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Reduction in human urinary MHPG excretion by guanethidine: Urinary MHPG as index of sympathetic nervous activity
Life Sciences, Vol . 24, pp . 1403-1406 Printed in the U.S .A .
Pergamon Press
RFDIICTION IN HUMAN URINARY MHPG E%CRETION BY GUANETHIDINS : URINARY MRPG AS INDER OF SYMPATHETIC NERVOUS ACTIVITY Joseph L. Izzo, Jr ., David Horwitz, a~ Harry R . Reiner Hypertension-Endocrine Branch, National Aeart, Lung, and Blood Institute, National Institutes of Health, Betheada, Maryland
20014
(Received in final form February 28, 1979) Summary The norepinephrine metabolites methoxyhydroxyphenyl glycol (MHPG) and vanillylmandelic acid (VMA) were measured in the urine of hypertenaive aubjecta before and during administration of guanethidine, a peripheral sympatholytic agent which does not cross the blood-brain barrier or deplete adrenal catecholaminea . Dosages of guanethidine (1 .2 mg/kg/day) sufficient to cause at least a 20 tort reduction in standing systolic blood pressure caused a mean 63X (maximum of 68X) reduction in urinary MHPG excretion (p~0 .01) while only causing a mean 37X (maximum of 44X) reduction (p<0 .005) in excretion of VMA. These results indicate that MHPG in human urine, ss in lower animals, is predominantly the product of peripheral sympathetic nervous system, rather than central nervous system norepinephrine metabolism . Urinary MHPG is more sensitive to specific sympatholytic therapy than is urinary VMA, and may be a useful index of sympathetic nervous activity . Controversy still exists about the origins of the norepinephrine metabolite methoxyhydroxyphenyl glycol (MHPG) in animal and human urine . Radiolabelled precursor studies have suggested that 65 to 90X of human urinary MHPG arises from the central nervous system (CNS) (1) . Other observations in mice (2) and rata (2,3,4) suggest that no more than 25 to 30X of urinary MHPG arisen from the CNS . Primate data are unclear ; in monkeys, between 20 and 65X of urinary MHPG is thought to be of CNS origin (5) . Resolution of this "central/peripheral" question is of interest because urinary MHPG has been proposed ss a marker o£ CNS norepinephrine metabolism (1,6) . However, if human urinary ?ff~G follows the pattern of lower animals and predominantly reflects peripheral norepinephrine turnover, it could be a useful marker of sympathetic nervous tone . Thin study reports changes in MHPG and vanillylmandelic acid (VMA) excretion in hypertensioe aubjecta treated with guanethidine, a peripheral sympatholytic agent, and draws inferences about the central vs . peripheral origins of human urinary MHPG . Methode Hypertenaive aubjecta, who had received no medications for at least two weeks, and in whom eecondary forms of hypertension were excluded, gave informed consent and were admitted to the Clinical Center for study . Diets were controlled for sodium and potassium intake (109 mEq and 100 mEq/day respectively) . Caffeine and foods high in monoaminea or monoamine metabolites were restricted . Subjects remained ambulatory but refrained from atremioua exercise . Blood pressure was measured at least 4 times daily by the arm cuff method with aubjecta in the supine 0024-3205/79/151403-0402 .00/0
1404
Sympatholytic Therapy Lowers Urinary MHPG
Vol, 24, No . 15, 1979
a~ standing positions . Consecutive 24-hour urine samples were collected and refrigerated ; each voided sample was split, with half added to a container with no preservative (for MHPG) and the other half preserved with HC1 (for VMA) . Following completion of each 24-hour collection, aliquots for MHPG were frozen at -20C, and aliquots for VMA were stored at 4 ° C until analysis . After an equilibration period of 5 to 7 days (control period), each subject received oral guanethidine daily for 6 to 10 days (treatment period) until there was at least a 20 torr reduction in systolic blood pressure (subject standing), Mean systolic and diastolic blood pressures and t4HPG and VMA excretion rates for the last two days each of the control and treatment periods were compared in each subject by paired t-teat . MHPG was measured by a method modified from Dekirmenjian and Maas (7), Urine samples were treated with B-glucuronidase/aryl sulfatase (Calbiochem) to hydrolyze conjugates, and extracted at pA 6 .2 with ethyl acetate (Fisher Chemical Co .) . Residues of these extracts were reacted with trifluoroacetic anhydride (Aldrich Chemical Co .) and the MHPG-traf luoroeaters were chromatographed on a 6-foot 3X SP-2401 column (Supelco, Inc .) at 160°C in a Hewlett-Packard 5830A gas chromatograph equipped with a nickel pulsed-electron-capture detector . Samples were quantitated by an internal standard method (MHPG-piperazine salt, Sigma Chemical Co .) VMA was determined by the method of Pisano (8) . Results Six subjects were studied as outlined, but the appearance of side effects (dizziness) in one subject prevented the desired 20 mm reduction in standing systolic blood pressure . Another subject withdrew from the study (for unrelated reasons) before completion . Both of these subjects demonstrated intermediate reductions in VMA and MHPG excretion and in blood pressure when compared to the data for the remaining 4 aubjecta as shown in Table I . Eden including the two incomplete studies (n-6), mean reduction in arterial pressure was 16%, mean reduction in MHPG excretion was 54%, and mean reduction in VMA excretion was 33X (statistical significances unchanged) . Data on the two partial studies were excluded from Table I in order to more clearly demonstrate the magnitude of reduction in metabolite excretion accompanying a therapeutic dose of guanethidine . Physiologic effects (lowering of blood pressure) occured simultaneously with biochemical effects (lowering of metabolite excretion rates) in all aubjecta . Two subjects were followed sequentially after withdrawal of guanethidine ; blood pressure and metabolite excretion rates returned to pre-treatment levels in about 7 days, Mean dosage of guanethidine was 99 ± 23 mg/day, or 1 .20 ± 0 .24 mg/kg/day ; mean duration of therapy was 8 .3 t 1 .0 day (all x ± SFSS, n=4) . Serum creatinine before guanethidine therapy was 1,1 ± 0 .13 mg/dl, and during guanethidine therapy 1 .2 ± x .18 mg/dl (all mean ± S .D ., n-4) . Discussion VMA and MHPG are the two major norepinephrine metabolites found in human urine . Values from this laboratory agree well with those reported by others (7-9) . The reader ie referred to more detailed descriptions of formation and excretion pathways for VMA and MHPG (lO), but selected points deserve mention here . Formation of either metabolite requires methylation by catechol-O~methyl transferase and oxidative deamination by monoamine oxidase (MAO) . The aldehyde intermediates produced by itAO are then oxidized to VMA or reduced to MHPG depending on the tissue . Both norepinephrine and epinephrine can be converted to both VMA and MHPG, but the major source of epinephrine, the adrenal medulla, contributes insignificantly to urinary MHPG (11) . MHPG is known to be the predominant nore-
Vol . 24, No . 15, 1979
Sympatholytic Therapy Lowers Urinary MHPG
1405
TABLE I Effect of Guanethidine on Blood Pressure and Norepinephrine Metabolite Excretion in Hypertenaive Subjects' Standing Blood Pressure (torr)
MHPG Excretion (mg/day)
VMA Excretion (mg/day)
CONTROL (C)
138 ± 8 97±6
2 .0 ± 0 .5
4 .2 ± 0 .4
TREATMENT (T)
111 ± 5 76±3
0 .71 ± 0 .15
2 .8 ± 0 .4
** -63 ± 5 (range 50-68)
** -37 ± 7 (range 20-44)
z CHANGE (T compared to C) * P
-20 ± 2 -22 ± 3 <0 .002 <0 .005
~ 0.01
<0 .005
t n=4, results expressed as mean ± SEtI . See text for full explanation . * Comparison of T to C by paired Student t-test . ** Different at p<0 .05 by unpaired Student t-teat .
niaephrine metabolite in both brain (12) and vascular sympathetic neurons (13), but VMA, a minor metabolite of central norepinephrine (12), is essentially peripheral in origin . Although MHPG arises from both central and peripheral (extra-adrenal) norepinephrine, predominance of one source over the other would justify correlation of urinary MHPG excretion with either behavioral or somatic pathophysiological events . Guanethidine would seem to be an ideal tool for investigation of the origins of human urinary MHPG . Thie peripheral sympatholytic agent depletes poatganglionic sympathetic norepinephrine stores (14), but does not cross the blood-brain harrier (15) . Brain and adrenal catecholamines are resistant to depletion by guanethidine, even in doses 30 to 100-fold greater than those employed here (15) . Guanethidine is not known to affect transport processes, and does not inhibit tissue MAO (16) or methyl tranaferasea (17), or interfere with assay methods for either VMA or MHPG (18) . Thin study was designed to achieve a moderate physiologic response (20 torr reduction in systolic blood pressure ; subject standing), not to maximally suppress s"~mpathetic function or MHPG excretion . Consequently, MHPG excretion in these subjects probably could have been reduced further than 687, by higher doses of guanethidine . No data are available on the effect of guanethidine on urinary t~IHPG in normal subjects, but debrisoquine, a drug related to guanethidine, caused marked reductions in MHPG excretion in normotensive rata (4) . It ie uncertain whether urinary VMA is sufficiently sensitive or specific to be used as a clinical marker of sympathetic function (10) . However, urinary MRPG responded more markedly than urinary VMA (633 va . 37°~ reduction, p<0 .05) to specific sympatholytic therapy . Additional evidence in support of the greater specificity of MHPG for sympathetic function is provided by preliminary observations in a patient with severe idiopathic orthoatatic hypotension, anhydrosis,
1406
Sympatholytic Therapy Lowers Urinary 1~4-IPG
Vol . 24, No . 15, 1979
impotence, no signs of central nervous system disease, and low plasma norepiaephrine which did not respond to postural change . In this man, VMA excretion (1 .6 mg/day) was about one-third of the normal mean for this laboratory, but MHPG excretion (<0 .2 mg/day) was about one-tenth of the normal mean (18) . Thus, present evidence indicates that in humane, as in lower animals, urinary MHPG arises predominantly from peripheral (sympathetic nervous system) norepinephrine . This is in contrast to indirect data (1), which may have underestimated MHPG is more stable than norepinephrine, the size of the peripheral MHPG pool . can be more easily measured than norepinephrine, and offers a time-integrated index of sympathetic norepinephrine turnover which may be a useful complement to the "point-in-time" measurements of plasma norepinephrine . Acknow ledgement The authors are grateful for the helpful suggestions and encouragement of Drs. Walter Lovenberg and John Pisano ; the technical assistance of Mr . Rodman Turner, Mrs . Marian Warner, and Miss Ethel Boykine ; and the secretarial assistance of Mrs . Peggy Davis . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .
M .H . EBERT and I .J . KOPIN, Trana . Am . Assoc . Phys . _88 256 (1975) . D .R . HOWLETT, F .A . JENNER, and S .R . NAHORSKI, J. Pharm. Pharmac . _27 : 449 (1975) . R . HOELDTKE, M . ROGAWSRI, and R.J . WURTMAN, Br . J . Pharm . _50 265 (1974) . F . KAROUM, R. WYATT, and E . COSTA, Neuropharm . _13 165 (1974) . J .W . MAAS, H. DEKIRMENJIAN, D. CARVER, et al ., Eur . J . Pharm. _23 121 (1973) . W.E . BUNNEY, Jr ., et al ., Ann . Int . Med . _87 319 (1977) . H . DEKIRMENJIAN and J .W . MAAS, Analyt . Biochem . _35 113 (1970) . J .J . PISANO, J .R . CROUT, and D. ABRAHAM, Clin . Chien. Acts _7 285 (1962) . J .L . IZZO, Jr ., D. HORWITZ, J .J . PISANO, and H.R . KEISER, Clin . Rea . _26 364 (1978) . I .J . KOPIN, Clin . Endo . Metab. _6 525 (1977) . F . RAROUM and E . COSTA, Biochem . Pharm . _23 533 (1974) . S .M . SCHANBERG, J .J . SCHILDKRAUT, G.R . BltEESE, AND I.J . ROPIN, Biochem. Pharm. _17 247 (1968) . S .M . MULDOON, P .M . VANHOUTTE, AND G.M . TYCE, Am . J . Physiol _234 H235 (1978) . R . CASS, R . KUNTZMAN, and B . B. BRODIE, Proc . Soc . Exp"". Biol . Med . _103 871 (1960) . R. KUNTZafAN, E . COSTA, G .L . CESSA, and B. B. BRODIE, Life Sci . _3 65 (1962) . T .L . CHRUSCIEL, Int . J . Neuropharm . _1 137 (1962) . E .T . ABBS, Life Sci. _1 99 (1962) . J .L . IZZO, Jr ., unpublished observations .
Pergamon Press
RFDIICTION IN HUMAN URINARY MHPG E%CRETION BY GUANETHIDINS : URINARY MRPG AS INDER OF SYMPATHETIC NERVOUS ACTIVITY Joseph L. Izzo, Jr ., David Horwitz, a~ Harry R . Reiner Hypertension-Endocrine Branch, National Aeart, Lung, and Blood Institute, National Institutes of Health, Betheada, Maryland
20014
(Received in final form February 28, 1979) Summary The norepinephrine metabolites methoxyhydroxyphenyl glycol (MHPG) and vanillylmandelic acid (VMA) were measured in the urine of hypertenaive aubjecta before and during administration of guanethidine, a peripheral sympatholytic agent which does not cross the blood-brain barrier or deplete adrenal catecholaminea . Dosages of guanethidine (1 .2 mg/kg/day) sufficient to cause at least a 20 tort reduction in standing systolic blood pressure caused a mean 63X (maximum of 68X) reduction in urinary MHPG excretion (p~0 .01) while only causing a mean 37X (maximum of 44X) reduction (p<0 .005) in excretion of VMA. These results indicate that MHPG in human urine, ss in lower animals, is predominantly the product of peripheral sympathetic nervous system, rather than central nervous system norepinephrine metabolism . Urinary MHPG is more sensitive to specific sympatholytic therapy than is urinary VMA, and may be a useful index of sympathetic nervous activity . Controversy still exists about the origins of the norepinephrine metabolite methoxyhydroxyphenyl glycol (MHPG) in animal and human urine . Radiolabelled precursor studies have suggested that 65 to 90X of human urinary MHPG arises from the central nervous system (CNS) (1) . Other observations in mice (2) and rata (2,3,4) suggest that no more than 25 to 30X of urinary MHPG arisen from the CNS . Primate data are unclear ; in monkeys, between 20 and 65X of urinary MHPG is thought to be of CNS origin (5) . Resolution of this "central/peripheral" question is of interest because urinary MHPG has been proposed ss a marker o£ CNS norepinephrine metabolism (1,6) . However, if human urinary ?ff~G follows the pattern of lower animals and predominantly reflects peripheral norepinephrine turnover, it could be a useful marker of sympathetic nervous tone . Thin study reports changes in MHPG and vanillylmandelic acid (VMA) excretion in hypertensioe aubjecta treated with guanethidine, a peripheral sympatholytic agent, and draws inferences about the central vs . peripheral origins of human urinary MHPG . Methode Hypertenaive aubjecta, who had received no medications for at least two weeks, and in whom eecondary forms of hypertension were excluded, gave informed consent and were admitted to the Clinical Center for study . Diets were controlled for sodium and potassium intake (109 mEq and 100 mEq/day respectively) . Caffeine and foods high in monoaminea or monoamine metabolites were restricted . Subjects remained ambulatory but refrained from atremioua exercise . Blood pressure was measured at least 4 times daily by the arm cuff method with aubjecta in the supine 0024-3205/79/151403-0402 .00/0
1404
Sympatholytic Therapy Lowers Urinary MHPG
Vol, 24, No . 15, 1979
a~ standing positions . Consecutive 24-hour urine samples were collected and refrigerated ; each voided sample was split, with half added to a container with no preservative (for MHPG) and the other half preserved with HC1 (for VMA) . Following completion of each 24-hour collection, aliquots for MHPG were frozen at -20C, and aliquots for VMA were stored at 4 ° C until analysis . After an equilibration period of 5 to 7 days (control period), each subject received oral guanethidine daily for 6 to 10 days (treatment period) until there was at least a 20 torr reduction in systolic blood pressure (subject standing), Mean systolic and diastolic blood pressures and t4HPG and VMA excretion rates for the last two days each of the control and treatment periods were compared in each subject by paired t-teat . MHPG was measured by a method modified from Dekirmenjian and Maas (7), Urine samples were treated with B-glucuronidase/aryl sulfatase (Calbiochem) to hydrolyze conjugates, and extracted at pA 6 .2 with ethyl acetate (Fisher Chemical Co .) . Residues of these extracts were reacted with trifluoroacetic anhydride (Aldrich Chemical Co .) and the MHPG-traf luoroeaters were chromatographed on a 6-foot 3X SP-2401 column (Supelco, Inc .) at 160°C in a Hewlett-Packard 5830A gas chromatograph equipped with a nickel pulsed-electron-capture detector . Samples were quantitated by an internal standard method (MHPG-piperazine salt, Sigma Chemical Co .) VMA was determined by the method of Pisano (8) . Results Six subjects were studied as outlined, but the appearance of side effects (dizziness) in one subject prevented the desired 20 mm reduction in standing systolic blood pressure . Another subject withdrew from the study (for unrelated reasons) before completion . Both of these subjects demonstrated intermediate reductions in VMA and MHPG excretion and in blood pressure when compared to the data for the remaining 4 aubjecta as shown in Table I . Eden including the two incomplete studies (n-6), mean reduction in arterial pressure was 16%, mean reduction in MHPG excretion was 54%, and mean reduction in VMA excretion was 33X (statistical significances unchanged) . Data on the two partial studies were excluded from Table I in order to more clearly demonstrate the magnitude of reduction in metabolite excretion accompanying a therapeutic dose of guanethidine . Physiologic effects (lowering of blood pressure) occured simultaneously with biochemical effects (lowering of metabolite excretion rates) in all aubjecta . Two subjects were followed sequentially after withdrawal of guanethidine ; blood pressure and metabolite excretion rates returned to pre-treatment levels in about 7 days, Mean dosage of guanethidine was 99 ± 23 mg/day, or 1 .20 ± 0 .24 mg/kg/day ; mean duration of therapy was 8 .3 t 1 .0 day (all x ± SFSS, n=4) . Serum creatinine before guanethidine therapy was 1,1 ± 0 .13 mg/dl, and during guanethidine therapy 1 .2 ± x .18 mg/dl (all mean ± S .D ., n-4) . Discussion VMA and MHPG are the two major norepinephrine metabolites found in human urine . Values from this laboratory agree well with those reported by others (7-9) . The reader ie referred to more detailed descriptions of formation and excretion pathways for VMA and MHPG (lO), but selected points deserve mention here . Formation of either metabolite requires methylation by catechol-O~methyl transferase and oxidative deamination by monoamine oxidase (MAO) . The aldehyde intermediates produced by itAO are then oxidized to VMA or reduced to MHPG depending on the tissue . Both norepinephrine and epinephrine can be converted to both VMA and MHPG, but the major source of epinephrine, the adrenal medulla, contributes insignificantly to urinary MHPG (11) . MHPG is known to be the predominant nore-
Vol . 24, No . 15, 1979
Sympatholytic Therapy Lowers Urinary MHPG
1405
TABLE I Effect of Guanethidine on Blood Pressure and Norepinephrine Metabolite Excretion in Hypertenaive Subjects' Standing Blood Pressure (torr)
MHPG Excretion (mg/day)
VMA Excretion (mg/day)
CONTROL (C)
138 ± 8 97±6
2 .0 ± 0 .5
4 .2 ± 0 .4
TREATMENT (T)
111 ± 5 76±3
0 .71 ± 0 .15
2 .8 ± 0 .4
** -63 ± 5 (range 50-68)
** -37 ± 7 (range 20-44)
z CHANGE (T compared to C) * P
-20 ± 2 -22 ± 3 <0 .002 <0 .005
~ 0.01
<0 .005
t n=4, results expressed as mean ± SEtI . See text for full explanation . * Comparison of T to C by paired Student t-test . ** Different at p<0 .05 by unpaired Student t-teat .
niaephrine metabolite in both brain (12) and vascular sympathetic neurons (13), but VMA, a minor metabolite of central norepinephrine (12), is essentially peripheral in origin . Although MHPG arises from both central and peripheral (extra-adrenal) norepinephrine, predominance of one source over the other would justify correlation of urinary MHPG excretion with either behavioral or somatic pathophysiological events . Guanethidine would seem to be an ideal tool for investigation of the origins of human urinary MHPG . Thie peripheral sympatholytic agent depletes poatganglionic sympathetic norepinephrine stores (14), but does not cross the blood-brain harrier (15) . Brain and adrenal catecholamines are resistant to depletion by guanethidine, even in doses 30 to 100-fold greater than those employed here (15) . Guanethidine is not known to affect transport processes, and does not inhibit tissue MAO (16) or methyl tranaferasea (17), or interfere with assay methods for either VMA or MHPG (18) . Thin study was designed to achieve a moderate physiologic response (20 torr reduction in systolic blood pressure ; subject standing), not to maximally suppress s"~mpathetic function or MHPG excretion . Consequently, MHPG excretion in these subjects probably could have been reduced further than 687, by higher doses of guanethidine . No data are available on the effect of guanethidine on urinary t~IHPG in normal subjects, but debrisoquine, a drug related to guanethidine, caused marked reductions in MHPG excretion in normotensive rata (4) . It ie uncertain whether urinary VMA is sufficiently sensitive or specific to be used as a clinical marker of sympathetic function (10) . However, urinary MRPG responded more markedly than urinary VMA (633 va . 37°~ reduction, p<0 .05) to specific sympatholytic therapy . Additional evidence in support of the greater specificity of MHPG for sympathetic function is provided by preliminary observations in a patient with severe idiopathic orthoatatic hypotension, anhydrosis,
1406
Sympatholytic Therapy Lowers Urinary 1~4-IPG
Vol . 24, No . 15, 1979
impotence, no signs of central nervous system disease, and low plasma norepiaephrine which did not respond to postural change . In this man, VMA excretion (1 .6 mg/day) was about one-third of the normal mean for this laboratory, but MHPG excretion (<0 .2 mg/day) was about one-tenth of the normal mean (18) . Thus, present evidence indicates that in humane, as in lower animals, urinary MHPG arises predominantly from peripheral (sympathetic nervous system) norepinephrine . This is in contrast to indirect data (1), which may have underestimated MHPG is more stable than norepinephrine, the size of the peripheral MHPG pool . can be more easily measured than norepinephrine, and offers a time-integrated index of sympathetic norepinephrine turnover which may be a useful complement to the "point-in-time" measurements of plasma norepinephrine . Acknow ledgement The authors are grateful for the helpful suggestions and encouragement of Drs. Walter Lovenberg and John Pisano ; the technical assistance of Mr . Rodman Turner, Mrs . Marian Warner, and Miss Ethel Boykine ; and the secretarial assistance of Mrs . Peggy Davis . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .
M .H . EBERT and I .J . KOPIN, Trana . Am . Assoc . Phys . _88 256 (1975) . D .R . HOWLETT, F .A . JENNER, and S .R . NAHORSKI, J. Pharm. Pharmac . _27 : 449 (1975) . R . HOELDTKE, M . ROGAWSRI, and R.J . WURTMAN, Br . J . Pharm . _50 265 (1974) . F . KAROUM, R. WYATT, and E . COSTA, Neuropharm . _13 165 (1974) . J .W . MAAS, H. DEKIRMENJIAN, D. CARVER, et al ., Eur . J . Pharm. _23 121 (1973) . W.E . BUNNEY, Jr ., et al ., Ann . Int . Med . _87 319 (1977) . H . DEKIRMENJIAN and J .W . MAAS, Analyt . Biochem . _35 113 (1970) . J .J . PISANO, J .R . CROUT, and D. ABRAHAM, Clin . Chien. Acts _7 285 (1962) . J .L . IZZO, Jr ., D. HORWITZ, J .J . PISANO, and H.R . KEISER, Clin . Rea . _26 364 (1978) . I .J . KOPIN, Clin . Endo . Metab. _6 525 (1977) . F . RAROUM and E . COSTA, Biochem . Pharm . _23 533 (1974) . S .M . SCHANBERG, J .J . SCHILDKRAUT, G.R . BltEESE, AND I.J . ROPIN, Biochem. Pharm. _17 247 (1968) . S .M . MULDOON, P .M . VANHOUTTE, AND G.M . TYCE, Am . J . Physiol _234 H235 (1978) . R . CASS, R . KUNTZMAN, and B . B. BRODIE, Proc . Soc . Exp"". Biol . Med . _103 871 (1960) . R. KUNTZafAN, E . COSTA, G .L . CESSA, and B. B. BRODIE, Life Sci . _3 65 (1962) . T .L . CHRUSCIEL, Int . J . Neuropharm . _1 137 (1962) . E .T . ABBS, Life Sci. _1 99 (1962) . J .L . IZZO, Jr ., unpublished observations .