs. Niall, Tregear and Potts, Endocrine Unit, Massachusetts General Hospital, Boston, U.S.A. Cyclic AMP and ATP were purchased from the Boehringer Corp., London Ltd., London W5, U.K. and [3H]-8-cyclic AMP (27.5 Ci/mmol) from the Radiochemical Centre, Amersham, Bucks, U.K. Procedure
Human brains were obtained at post-mortem from patients who died of coronary thrombosis. The brains were removed within 12 hr after death and dissected immediately. Adenylate cyclase activity was assayed by the method of ALBANO, MAUDSLEY, BROWN * Present address: Department of Radioisotopes. St. Vincent’s Hospital. Dublin 4, Eire. 61.5
M. J.
616
DUFFY. J. WONG and
D.
POWELL
and BARNES,(1973) as modified by DUFFY and POWELL(1975). The brain regions were homogenised in an ice-cold medium (I: 3 w/v) containing 0.25 M sucrose, 25 mM KCl, 5 mM MgCl, and 50 mM Tris-HCl buffer (pH 7.4) using 10 strokes with a hand operated Potter-Elvehjem homogeniser. The homogenate was filtered through 2 layers of cheese cloth and centrifuged at 16009 for 10 min at O-4°C. The pellet was used as the enzyme source. The standard adenylate cyclase assay system contained, in a final volume of 0.4 ml, (final concentrations) 2 mM ATP, 3 mM MgClz 10 mM NaCl, 10 mM KCl, 6 mM theophylline. 50mh4 Tris-HCl buffer (pH 7.4) and was with or without 1 ,UM substance P (1 PM substance P was found to give maximal stimulation of whole brain adenylate cyclase activity; DUFFY and POWELL, 1975). The reaction was started by the addition of enzyme preparation (1 mg protein) and incubation was carried out in a shaking water bath at 37°C for 1Omin. Enzyme activity was terminated by placing the incubation tubes in a boiling water bath for 3 min followed by freezing and thawing and centrifugation at 2000g for 10 min to remove insoluble material. Samples were then diluted appropriately for assay of cyclic AMP content by saturation analysis (BROWN,ALBANO,EKINSand SGHERZI,1971). Substance P was determined by radioimmunoassay as described by POWELL et al. (1973). Since the peptide contains no amino acid residues suitable for iodination, a substance P derivative was used with a tyrosyl residue substituted for the phenylalanine at position 8. The tyrosyl-8 derivitative was labelled with [’ 251] using ‘Chloramine T’ and separated from the free iodine by adsorption to microfine silica. After incubation of antiserum and synthetic substance P standard (or unknown sample) for 24 hours, radioactive peptide was added and incubation continued for another 48 hours. Antibodybound label was then separated from free label in the incubate by the addition of dextran coated charcoal. Substance P was extracted from the brain areas and purified to the stage before chromatography according to the method of CHANG and LEEMAN (1970). The recovery value was 48”/, + 2.3 (S.E.M. of 9 experiments). Protein was determined by the method of LOWRY,ROSEBROUGH, FARR and RANDALL (1951) using bovine serum albumin as standard. RESULTS Concentration of’ substance P in diflerent areas
qf the humarz brain
As shown in Table 1, immunoreactive substance P was found in all areas of the human brain investigated. However the concentration of the peptide varied very widely from region to region. Highest amounts were present in the substantia nigra, pineal gland and the hypothalamus with lowest amounts occurring in the frontal and temporal lobes. Table
I, Distribution
of substance
P, basal adenylate cyclase activity and substance cyclase in different areas of human brain Adqlate
P-stimulated
adenylate
cyclase act,wty
Substance P (1 i(M)
Stimulatmn by su bstancc P ‘i0
Frontal lobe (GM) Frontal lobe (WM) Parietal Lobe (GM) Parietal Lobe (WM) Temporal Lobe (GM) Temporal lobe (WM) Occq,tal lobe (GM) Occ~p,tal lobe (WM) Cerebellum fol~a Cerebellum (WM) Hypothalmus Substantna mpra Pmeal gland
Values are means + SE. Number of brains examined are given in parentheses. Substance P determinations were carried out in triplicate and adenylate cyclase activity measurements in duplicate. Significance of stimulation was calculated using paired Students t-test. GM: grey matter; WM: white matter.
Substance P and adenylate cyclase activity
Stimulation
of adenylate
cyclase
activity
by substance
617
P in different areas of human brain
As shown in Table 1, the basal adenylate cyclase activity also varied throughout the various brain regions; highest activity was present in the hypothalamus and lowest in the temporal lobe white matter. Synthetic substance P (1 PM) significantly stimulated the basal adenylate cyclase activity in all the areas. However the range of stimulation (48-82x) did not vary significantly from one brain area to another and there was no evidence of any correlation between the percentage stimulation of adenylate cyclase activity by synthetic substance P and endogenous concentration of the peptide. As a control, substance P (1 PM) had no stimulatory effect on adenylate cyclase activity in human liver prepared and assayed as described above for brain. DISCUSSION The detection of a relatively high concentration of substance P in the substantia nigra and hypothalamus using radioimmunoassay in this investigation is in agreement with previous findings using the less sensitive and less specific bioassay (ZETLER, 1970). However, the presence of the peptide in the pineal gland had not previously been reported and ZELTER(1970) using the bioassay failed to detect substance P in the cerebellum. The regional distribution of basal adenylate cyclase activities in human brain described here is in general agreement with previous findings (WILLIAMS,LKTLE and ENSINCK, 1969). The stimulation of the basal adenylate cyclase activity by substance P is one of the first cases to be reported of stimulation of the brain cyclase by a naturally occurring peptide. Glucagon and insulin, which are not thought to occur in brain, have previously been shown to have no effect on brain adenylate cyclase activity (WILLIAMSet al., 1969). Interestingly, catecholamines and prostaglandins, which are also found in brain, also stimulate brain adenylate cyclase activity in vitro (VON HUNGEN and ROBERTS,1973; COLLIERand ROY, 1974; DUFFY and POWELL, 1975). Finally this report is one of the first describing an action of synthetic substance P at a molecular level. Whether the stimulation of brain adenylate cyclase activity by the peptide is a physiological or pharmacological effect remains to be determined.
REFERENCES ALBANO,J., MAUDSLEY,D. V., BROWN, B. L. and BARNES,G. D. (1973). A simplified procedure determination of adenylate cyclase activity. Bioclzem. Sec. Trans. 1: 477-479.
for the
618
M. J. DUFFY, J. WONG and D. POWELL
VOI\ HUNGEN, K. and ROBERTS,S. (1973). Cathecholamine and Ca’+ activation of adenylate cyclase systems in synaptosomal fractions from rat cerebral cortex. Nature, New BioL 242: 58-60. WILLIAMS,R., LITTLE,S. and ENSINCK,J. (1969). Adenyl cyclase and phosphodiesterase activities in brain areas of man, monkey and rat. Am. J. Med. Sci. 258: 190-202. ZETLER.G. (1970). Biologically active peptides (substance P). In: Handbook gf Ncurochrrtristry. Vol. 4. (LAJTHA. A., Ed.), pp. 135-148, Plenum, New York.
1975)
Summary-Substance P and adenylate cyclase activity were measured in different areas of human brain. Highest levels of both substance P and basal adenyiate cycla.seactivity were found in the hypothalmus, pineal gland and substantia nigra. Substance P was found to stimulate adenylate cyclase activity in all brain areas but not in liver tissue.
Substance P is an undecapeptide found in high concentrations in brain and intestine. The peptide has been purified from bovine hypothalamus (CHANG and LEEMAN, 1970), its amino acid sequence determined (CHANG, LEEMAN and NIALL, 1971) and synthesised by the solid phase procedure (TREGEAR, NIALL, POTTS, LEEMAN and CHANG, 1971). The synthetic product was found to possess equipotent activity in multiple bioassays and in immunoas~ys when compared with natural bovine substance P (TREGBAR ef at., 1971; POWELL, LEEHAN, TREGEAR, NIALL and POTTS, 1973}, suggesting identity between the synthetic and natural substance. A sensitive and specific radioimmunoassay for measuring the peptide was developed (POWELL et al., 1973) and this has shown immunochemical similarity between substance P in bovine, rat and human brains. The peptide causes a wide variety of effects both in vitro and in viva, which include excitation of neurones (KRNJEVI~: and MORRIS, 1974) stimulation of gut contraction and salivation (CHANG and LEEMAN, 1970) antagonism to morphine analgesia (STERN and HADZOVIC, 1973) potentiation of LSD-induced tremor (STERN, 1973) and stimulation of Na’ excretion in the kidney (MILLS, MACFARLANE and WARD, 1974). The physiological function of substance P is unknown. Its high concentration in brain and its uneven distribution in this tissue as determined by bioassay and by immunoassay suggests that the peptide might be a neurotransmitter (LEMBECK, 1953; PowELL et al., 1973). Since cyclic AMP is also thought to influence neurotransmission (DRUMMOND,1973) we decided to investigate the effect of substance P on cyclic AMP formation in human brain. We also decided to compare the distribution of substance P in brain with the distribution of basal adenylate cyclase activity and the extent of activation of adenylate cyclase activity by substance P in different brain regions. MATERIALS
AND METHODS
Drugs
Synthetic substance P and tyrosyl-8 substance P were kindly provided by Drs. Niall, Tregear and Potts, Endocrine Unit, Massachusetts General Hospital, Boston, U.S.A. Cyclic AMP and ATP were purchased from the Boehringer Corp., London Ltd., London W5, U.K. and [3H]-8-cyclic AMP (27.5 Ci/mmol) from the Radiochemical Centre, Amersham, Bucks, U.K. Procedure
Human brains were obtained at post-mortem from patients who died of coronary thrombosis. The brains were removed within 12 hr after death and dissected immediately. Adenylate cyclase activity was assayed by the method of ALBANO, MAUDSLEY, BROWN * Present address: Department of Radioisotopes. St. Vincent’s Hospital. Dublin 4, Eire. 61.5
M. J.
616
DUFFY. J. WONG and
D.
POWELL
and BARNES,(1973) as modified by DUFFY and POWELL(1975). The brain regions were homogenised in an ice-cold medium (I: 3 w/v) containing 0.25 M sucrose, 25 mM KCl, 5 mM MgCl, and 50 mM Tris-HCl buffer (pH 7.4) using 10 strokes with a hand operated Potter-Elvehjem homogeniser. The homogenate was filtered through 2 layers of cheese cloth and centrifuged at 16009 for 10 min at O-4°C. The pellet was used as the enzyme source. The standard adenylate cyclase assay system contained, in a final volume of 0.4 ml, (final concentrations) 2 mM ATP, 3 mM MgClz 10 mM NaCl, 10 mM KCl, 6 mM theophylline. 50mh4 Tris-HCl buffer (pH 7.4) and was with or without 1 ,UM substance P (1 PM substance P was found to give maximal stimulation of whole brain adenylate cyclase activity; DUFFY and POWELL, 1975). The reaction was started by the addition of enzyme preparation (1 mg protein) and incubation was carried out in a shaking water bath at 37°C for 1Omin. Enzyme activity was terminated by placing the incubation tubes in a boiling water bath for 3 min followed by freezing and thawing and centrifugation at 2000g for 10 min to remove insoluble material. Samples were then diluted appropriately for assay of cyclic AMP content by saturation analysis (BROWN,ALBANO,EKINSand SGHERZI,1971). Substance P was determined by radioimmunoassay as described by POWELL et al. (1973). Since the peptide contains no amino acid residues suitable for iodination, a substance P derivative was used with a tyrosyl residue substituted for the phenylalanine at position 8. The tyrosyl-8 derivitative was labelled with [’ 251] using ‘Chloramine T’ and separated from the free iodine by adsorption to microfine silica. After incubation of antiserum and synthetic substance P standard (or unknown sample) for 24 hours, radioactive peptide was added and incubation continued for another 48 hours. Antibodybound label was then separated from free label in the incubate by the addition of dextran coated charcoal. Substance P was extracted from the brain areas and purified to the stage before chromatography according to the method of CHANG and LEEMAN (1970). The recovery value was 48”/, + 2.3 (S.E.M. of 9 experiments). Protein was determined by the method of LOWRY,ROSEBROUGH, FARR and RANDALL (1951) using bovine serum albumin as standard. RESULTS Concentration of’ substance P in diflerent areas
qf the humarz brain
As shown in Table 1, immunoreactive substance P was found in all areas of the human brain investigated. However the concentration of the peptide varied very widely from region to region. Highest amounts were present in the substantia nigra, pineal gland and the hypothalamus with lowest amounts occurring in the frontal and temporal lobes. Table
I, Distribution
of substance
P, basal adenylate cyclase activity and substance cyclase in different areas of human brain Adqlate
P-stimulated
adenylate
cyclase act,wty
Substance P (1 i(M)
Stimulatmn by su bstancc P ‘i0
Frontal lobe (GM) Frontal lobe (WM) Parietal Lobe (GM) Parietal Lobe (WM) Temporal Lobe (GM) Temporal lobe (WM) Occq,tal lobe (GM) Occ~p,tal lobe (WM) Cerebellum fol~a Cerebellum (WM) Hypothalmus Substantna mpra Pmeal gland
Values are means + SE. Number of brains examined are given in parentheses. Substance P determinations were carried out in triplicate and adenylate cyclase activity measurements in duplicate. Significance of stimulation was calculated using paired Students t-test. GM: grey matter; WM: white matter.
Substance P and adenylate cyclase activity
Stimulation
of adenylate
cyclase
activity
by substance
617
P in different areas of human brain
As shown in Table 1, the basal adenylate cyclase activity also varied throughout the various brain regions; highest activity was present in the hypothalamus and lowest in the temporal lobe white matter. Synthetic substance P (1 PM) significantly stimulated the basal adenylate cyclase activity in all the areas. However the range of stimulation (48-82x) did not vary significantly from one brain area to another and there was no evidence of any correlation between the percentage stimulation of adenylate cyclase activity by synthetic substance P and endogenous concentration of the peptide. As a control, substance P (1 PM) had no stimulatory effect on adenylate cyclase activity in human liver prepared and assayed as described above for brain. DISCUSSION The detection of a relatively high concentration of substance P in the substantia nigra and hypothalamus using radioimmunoassay in this investigation is in agreement with previous findings using the less sensitive and less specific bioassay (ZETLER, 1970). However, the presence of the peptide in the pineal gland had not previously been reported and ZELTER(1970) using the bioassay failed to detect substance P in the cerebellum. The regional distribution of basal adenylate cyclase activities in human brain described here is in general agreement with previous findings (WILLIAMS,LKTLE and ENSINCK, 1969). The stimulation of the basal adenylate cyclase activity by substance P is one of the first cases to be reported of stimulation of the brain cyclase by a naturally occurring peptide. Glucagon and insulin, which are not thought to occur in brain, have previously been shown to have no effect on brain adenylate cyclase activity (WILLIAMSet al., 1969). Interestingly, catecholamines and prostaglandins, which are also found in brain, also stimulate brain adenylate cyclase activity in vitro (VON HUNGEN and ROBERTS,1973; COLLIERand ROY, 1974; DUFFY and POWELL, 1975). Finally this report is one of the first describing an action of synthetic substance P at a molecular level. Whether the stimulation of brain adenylate cyclase activity by the peptide is a physiological or pharmacological effect remains to be determined.
REFERENCES ALBANO,J., MAUDSLEY,D. V., BROWN, B. L. and BARNES,G. D. (1973). A simplified procedure determination of adenylate cyclase activity. Bioclzem. Sec. Trans. 1: 477-479.
for the
618
M. J. DUFFY, J. WONG and D. POWELL
VOI\ HUNGEN, K. and ROBERTS,S. (1973). Cathecholamine and Ca’+ activation of adenylate cyclase systems in synaptosomal fractions from rat cerebral cortex. Nature, New BioL 242: 58-60. WILLIAMS,R., LITTLE,S. and ENSINCK,J. (1969). Adenyl cyclase and phosphodiesterase activities in brain areas of man, monkey and rat. Am. J. Med. Sci. 258: 190-202. ZETLER.G. (1970). Biologically active peptides (substance P). In: Handbook gf Ncurochrrtristry. Vol. 4. (LAJTHA. A., Ed.), pp. 135-148, Plenum, New York.
