Brain Research, 5(J7 (1990) 341-343 Elsevier
341
BRES 23923
Angiotensin II inhibits acetylcholine release from human temporal cortex" implications for cognition J.M. Barnes ~, N.M. Barnes ~, B. Costall ~, Z.P. Horovitz 2, J.W. Ironside 3, R.J. Naylor ~ and T.J. Williams ~ 1Postgraduate Studies in Pharmacology, The School of Pharmacy, University of Bradford, Bradford (U. K. ), 2The Squibb Institute for Medical Research, Princeton, NJ (U.S.A.) and ¢Department of Pathology. University of Leeds, Leeds (U. K. )
(Accepted 3 October 1989) Key words': Angiotensin II; Acetylcholine; Human temporal cortex
Angiotensin II was shown to inhibit potassium-stimulated release of [3H]acetylcholine from slices of fresh human temporal cortex, obtained at surgery, and subsequently loaded with [3H]choline for the biochemical analyses. The inhibitory effect of angiotensin I1 was antagonised by the specific angiotensin II receptor antagonist [l-sarcosine, 8-threonine]-angiotensin II. High affinity binding sites were identified in the human temporal cortex using [125I]angiotensin II, and may provide the functional site of action of angiotensin II to modify [:~H]acetylcholine release. Angiotensin converting enzyme ( A C E ) is a dipeptidylcarboxypeptidase (EC 3.4.15.1) which hydrolytically converts angiotensin I to form the active octapeptide, angiotensin II. The potential benefit of the A C E inhibitors to influence mental functioning was brought to light by the 'quality of life' study by Croog and colleagues 7. Performance in tasks such as 'trail making' was improved, and other authors provided anecdotal reports of improved mood of patients receiving captopril s'15. These reports found scientific support in a battery of tests for cognitive performance 5"6"~2"~3. These findings together have prompted careful clinical trials on the A C E inhibitors in situations of impaired cognitive performance, although the explanation for the mechanism whereby agents traditionally used to control cardiovascular problems could improve mental functioning remains to be determined. A clue to a neurochemical correlate was derived from studies showing that angiotensin II was potent to inhibit the 'in vitro' release of acetylcholine from rat entorhinal cortex z. If this inhibitory action could be prevented by the A C E inhibitors, then our present knowledge of cortical acetylcholine involvement with cognition l~ 11 would link such a response with potential to improve cognitive performance. However, hypotheses based on animal work may not reflect the human situation, and the present work shows that angiotensin lI can also inhibit the release of acetylcholine from human temporal cortex. Temporal cortex from the subdominant hemisphere was obtained during surgical removal of a large meningioma from a 47-year-old woman who had not received any drug treatment which may influence the
angiotensin system prior to surgery. Histological examination of temporal cortex immediately adjacent to the assayed specimen showed no evidence of neoplastic infiltration or any other significant abnormality. For release studies the tissue was used within 1 h of excision (maintained meanwhile at approximately 4 °C in isotonic saline), using techniques previously described 2. Briefly, 200 ~1 of settled brain slices ([).35 x 0.35 m m x thickness of cortical ribbon), preloaded with [3H]choline (15 Ci/mmol, A m e r s h a m ) , were loaded into each of 20 Swinnex perfusion chambers, each chamber constituting a separate channel. The tissue was perfused at a rate of 0.5 ml/min with Krebs' buffer containing 1.0 u M hemicholinium-3 (Sigma). After a 30-min washout period, 4-min fractions of perfusate were collected for 80 rain. After 12 min ($1) and 48 rain ($2) the tissue was stimulated by a 4-min pulse of elevated (20.0 mM) potassium chloride. In drug-treated channels, 1.0 u M angiotensin II (Asp-Arg-Val-Tyr-lle-His-Pro-Phe, Sigma), 0.1 /~M [lsarcosine, 8-threonine]angiotensin II (Sigma), separately or combined, were perfused 20 min prior to, during and subsequent to the S2 stimulation. The tritium content in the 4-min fractions, and that remaining in the tissue, were assayed by liquid scintillation spectroscopy (Tri-Carb 1900CA, efficiency 47%). Disintegrations per min were converted to fractional release by dividing by the total amount of radioactivity present in the tissue at the end of each 4-min collection period. The $2/S j ratio was calculated. Angiotensin ll recognition sites were quantified by saturation analysis with [1251]angiotensin II (9 concentrations, 0.02-4.0 nM, 1988 Ci/mmol, Amersham). For
Correspondence: N.M. Barnes, Postgraduate Studies in Pharmacology, The School of Pharmacy, University of Bradlk3rd, Bradford BD7 1DP, U.K.
0006-8993/90/$03.50 © 199(t Elsevier Scicncc Publishers B.V. (Biomedical Division)
342 binding studies tissue was homogenised in 40 vols. (wt/vol) ice-cold 50.0 mM HEPES buffer (pH 7.4) using a Polytron blender (setting 7 for 15 s). The homogenate was centrifuged at 48,000 g for 10 min at 4 °C and the pellet washed twice by resuspension and centrifugation. The resulting pellet was resuspended and incubated at 37 °C for 30 min in 40 vols. 50.0 mM HEPES buffer containing 5.0 mM NazEDTA and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4 before being centrifuged at 48,000 g for 10 min at 4 °C. The final pellet was resuspended in 20 vols. incubation buffer (50,0 mM HEPES buffer containing 5.0 mM Na2EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10.0 ~tM glucagon, 5.0 mM dithiothreitol and 0.5% (wt/vol) bovine serum albumin, pH 7.4) to form the binding homogenate. To initiate binding 250-ktl aliquots of human temporal cortex homogenate (or incubation buffer for filter blanks) were added in triplicate to 750 ~i of incubation buffer containing [12SI]angiotensin II in the presence and absence of 10.0/~M [1-sarcosine, 8-threonine]angiotensin II to define specific binding. The binding was allowed to proceed at 37 °C for 45 min before termination by rapid filtration through pre-soaked ( 0 . 1 % vol/vol polyethyieneimine) Whatman GF/B filters followed by washing with 10 ml ice-cold 0.9% (wt/vol) sodium chloride. Bound radioactivity was assayed by liquid scintillation spectroscopy at an efficiency of approximately 78%. To determine the angiotensin converting enzyme activity, the tissue was homogenised in 0.1 M dipotassium phosphate-0.3 M sodium chloride buffer, pH 8.3, using an ultrasonic homogeniser (70% power for 5 s, Soniprep 150 MSE). To initiate the enzyme reaction, 50 /A homogenate was added to 200/zl incubation buffer (5.0 mM hippuryl-L-histidyl-L-ieucine, final concentration, in 0.1 M dipotassium phosphate-0.3 M sodium chloride, pH 8.3) and incubated at 37 °C (or on ice for blanks) for 30 min. The reaction was terminated by the addition of 50 /~1 perchloric acid (final concentration 0.4 M) and the samples centrifuged at 15,000 g for 3 min at room temperature. 100 ~1 of the supernatant was added to 500 ktl 0.38 M sodium hydroxide followed by 50 ktl 0phthaldialdehyde (1.0% wt/vol in methanol) and 30 ~1 2.5 M phosphoric acid 10 min later. Fluorescence was then measured at an excitation wavelength of 360 nM and an emission wavelength of 500 riM. Enzymatic activity was expressed as product liberated above blank levels per mg protein. Protein assays were performed by the method of Bradford 4 using bovine serum albumin as standard. In the release studies, 4-rain pulses of 20.0 mM potassium chloride evoked the release of tritium from slices of human temporal cortex. The evoked release was approximately 2-3 times the basal release (basal fractional release approximately 2% per 4 min), The size of
1.2
1.0
0.8
~o 0.6
0.4
0.2
0.0
C
AT II
ANTAG
AT
II
+
ANTAG
Fig. 1. Modulation of potassium-stimulated [3H]acetylcholine release from slices of fresh human temporal cortex by angiotensin II (1.0 /~M, AT II) and interaction with [l-sarcosine, 8-threonine]angiotensin II (0.1/~M, ANTAG). Each value is the mean + S.E.M. of 3-5 channels. C, potassium only treatment; AT II, angiotensin II (1.0/~M); ANTAG, [1-sarcosine, 8-threonine]angiotensin II (0.1 ktM); AT II + ANTAG, angiotensin II (1.0/~M) and [1-sarcosine, 8-threonine]angiotensin II (0.1 ~tM). P < 0.05 (ANOVA). Significant difference between control and angiotensin 1I (**P < 0.01) and significant difference between angiotensin II and angiotensin II + antagonist (++P < 0.01), Dunnett's t-test.
the evoked release in response to the second stimulation ($2) was similar to that obtained from the first ($t). The resultant SJS 1 ratio for the potassium only treatment was 0.998 + 0.079 (mean + S.E.M., 4 channels). Angiotensin 1I (1.0 ~M) significantly inhibited potassium-stimulated tritium release by some 45% ($2/$1, ratio = 0:556 + 0.086, mean _+ S.E.M., 5 channels, P < 0.01, Dunnett's t-test, Fig. 1) whilst the angiotensin II receptor antagonist [1-sarcosine, 8-threonine]angiotensin I1 (0.1 /~M) when superfused alone failed to alter the $2/S t ratio (0.947 + 0.047, mean + S.E.M., 3 channels, Fig. 1). However, when superfused in combination with angiotensin II (i.0 /tM) the antagonist antagonised the inhibitory action of angiotensin II back to control values ($2/S1, ratio = 1.18 + 0.067, mean + S.E.M., 3 channels, Fig. 1). [1251]Angiotensin II binding studies identified a saturable specific binding site (defined by 10.0 /~M [1sarcosine, 8-threonine]angiotensin II). Following Scatchard transformation of the specific binding, it was apparent that [125I]angiotensin I1 was labelling a homogenous population of binding sites with high affinity (KD = 1.02 nM, B ..... = 8.6 fmol/mg protein, Fig. 2). Angiotensin converting enzyme activity in this human
343
t e m p o r a l cortex was shown to be 1.03 nmol/min/mg p r o t e i n (mean of triplicate determination). Using human temporal cortex tissue we have extended our previous findings that angiotensin II can inhibit potassium-stimulated [3H]acetylcholine release from rat cortical tissue 2. Furthermore, the specificity of this response has been confirmed using the specific angiotensin II receptor antagonist, [1-sarcosine, 8-threonine]angiotensin II 2. The failure of the antagonist alone to modify the release of [3H]acetylcholine reflects the absence of an endogenous angiotensin II tone in the 'in vitro' preparation. M o d u l a t i o n of acetylcholine release by c o m p o n e n t s of
the angiotensin system is further s u p p o r t e d by the work of Usinger and colleagues ~3. These workers found a decrease in central acetylcholine levels in the rat (reported as being due to an increase in release) following peripheral administration of an A C E inhibitor. This in combination with an increase in c G M P (possibly indicative of stimulation of guanylate cyclase by released acetylcholine ~9) indicates that in these studies the net effect of A C E inhibition is an increase in acetylcholine release. The physiological relevance of these findings gains support from the data that o t h e r c o m p o n e n t s of the angiotensin system are present in the human t e m p o r a l cortex, namely angiotensin-converting enzyme activity and high affinity binding sites for [125I]angiotensin 1I. Sites labelled with [125I]angiotensin II have previously been subject to a pharmacological characterisation, which confirmed that they do indeed represent the recognition site of the angiotensin II receptor -~. Such sites m a y provide the functional site of action of angiotensin II to modify acetylcholine release. The finding that angiotensin II can inhibit acetylcholine release from h u m a n t e m p o r a l cortex, with the knowledge that angiotensin II f o r m a t i o n can be inhibited by the angiotensin-converting enzyme inhibitors, suggests that this m a y provide a neurochemical correlate to the m o o d elevating and cognitive enhancing p r o p e r t i e s of the A C E inhibitors 5-s'~2'13"=5. This also lends further support to the d e v e l o p m e n t of A C E inhibitors as cognitive enhancing agents, particularly when A C E activity has been found to be elevated in the C S F ~4 and several brain regions ~ of patients with A l z h e i m e r ' s disease.
1 Arregui, A., Perry, E.K., Rossor, M. and Tomlinson, B.E,, Angiotensin-converting enzyme in Alzheimer's disease: increased activity in caudate nucleus and cortical areas, J. Neurochern., 38 (1982) 1490-1493. 2 Barnes, J.M., Barnes, N.M., Costall, B., Horovitz, Z.P. and Naylor, R.J., Angiotensin II inhibits the release of [3H]acetylcholine from rat entorhinal cortex 'in vitro', Brain Research. 491 (1989) 136-143. 3 Bennett, J.P. and Snyder, S.H., Angiotensin II binding to mammalian brain membranes, J. Biol. Chem., 251 (1976) 7423-7430. 4 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding, Anal. Biochem., 72 (1976) 248-254. 5 Costall, B., Coughlan, J., Horovitz, Z.P., Kelly, M.E., Naylor, R.J. and Tomkins, D.M., The effects of ACE inhibitors captopril and SQ29,852 in rodent tests of cognition, Pharmacol. Biochem. Behav., 33 (1989) 573-579. 6 Costall, B., Horovitz, Z.P., Kelly, M.E., Naylor, R.J. and Tomkins, D.M., Captopril improves basic performance and antagonises scopolamine impairment in a mouse habituation test, Br. J. Pharmacol., 95 (Suppl.) (1988) 882P. 7 Croog, S.H., Levine, S., Testa, M.A., et al., The effects of antihypertensive therapy on the quality of life, New Eng. J. Med., 314 (1986) 1657-1664. 8 Deiken, R.F., Captopril treatment of depression, Biol. Psychiat., 21 (1986) 1428-1452. 9 Lenox, R.H., Kant, G.J. and Meyerhoff, J.L., Regional
sensitivity of cyclic AMP and cyclic GMP in rat brain to central cholinergic stimulation, Life Sci., 26 (1980) 2201-2209. Rossor, M., The neurochemistry of cortical dementias. In S.M. Stahl, S.D. Iversen and E.C. Goodman (Eds.), Cognitive Neurochemistry, Oxford University Press, U.K., 1987, pp. 233-247. Rupniak, N.M.J. and Iversen, S.D., Primate models of senile dementia. In S.M. Stahl, S.D. Iversen and E.C. Goodman (Eds.), Cognitive Neurochemistry, Oxford University Press, U.K., 1987, pp. 57-72. Sudilovsky, A., Turnbull, B.A. and Gershon, S., Angiotensinconverting enzyme inhibition and extinction of shuttle box avoidance behaviour in the rat, 27th Annual Meeting of the American College of Neuropsychopharmacology, Abstract 130, 1988. Usinger, P., Hock, F.J., Wiemer, G., Gerhards, H.J., Henning, R. and Urbach, H., Hoe 288: indications on the memoryenhancing effects of a peptidase inhibitor, Drug Dev. Res., 14 (1988) 315--324, Zubenko, G.S., Marquis, J.K., Volicer, L., Direnfeld, L.K., Langlais, P.J. and Nixon, R.A., Cerebrospinal fluid levels of angiotensin-converting enzyme, acetylcholinesterase and dopamine metabolities in dementia associated with Alzheimer's disease and Parkinson's disease: a correlative study, Biol. Psychol., 21 (1986) 1365-1381. Zubenko, G.A. and Nixon, R.A., Mood-elevating effect of captopril in depressed patients, Am. J. Psychiat., 141 (1984) 110-111.
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Fig. 2. The binding of [~zSI]angiotensin II (0.02-4.0 nM) in the absence (total binding, []) and presence (non-specific binding, []) o f [ 1-sarcosine, 8-threonine]angiotensin II (10.0 ~M) to homogenate of human temporal cortex. The specific binding (B) is subject (inset) to Scatchard transformation. Results are presented where each value is the mean of a triplicate determination. (Bound, fmol/mg protein; B/F, fmol/mg protein/nM.)
10
11
12
13
14
15
341
BRES 23923
Angiotensin II inhibits acetylcholine release from human temporal cortex" implications for cognition J.M. Barnes ~, N.M. Barnes ~, B. Costall ~, Z.P. Horovitz 2, J.W. Ironside 3, R.J. Naylor ~ and T.J. Williams ~ 1Postgraduate Studies in Pharmacology, The School of Pharmacy, University of Bradford, Bradford (U. K. ), 2The Squibb Institute for Medical Research, Princeton, NJ (U.S.A.) and ¢Department of Pathology. University of Leeds, Leeds (U. K. )
(Accepted 3 October 1989) Key words': Angiotensin II; Acetylcholine; Human temporal cortex
Angiotensin II was shown to inhibit potassium-stimulated release of [3H]acetylcholine from slices of fresh human temporal cortex, obtained at surgery, and subsequently loaded with [3H]choline for the biochemical analyses. The inhibitory effect of angiotensin I1 was antagonised by the specific angiotensin II receptor antagonist [l-sarcosine, 8-threonine]-angiotensin II. High affinity binding sites were identified in the human temporal cortex using [125I]angiotensin II, and may provide the functional site of action of angiotensin II to modify [:~H]acetylcholine release. Angiotensin converting enzyme ( A C E ) is a dipeptidylcarboxypeptidase (EC 3.4.15.1) which hydrolytically converts angiotensin I to form the active octapeptide, angiotensin II. The potential benefit of the A C E inhibitors to influence mental functioning was brought to light by the 'quality of life' study by Croog and colleagues 7. Performance in tasks such as 'trail making' was improved, and other authors provided anecdotal reports of improved mood of patients receiving captopril s'15. These reports found scientific support in a battery of tests for cognitive performance 5"6"~2"~3. These findings together have prompted careful clinical trials on the A C E inhibitors in situations of impaired cognitive performance, although the explanation for the mechanism whereby agents traditionally used to control cardiovascular problems could improve mental functioning remains to be determined. A clue to a neurochemical correlate was derived from studies showing that angiotensin II was potent to inhibit the 'in vitro' release of acetylcholine from rat entorhinal cortex z. If this inhibitory action could be prevented by the A C E inhibitors, then our present knowledge of cortical acetylcholine involvement with cognition l~ 11 would link such a response with potential to improve cognitive performance. However, hypotheses based on animal work may not reflect the human situation, and the present work shows that angiotensin lI can also inhibit the release of acetylcholine from human temporal cortex. Temporal cortex from the subdominant hemisphere was obtained during surgical removal of a large meningioma from a 47-year-old woman who had not received any drug treatment which may influence the
angiotensin system prior to surgery. Histological examination of temporal cortex immediately adjacent to the assayed specimen showed no evidence of neoplastic infiltration or any other significant abnormality. For release studies the tissue was used within 1 h of excision (maintained meanwhile at approximately 4 °C in isotonic saline), using techniques previously described 2. Briefly, 200 ~1 of settled brain slices ([).35 x 0.35 m m x thickness of cortical ribbon), preloaded with [3H]choline (15 Ci/mmol, A m e r s h a m ) , were loaded into each of 20 Swinnex perfusion chambers, each chamber constituting a separate channel. The tissue was perfused at a rate of 0.5 ml/min with Krebs' buffer containing 1.0 u M hemicholinium-3 (Sigma). After a 30-min washout period, 4-min fractions of perfusate were collected for 80 rain. After 12 min ($1) and 48 rain ($2) the tissue was stimulated by a 4-min pulse of elevated (20.0 mM) potassium chloride. In drug-treated channels, 1.0 u M angiotensin II (Asp-Arg-Val-Tyr-lle-His-Pro-Phe, Sigma), 0.1 /~M [lsarcosine, 8-threonine]angiotensin II (Sigma), separately or combined, were perfused 20 min prior to, during and subsequent to the S2 stimulation. The tritium content in the 4-min fractions, and that remaining in the tissue, were assayed by liquid scintillation spectroscopy (Tri-Carb 1900CA, efficiency 47%). Disintegrations per min were converted to fractional release by dividing by the total amount of radioactivity present in the tissue at the end of each 4-min collection period. The $2/S j ratio was calculated. Angiotensin ll recognition sites were quantified by saturation analysis with [1251]angiotensin II (9 concentrations, 0.02-4.0 nM, 1988 Ci/mmol, Amersham). For
Correspondence: N.M. Barnes, Postgraduate Studies in Pharmacology, The School of Pharmacy, University of Bradlk3rd, Bradford BD7 1DP, U.K.
0006-8993/90/$03.50 © 199(t Elsevier Scicncc Publishers B.V. (Biomedical Division)
342 binding studies tissue was homogenised in 40 vols. (wt/vol) ice-cold 50.0 mM HEPES buffer (pH 7.4) using a Polytron blender (setting 7 for 15 s). The homogenate was centrifuged at 48,000 g for 10 min at 4 °C and the pellet washed twice by resuspension and centrifugation. The resulting pellet was resuspended and incubated at 37 °C for 30 min in 40 vols. 50.0 mM HEPES buffer containing 5.0 mM NazEDTA and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4 before being centrifuged at 48,000 g for 10 min at 4 °C. The final pellet was resuspended in 20 vols. incubation buffer (50,0 mM HEPES buffer containing 5.0 mM Na2EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10.0 ~tM glucagon, 5.0 mM dithiothreitol and 0.5% (wt/vol) bovine serum albumin, pH 7.4) to form the binding homogenate. To initiate binding 250-ktl aliquots of human temporal cortex homogenate (or incubation buffer for filter blanks) were added in triplicate to 750 ~i of incubation buffer containing [12SI]angiotensin II in the presence and absence of 10.0/~M [1-sarcosine, 8-threonine]angiotensin II to define specific binding. The binding was allowed to proceed at 37 °C for 45 min before termination by rapid filtration through pre-soaked ( 0 . 1 % vol/vol polyethyieneimine) Whatman GF/B filters followed by washing with 10 ml ice-cold 0.9% (wt/vol) sodium chloride. Bound radioactivity was assayed by liquid scintillation spectroscopy at an efficiency of approximately 78%. To determine the angiotensin converting enzyme activity, the tissue was homogenised in 0.1 M dipotassium phosphate-0.3 M sodium chloride buffer, pH 8.3, using an ultrasonic homogeniser (70% power for 5 s, Soniprep 150 MSE). To initiate the enzyme reaction, 50 /A homogenate was added to 200/zl incubation buffer (5.0 mM hippuryl-L-histidyl-L-ieucine, final concentration, in 0.1 M dipotassium phosphate-0.3 M sodium chloride, pH 8.3) and incubated at 37 °C (or on ice for blanks) for 30 min. The reaction was terminated by the addition of 50 /~1 perchloric acid (final concentration 0.4 M) and the samples centrifuged at 15,000 g for 3 min at room temperature. 100 ~1 of the supernatant was added to 500 ktl 0.38 M sodium hydroxide followed by 50 ktl 0phthaldialdehyde (1.0% wt/vol in methanol) and 30 ~1 2.5 M phosphoric acid 10 min later. Fluorescence was then measured at an excitation wavelength of 360 nM and an emission wavelength of 500 riM. Enzymatic activity was expressed as product liberated above blank levels per mg protein. Protein assays were performed by the method of Bradford 4 using bovine serum albumin as standard. In the release studies, 4-rain pulses of 20.0 mM potassium chloride evoked the release of tritium from slices of human temporal cortex. The evoked release was approximately 2-3 times the basal release (basal fractional release approximately 2% per 4 min), The size of
1.2
1.0
0.8
~o 0.6
0.4
0.2
0.0
C
AT II
ANTAG
AT
II
+
ANTAG
Fig. 1. Modulation of potassium-stimulated [3H]acetylcholine release from slices of fresh human temporal cortex by angiotensin II (1.0 /~M, AT II) and interaction with [l-sarcosine, 8-threonine]angiotensin II (0.1/~M, ANTAG). Each value is the mean + S.E.M. of 3-5 channels. C, potassium only treatment; AT II, angiotensin II (1.0/~M); ANTAG, [1-sarcosine, 8-threonine]angiotensin II (0.1 ktM); AT II + ANTAG, angiotensin II (1.0/~M) and [1-sarcosine, 8-threonine]angiotensin II (0.1 ~tM). P < 0.05 (ANOVA). Significant difference between control and angiotensin 1I (**P < 0.01) and significant difference between angiotensin II and angiotensin II + antagonist (++P < 0.01), Dunnett's t-test.
the evoked release in response to the second stimulation ($2) was similar to that obtained from the first ($t). The resultant SJS 1 ratio for the potassium only treatment was 0.998 + 0.079 (mean + S.E.M., 4 channels). Angiotensin 1I (1.0 ~M) significantly inhibited potassium-stimulated tritium release by some 45% ($2/$1, ratio = 0:556 + 0.086, mean _+ S.E.M., 5 channels, P < 0.01, Dunnett's t-test, Fig. 1) whilst the angiotensin II receptor antagonist [1-sarcosine, 8-threonine]angiotensin I1 (0.1 /~M) when superfused alone failed to alter the $2/S t ratio (0.947 + 0.047, mean + S.E.M., 3 channels, Fig. 1). However, when superfused in combination with angiotensin II (i.0 /tM) the antagonist antagonised the inhibitory action of angiotensin II back to control values ($2/S1, ratio = 1.18 + 0.067, mean + S.E.M., 3 channels, Fig. 1). [1251]Angiotensin II binding studies identified a saturable specific binding site (defined by 10.0 /~M [1sarcosine, 8-threonine]angiotensin II). Following Scatchard transformation of the specific binding, it was apparent that [125I]angiotensin I1 was labelling a homogenous population of binding sites with high affinity (KD = 1.02 nM, B ..... = 8.6 fmol/mg protein, Fig. 2). Angiotensin converting enzyme activity in this human
343
t e m p o r a l cortex was shown to be 1.03 nmol/min/mg p r o t e i n (mean of triplicate determination). Using human temporal cortex tissue we have extended our previous findings that angiotensin II can inhibit potassium-stimulated [3H]acetylcholine release from rat cortical tissue 2. Furthermore, the specificity of this response has been confirmed using the specific angiotensin II receptor antagonist, [1-sarcosine, 8-threonine]angiotensin II 2. The failure of the antagonist alone to modify the release of [3H]acetylcholine reflects the absence of an endogenous angiotensin II tone in the 'in vitro' preparation. M o d u l a t i o n of acetylcholine release by c o m p o n e n t s of
the angiotensin system is further s u p p o r t e d by the work of Usinger and colleagues ~3. These workers found a decrease in central acetylcholine levels in the rat (reported as being due to an increase in release) following peripheral administration of an A C E inhibitor. This in combination with an increase in c G M P (possibly indicative of stimulation of guanylate cyclase by released acetylcholine ~9) indicates that in these studies the net effect of A C E inhibition is an increase in acetylcholine release. The physiological relevance of these findings gains support from the data that o t h e r c o m p o n e n t s of the angiotensin system are present in the human t e m p o r a l cortex, namely angiotensin-converting enzyme activity and high affinity binding sites for [125I]angiotensin 1I. Sites labelled with [125I]angiotensin II have previously been subject to a pharmacological characterisation, which confirmed that they do indeed represent the recognition site of the angiotensin II receptor -~. Such sites m a y provide the functional site of action of angiotensin II to modify acetylcholine release. The finding that angiotensin II can inhibit acetylcholine release from h u m a n t e m p o r a l cortex, with the knowledge that angiotensin II f o r m a t i o n can be inhibited by the angiotensin-converting enzyme inhibitors, suggests that this m a y provide a neurochemical correlate to the m o o d elevating and cognitive enhancing p r o p e r t i e s of the A C E inhibitors 5-s'~2'13"=5. This also lends further support to the d e v e l o p m e n t of A C E inhibitors as cognitive enhancing agents, particularly when A C E activity has been found to be elevated in the C S F ~4 and several brain regions ~ of patients with A l z h e i m e r ' s disease.
1 Arregui, A., Perry, E.K., Rossor, M. and Tomlinson, B.E,, Angiotensin-converting enzyme in Alzheimer's disease: increased activity in caudate nucleus and cortical areas, J. Neurochern., 38 (1982) 1490-1493. 2 Barnes, J.M., Barnes, N.M., Costall, B., Horovitz, Z.P. and Naylor, R.J., Angiotensin II inhibits the release of [3H]acetylcholine from rat entorhinal cortex 'in vitro', Brain Research. 491 (1989) 136-143. 3 Bennett, J.P. and Snyder, S.H., Angiotensin II binding to mammalian brain membranes, J. Biol. Chem., 251 (1976) 7423-7430. 4 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding, Anal. Biochem., 72 (1976) 248-254. 5 Costall, B., Coughlan, J., Horovitz, Z.P., Kelly, M.E., Naylor, R.J. and Tomkins, D.M., The effects of ACE inhibitors captopril and SQ29,852 in rodent tests of cognition, Pharmacol. Biochem. Behav., 33 (1989) 573-579. 6 Costall, B., Horovitz, Z.P., Kelly, M.E., Naylor, R.J. and Tomkins, D.M., Captopril improves basic performance and antagonises scopolamine impairment in a mouse habituation test, Br. J. Pharmacol., 95 (Suppl.) (1988) 882P. 7 Croog, S.H., Levine, S., Testa, M.A., et al., The effects of antihypertensive therapy on the quality of life, New Eng. J. Med., 314 (1986) 1657-1664. 8 Deiken, R.F., Captopril treatment of depression, Biol. Psychiat., 21 (1986) 1428-1452. 9 Lenox, R.H., Kant, G.J. and Meyerhoff, J.L., Regional
sensitivity of cyclic AMP and cyclic GMP in rat brain to central cholinergic stimulation, Life Sci., 26 (1980) 2201-2209. Rossor, M., The neurochemistry of cortical dementias. In S.M. Stahl, S.D. Iversen and E.C. Goodman (Eds.), Cognitive Neurochemistry, Oxford University Press, U.K., 1987, pp. 233-247. Rupniak, N.M.J. and Iversen, S.D., Primate models of senile dementia. In S.M. Stahl, S.D. Iversen and E.C. Goodman (Eds.), Cognitive Neurochemistry, Oxford University Press, U.K., 1987, pp. 57-72. Sudilovsky, A., Turnbull, B.A. and Gershon, S., Angiotensinconverting enzyme inhibition and extinction of shuttle box avoidance behaviour in the rat, 27th Annual Meeting of the American College of Neuropsychopharmacology, Abstract 130, 1988. Usinger, P., Hock, F.J., Wiemer, G., Gerhards, H.J., Henning, R. and Urbach, H., Hoe 288: indications on the memoryenhancing effects of a peptidase inhibitor, Drug Dev. Res., 14 (1988) 315--324, Zubenko, G.S., Marquis, J.K., Volicer, L., Direnfeld, L.K., Langlais, P.J. and Nixon, R.A., Cerebrospinal fluid levels of angiotensin-converting enzyme, acetylcholinesterase and dopamine metabolities in dementia associated with Alzheimer's disease and Parkinson's disease: a correlative study, Biol. Psychol., 21 (1986) 1365-1381. Zubenko, G.A. and Nixon, R.A., Mood-elevating effect of captopril in depressed patients, Am. J. Psychiat., 141 (1984) 110-111.
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Fig. 2. The binding of [~zSI]angiotensin II (0.02-4.0 nM) in the absence (total binding, []) and presence (non-specific binding, []) o f [ 1-sarcosine, 8-threonine]angiotensin II (10.0 ~M) to homogenate of human temporal cortex. The specific binding (B) is subject (inset) to Scatchard transformation. Results are presented where each value is the mean of a triplicate determination. (Bound, fmol/mg protein; B/F, fmol/mg protein/nM.)
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