Journal of the Autonomic Nervous System, 39 (1992) 159-168
159
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01286
Effect of L-threo-3,4-dihydroxyphenylserine on muscle sympathetic nerve activity in humans S a t o s h i I w a s e a, T a d a a k i M a n o a, M a s a n a r i K u n i m o t o b a n d M i t s u r u Saito a a Department of Autonomic and Behavioral Neurosciences, Division of Higher Nervous Control, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan and b Department of Neurology, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo, Japan (Received 22 October 1991) (Revision received 23 March 1992) (Accepted 24 March 1992)
Key words: Muscle sympathetic nerve activity; L-threo-3,4-Dihydroxyphenylserine; Microneurography; Norepinephrine; Positional change; Orthostatic hypotension
Abstract To clarify the effect of L-threo-3,4-dihydroxyphenyiserine (L-threo-DOPS), a precursor of norepinephrine (NE), the effect of this drug on microneurographically recorded muscle sympathetic nerve activity (MSA) from the tibial nerve was analyzed in ten healthy male volunteers. A single dose of 600 mg of L-threo-DOPS was orally administered and the effect of this norepinephrine precursor on MSA at resting and at upright standing positions, as well as the MSA responsiveness to head-up tilting was examined by comparing the data obtained after administration of the drug with those obtained under control conditions. The plasma NE
levels were determined in two subjects. The results were as follows: (1) resting MSA increased significantly 80 min after administration of L-threo-DOPS and was accompanied by an increase in plasma NE levels; (2) standing MSA when treated with the drug was not significantlydifferent from values obtained under control conditions; and (3) MSA responsiveness to orthostasis was reduced after L-threo-DOPS. We conclude from the activation of MSA by L-threo-DOPS that this drug raised blood pressure not only through an increase in metabolized plasma NE levels, but also through the enhancement of MSA by activation of descending noradrenergic or adrenergic pathways proximal to the recording site of the sympathetic discharge.
Introduction Sympathetic outflow to muscles (muscle sympathetic nerve activity, MSA) plays an important role in controlling systemic blood pressure and in hemodynamic regulation [19,20]. We have reported that this activity is enhanced by postural changes, and it is linearly correlated to the longi-
Correspon'dence to: S. Iwase, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-01, Japan.
tudinal body axis component of gravity (+Gz) [5,6]. M S A is suppressed by al-adrenergic agonists, e.g. norepinephrine, or phenylephrine [10]. L-threo-3,4-dihydroxyphenylserine (L-threoDOPS) is converted directly into L-norepinephrine (NE) by L-aromatic amino acid decarboxylase, both in the periphery and the brain [4,13,15]. The therapeutic mechanism of this drug has not been well clarified; however, there have been reports on the drug's efficacy in akinesia and frozen gait associated with Parkinson's disease [11], in orthostatic hypotension accompanying
160 Shy-Drager syndrome [16] and familial amyloid neuropathy [17]. We have administered L-threo-DOPS to a patient with Shy-Drager syndrome and observed that this drug enhances MSA in the patient who hardly discharged MSA, suggesting that the improvement of orthostatic hypotension was due, not to the NE derived from L-threo-DOPS in the periphery, but to the NE produced by conversion of L-threo-DOPS at sites proximal to the sympathetic ganglion [8]. In order to prove this tiypothesis, we administered the drug to healthy male volunteers and analyzed the changes of MSA.
Materials and Methods
Subjects The subjects were ten healthy men whose age ranged from 19-23 years. All subjects gave their oral and written informed consent before entering the study. The protocol for this study was approved by the Human Research Committee of Research Institute of Environmental Medicine, Nagoya University.
Recording of muscle sympathetic nerve activity The subjects were asked to lie on a tilting bed. Sympathetic nerve discharges were recorded by using a tungsten microelectrode with a diameter of 1 ~ m at the tip, and 100/zm at the shaft; the impedance was 3-5 MI2. The electrode was inserted into the muscle fascicle of the tibial nerve at the popliteal fossa through an opening in the bed at knee level. MSA identification was based on the following criteria: (1) appearance of afferent discharges when tapping or stretching the calf muscle but not when gently touching the skin; (2) spontaneously grouped burst discharges in synchrony with the heartbeat; (3) rhythmic discharges not affected by arousal stimuli; and (4) marked accentuation when performing Valsalva's maneuver. The sympathetic nerve signals were fed into a high impedance input preamplifier (DAM-6A) and a band-pass filter (500-5000 Hz, 24 dB/oct); then they were monitored on a cathode ray oscilloscope (Tektroniks 5113). All data were recorded
continuously, stored in a multichannel FM magnetic tape recorder (Sony-Magnescale KS-616U) and analyzed by off-line processing.
Quantification of muscle sympathetic nerve activity The full-wave rectified and integrated neurogram of MSA was quantified as the 'burst rate', i.e. the burst number per min. The burst rate in the supine rest position (0°) was designated as the resting MSA. The slope of the regression line established between the sine of the tilt angle and the burst rate at a certain tilt angle corresponded to the responsiveness of MSA to orthostasis. The burst rate of MSA at the upright standing position was designated as the standing MSA. These three parameters, resting MSA, standing MSA and MSA responsiveness to orthostasis were determined in the ten subjects under control conditions and after administering a single dose of the drug.
Other recordings The arterial blood pressure was measured at 1 min intervals on the left arm using an autosphygmomanometer (Nippon Colin, BP-203NP). The electrocardiogram (ECG) and respiration were also monitored (Nihon Kohden, MZE-6100). All data were recorded continuously and stored in a multichannel FM tape recorder (Sony-Magnescale, KS-616U).
Determination of norepinephrine Plasma norepinephrine (NE) levels could be determined in only two subjects due to technical limitations. A needle catheter (19 gauge scalp vein needle) was introduced into an antecubital vein of the left arm and attached to a three-way stopcock. The stopcock-catheter assembly was filled with a heparin solution (heparin with sterile saline, 100 /~/ml) to maintain catheter patency. Blood specimen was collected through this assembly and transferred to a chilled tube containing 0.1 M ascorbic acid and 0.2 M EDTA-disodium solutions (each 1% of the volume of the blood amount) and kept on ice until centrifugation. Blood samples were centrifuged (4000 rpm) at 4°C and 10 g for 10 min, then the plasma was separated. Half volume of 5% perchloric acid was
161
added to a measured amount of plasma. The mixture was triplicated, vortexed for 1 min and stored at -80°C until analysis. The frozen specimens were semi-thawed, centrifuged at 3000 rpm for 10 min, and the supernatant was analyzed by a fully automatic catecholamine analyzer (Toyo Soda). NE and epinephrine (EP) were separated by injecting lithium chloride solution (pH 3.75) into the column filled with cation exchange resin. The NE-containing fraction was then fed into a trihydroxyindole reaction unit and the NE concentration was determined by fluorescence at the wave length of 490-510 nm under excitation at 380-410 nm.
Protocols for the experimentalprocedure The experiments were carried out under control conditions and after administration of the
Before L-threo-DOPS I
I
I
I
I
I
I
drug, in each subject. In each experiment, three head-up tiltings were performed before, 2 h after, and 4 h after the administration of L-threo-DOPS. After the identification of MSA, the subjects were allowed to rest quietly in the supine position with the bed horizontally placed for 10 min to determine the resting MSA. The head of the bed was then gradually tilted upward to 10°, 20°, 40°, 60° and 90° (upright standing position) to perform the first tilt. The subjects were kept at each angle for 2 min to achieve stabilization, and then for at least another 3 min to record the MSA, ECG, blood pressure and respiration. After the recording at the upright position, the bed was placed back in horizontal position (0°), and the subjects were allowed to rest for 10 min. To examine the effect of the drug on MSA, a single dose of 600 mg L-threo-DOPS (Sumitomo Pharmaceutical Co.
4 hr after L-threo-DOPS
I
'
I
'
'
i
,
'
I'
'
>
MSA Neurograrn
8
Integrated Neurogram
ECa 5 8ec Fig. 1. Muscle sympathetic nerve activity (MSA) at rest before and 4 h after oral administration of 600 mg L-threo-DOPS. From top to bottom, the traces are: raw M S A neurogram; its full-wave rectified and integrated trace; and the electrocardiogram. The resting M S A burst rate increased after administration of 600 mg L-threo-DOPS.
162
for 2 h. The second tilt was performed 2 h after the administration of t_-threo-DOPS when the maximum plasma concentration of r_-threo-DOPS was attained. After 4 h of administration of Lthreo-DOPS, the third tilt was performed. The maximum concentration of derived NE from Lthreo-DOPS was now obtained [18]. Under control conditions, measurements were carried out in the same way as mentioned above except that the drug was not administered. Statistical analysis
Changes in resting MSA, standing MSA and MSA responsiveness to orthostasis between administered and untreated trials were analyzed by comparing them with the resting MSA before administration, which was defined as AMSA to standardize the individual differences, i.e. the values were compared after the subtraction of the values before the administration of the drug. Relationships between the sine function of the body tilt angle and MSA burst rate or plasma NE were determined using the linear regression analysis. The correlation coefficients (r) for the productmoment equation were also calculated.
Fig. 2. Changes in muscle sympathetic nerve activity after oral administration of L-threo-DOPS. The mean resting MSA ( f S.E.) until 110 min after 600 mg r_-threo-DOPS is shown at 10 min intervals. Approximately 80 min after administration, the resting MSA burst rate began to increase and it was significantly enhanced compared with the control values. Data at each point are expressed as mean f S.E. MSA burst rate in administered trial: -o--, MSA in untreated (control) trial: _ _ _._ _ _
Ltd., Japan), which corresponds to the total daily dose [HI, was administered orally and the subjects were asked to remain in the supine position
60 -
Before Administration
- -O--Untreated 50 -
trial
-•-L-three-DOPS
0
trial
z. ; P
50
/
z .g
2 h after Administration 60-
60
- -q - untreated tmi 1
-e-L-three-DOPS
50 -
tnal
- Q-Untreated
trial
-O-L-threeLIOPS
trial
=
40 -
30-
0. 111 = zo5 ; m 10 -
5
40-
v ki e
30-
al r
a 5
5 m
I. u
4 h atter Administration
a
o-
0
y=
,7.9+343x
20-
y = 0 6 + 45.2.x
10 -
4
1
Q
0-
P’
/ I
0.0
0 2
0 4
0 6
.
I
0.6
.
,
1 0
Sine Function of T’lltAngka
0 0 Sine
0.2
0 4
0.6
0 6
1.0
Function of Till Angles
0.0
0.2
0 4
0.6
0.6
1.0
Sine Function of nit Angbs
Fig. 3. Changes in muscle sympathetic nerve activity before and after oral administration of 600 mg r_-threo-DOPS during head-up tilting. Typical AMSA burst rates, which are defined as the difference from resting MSA, before and after (2 h and 4 h) in the administered (-•L-threo-DOPS), and untreated (- - -o- - - Control) trials are shown. There were no significant differences between the administered and untreated trials before and 2 h after r-threo-DOPS. However, significant elevation of MSA at supine position was observed in the administered trial 4 h after the administration.
163 Plasma norepinephrine levels, although in only two subjects, were analyzed under control conditions, before administration of the drug, and then 2 h and 4 h after administration. The heart rate and blood pressure measured under control conditions and after administration of the drug were averaged and compared in all ten subjects, especially 4 h after administration. Student's t-test was employed for the comparison of data.
(':2hrsafter) n=10
3C
E
t:
i
,o
p
159
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01286
Effect of L-threo-3,4-dihydroxyphenylserine on muscle sympathetic nerve activity in humans S a t o s h i I w a s e a, T a d a a k i M a n o a, M a s a n a r i K u n i m o t o b a n d M i t s u r u Saito a a Department of Autonomic and Behavioral Neurosciences, Division of Higher Nervous Control, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan and b Department of Neurology, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo, Japan (Received 22 October 1991) (Revision received 23 March 1992) (Accepted 24 March 1992)
Key words: Muscle sympathetic nerve activity; L-threo-3,4-Dihydroxyphenylserine; Microneurography; Norepinephrine; Positional change; Orthostatic hypotension
Abstract To clarify the effect of L-threo-3,4-dihydroxyphenyiserine (L-threo-DOPS), a precursor of norepinephrine (NE), the effect of this drug on microneurographically recorded muscle sympathetic nerve activity (MSA) from the tibial nerve was analyzed in ten healthy male volunteers. A single dose of 600 mg of L-threo-DOPS was orally administered and the effect of this norepinephrine precursor on MSA at resting and at upright standing positions, as well as the MSA responsiveness to head-up tilting was examined by comparing the data obtained after administration of the drug with those obtained under control conditions. The plasma NE
levels were determined in two subjects. The results were as follows: (1) resting MSA increased significantly 80 min after administration of L-threo-DOPS and was accompanied by an increase in plasma NE levels; (2) standing MSA when treated with the drug was not significantlydifferent from values obtained under control conditions; and (3) MSA responsiveness to orthostasis was reduced after L-threo-DOPS. We conclude from the activation of MSA by L-threo-DOPS that this drug raised blood pressure not only through an increase in metabolized plasma NE levels, but also through the enhancement of MSA by activation of descending noradrenergic or adrenergic pathways proximal to the recording site of the sympathetic discharge.
Introduction Sympathetic outflow to muscles (muscle sympathetic nerve activity, MSA) plays an important role in controlling systemic blood pressure and in hemodynamic regulation [19,20]. We have reported that this activity is enhanced by postural changes, and it is linearly correlated to the longi-
Correspon'dence to: S. Iwase, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-01, Japan.
tudinal body axis component of gravity (+Gz) [5,6]. M S A is suppressed by al-adrenergic agonists, e.g. norepinephrine, or phenylephrine [10]. L-threo-3,4-dihydroxyphenylserine (L-threoDOPS) is converted directly into L-norepinephrine (NE) by L-aromatic amino acid decarboxylase, both in the periphery and the brain [4,13,15]. The therapeutic mechanism of this drug has not been well clarified; however, there have been reports on the drug's efficacy in akinesia and frozen gait associated with Parkinson's disease [11], in orthostatic hypotension accompanying
160 Shy-Drager syndrome [16] and familial amyloid neuropathy [17]. We have administered L-threo-DOPS to a patient with Shy-Drager syndrome and observed that this drug enhances MSA in the patient who hardly discharged MSA, suggesting that the improvement of orthostatic hypotension was due, not to the NE derived from L-threo-DOPS in the periphery, but to the NE produced by conversion of L-threo-DOPS at sites proximal to the sympathetic ganglion [8]. In order to prove this tiypothesis, we administered the drug to healthy male volunteers and analyzed the changes of MSA.
Materials and Methods
Subjects The subjects were ten healthy men whose age ranged from 19-23 years. All subjects gave their oral and written informed consent before entering the study. The protocol for this study was approved by the Human Research Committee of Research Institute of Environmental Medicine, Nagoya University.
Recording of muscle sympathetic nerve activity The subjects were asked to lie on a tilting bed. Sympathetic nerve discharges were recorded by using a tungsten microelectrode with a diameter of 1 ~ m at the tip, and 100/zm at the shaft; the impedance was 3-5 MI2. The electrode was inserted into the muscle fascicle of the tibial nerve at the popliteal fossa through an opening in the bed at knee level. MSA identification was based on the following criteria: (1) appearance of afferent discharges when tapping or stretching the calf muscle but not when gently touching the skin; (2) spontaneously grouped burst discharges in synchrony with the heartbeat; (3) rhythmic discharges not affected by arousal stimuli; and (4) marked accentuation when performing Valsalva's maneuver. The sympathetic nerve signals were fed into a high impedance input preamplifier (DAM-6A) and a band-pass filter (500-5000 Hz, 24 dB/oct); then they were monitored on a cathode ray oscilloscope (Tektroniks 5113). All data were recorded
continuously, stored in a multichannel FM magnetic tape recorder (Sony-Magnescale KS-616U) and analyzed by off-line processing.
Quantification of muscle sympathetic nerve activity The full-wave rectified and integrated neurogram of MSA was quantified as the 'burst rate', i.e. the burst number per min. The burst rate in the supine rest position (0°) was designated as the resting MSA. The slope of the regression line established between the sine of the tilt angle and the burst rate at a certain tilt angle corresponded to the responsiveness of MSA to orthostasis. The burst rate of MSA at the upright standing position was designated as the standing MSA. These three parameters, resting MSA, standing MSA and MSA responsiveness to orthostasis were determined in the ten subjects under control conditions and after administering a single dose of the drug.
Other recordings The arterial blood pressure was measured at 1 min intervals on the left arm using an autosphygmomanometer (Nippon Colin, BP-203NP). The electrocardiogram (ECG) and respiration were also monitored (Nihon Kohden, MZE-6100). All data were recorded continuously and stored in a multichannel FM tape recorder (Sony-Magnescale, KS-616U).
Determination of norepinephrine Plasma norepinephrine (NE) levels could be determined in only two subjects due to technical limitations. A needle catheter (19 gauge scalp vein needle) was introduced into an antecubital vein of the left arm and attached to a three-way stopcock. The stopcock-catheter assembly was filled with a heparin solution (heparin with sterile saline, 100 /~/ml) to maintain catheter patency. Blood specimen was collected through this assembly and transferred to a chilled tube containing 0.1 M ascorbic acid and 0.2 M EDTA-disodium solutions (each 1% of the volume of the blood amount) and kept on ice until centrifugation. Blood samples were centrifuged (4000 rpm) at 4°C and 10 g for 10 min, then the plasma was separated. Half volume of 5% perchloric acid was
161
added to a measured amount of plasma. The mixture was triplicated, vortexed for 1 min and stored at -80°C until analysis. The frozen specimens were semi-thawed, centrifuged at 3000 rpm for 10 min, and the supernatant was analyzed by a fully automatic catecholamine analyzer (Toyo Soda). NE and epinephrine (EP) were separated by injecting lithium chloride solution (pH 3.75) into the column filled with cation exchange resin. The NE-containing fraction was then fed into a trihydroxyindole reaction unit and the NE concentration was determined by fluorescence at the wave length of 490-510 nm under excitation at 380-410 nm.
Protocols for the experimentalprocedure The experiments were carried out under control conditions and after administration of the
Before L-threo-DOPS I
I
I
I
I
I
I
drug, in each subject. In each experiment, three head-up tiltings were performed before, 2 h after, and 4 h after the administration of L-threo-DOPS. After the identification of MSA, the subjects were allowed to rest quietly in the supine position with the bed horizontally placed for 10 min to determine the resting MSA. The head of the bed was then gradually tilted upward to 10°, 20°, 40°, 60° and 90° (upright standing position) to perform the first tilt. The subjects were kept at each angle for 2 min to achieve stabilization, and then for at least another 3 min to record the MSA, ECG, blood pressure and respiration. After the recording at the upright position, the bed was placed back in horizontal position (0°), and the subjects were allowed to rest for 10 min. To examine the effect of the drug on MSA, a single dose of 600 mg L-threo-DOPS (Sumitomo Pharmaceutical Co.
4 hr after L-threo-DOPS
I
'
I
'
'
i
,
'
I'
'
>
MSA Neurograrn
8
Integrated Neurogram
ECa 5 8ec Fig. 1. Muscle sympathetic nerve activity (MSA) at rest before and 4 h after oral administration of 600 mg L-threo-DOPS. From top to bottom, the traces are: raw M S A neurogram; its full-wave rectified and integrated trace; and the electrocardiogram. The resting M S A burst rate increased after administration of 600 mg L-threo-DOPS.
162
for 2 h. The second tilt was performed 2 h after the administration of t_-threo-DOPS when the maximum plasma concentration of r_-threo-DOPS was attained. After 4 h of administration of Lthreo-DOPS, the third tilt was performed. The maximum concentration of derived NE from Lthreo-DOPS was now obtained [18]. Under control conditions, measurements were carried out in the same way as mentioned above except that the drug was not administered. Statistical analysis
Changes in resting MSA, standing MSA and MSA responsiveness to orthostasis between administered and untreated trials were analyzed by comparing them with the resting MSA before administration, which was defined as AMSA to standardize the individual differences, i.e. the values were compared after the subtraction of the values before the administration of the drug. Relationships between the sine function of the body tilt angle and MSA burst rate or plasma NE were determined using the linear regression analysis. The correlation coefficients (r) for the productmoment equation were also calculated.
Fig. 2. Changes in muscle sympathetic nerve activity after oral administration of L-threo-DOPS. The mean resting MSA ( f S.E.) until 110 min after 600 mg r_-threo-DOPS is shown at 10 min intervals. Approximately 80 min after administration, the resting MSA burst rate began to increase and it was significantly enhanced compared with the control values. Data at each point are expressed as mean f S.E. MSA burst rate in administered trial: -o--, MSA in untreated (control) trial: _ _ _._ _ _
Ltd., Japan), which corresponds to the total daily dose [HI, was administered orally and the subjects were asked to remain in the supine position
60 -
Before Administration
- -O--Untreated 50 -
trial
-•-L-three-DOPS
0
trial
z. ; P
50
/
z .g
2 h after Administration 60-
60
- -q - untreated tmi 1
-e-L-three-DOPS
50 -
tnal
- Q-Untreated
trial
-O-L-threeLIOPS
trial
=
40 -
30-
0. 111 = zo5 ; m 10 -
5
40-
v ki e
30-
al r
a 5
5 m
I. u
4 h atter Administration
a
o-
0
y=
,7.9+343x
20-
y = 0 6 + 45.2.x
10 -
4
1
Q
0-
P’
/ I
0.0
0 2
0 4
0 6
.
I
0.6
.
,
1 0
Sine Function of T’lltAngka
0 0 Sine
0.2
0 4
0.6
0 6
1.0
Function of Till Angles
0.0
0.2
0 4
0.6
0.6
1.0
Sine Function of nit Angbs
Fig. 3. Changes in muscle sympathetic nerve activity before and after oral administration of 600 mg r_-threo-DOPS during head-up tilting. Typical AMSA burst rates, which are defined as the difference from resting MSA, before and after (2 h and 4 h) in the administered (-•L-threo-DOPS), and untreated (- - -o- - - Control) trials are shown. There were no significant differences between the administered and untreated trials before and 2 h after r-threo-DOPS. However, significant elevation of MSA at supine position was observed in the administered trial 4 h after the administration.
163 Plasma norepinephrine levels, although in only two subjects, were analyzed under control conditions, before administration of the drug, and then 2 h and 4 h after administration. The heart rate and blood pressure measured under control conditions and after administration of the drug were averaged and compared in all ten subjects, especially 4 h after administration. Student's t-test was employed for the comparison of data.
(':2hrsafter) n=10
3C
E
t:
i
,o
p
