- Email: [email protected]
NaCl)-induced hypertension
NeurophormocologyVol. 27, No. 4, pp. 417-426, 1988 Printed in Great Britain
Ot28-3908/88$3.00+ 0.00 Pergamon Press plc
CENTRAL SEROTONERGIC ALTERATIONS IN DEOXYCORTICOSTERONE ACETATE/NaCl (DOCA/NaCl)-INDUCED HYPERTENSION R. DAWSON JR,’ S. NAGAMHAMA’and S. OPARIL~ ‘Department of Pharmacodynamics, College of Pharmacy, University of Florida, J. Hillis Miller Health Center, Box J-487, Gainesville, Florida 32610, U.S.A. and ‘Hypertension Research Program, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, U.S.A. (Accepted 6 October 1987)
Summary-The present study investigated the metabolism of serotonin (5-HT) in rats made hypertensive by treatment with DOCA/NaCl. 5Hydroxytryptamine and its major metabolite, 5-hydroxyindoleacetic acid (5HIAA) were significantly elevated in a number of regions of the brain in rats treated for 2 weeks with DOCA/NaCl. Elevations in levels of 5-HIAA were present with 4 weeks of treatment with DOCA/NaCl but levels of S-HT were not altered. No changes in the metabolism of 5-HT were detectable with 5 weeks of treatment with DOCA/NaCl. Levels of tryptophan were also elevated in a number of regions of the brain by treatment with DOCA/NaCI. Alterations in norepinephrine (NE) in the brainstem were present with 2, 4 or 5 weeks of treatment with DOCA/NaCl. The neurochemical effects of muscimol, a GABA agonist, were also investigated in rats treated with DOCA/NaCI for 4 weeks. Significant increases in levels of 5-HIAA and 5-HT were present in rats treated with DOCA/NaCI but not in controls, 15 min after intraventricular administration of muscimol (1.0 pg/300 g body weight). Sixty min after muscimol, 5-HIAA was increased in both rats treated with DOCA/NaCl and control rats, but 5-HT was only increased in the hypothalamus. Treatment with DOCA/NaCl produced changes in the metabolism of 5-HT that may be important in the genesis of hypertension, but are not required for the maintenance of elevated arterial pressure. A disturbance of GABA-5-HT interactions between GABA and S-HT in brainstem sites may also contribute to the pathogenesis of hypertension induced by DOCA/NaCl. Key words: muscimol.
hypertension,
deoxycorticosterone
Serotonin (5-HT) in both the central and peripheral nervous systems appears to be involved in cardiovascular regulation (Kuhn, Wolfe and Lovenberg, 1980; Wolfe, Kuhn and Lovenberg, 1985; Vanhoutte, 1985; Chalmers, 1975). Alterations in levels and metabolism of 5-HT are present in genetic and experimentally-induced forms of hypertension (Chemerinski, Ramirez and Enero, 1980; Echizen and Freed, 1984; Saavedra, 1981; Smith, Browning and Myers, 1979; Felten, Weyhenmeyer and Felten, 1984). The exact nature or locus of the abnormalities in the neurochemistry of serotonin in the central nervous system, associated with hypertension have remained elusive. The role of 5-HT in normal cardiovascular control has been difficult to assess and the question of whether serotonergic efferent pathways are excitatory and/or inhibitory to the sympathetic system in not completely resolved (Baum and Shropshire, 1975; Wolfe et al., 1985; McCall and Humphrey, 1982; Pilowsky, Kapoor, Minson, West and Chalmers, 1986). Much of the difficulty in interpreting the role of 5-HT in cardiovascular control arises from the complexity of 5-HT neuronal pathways and the ana-
acetate,
serotonin,
5-hydroxyindoleacetic
acid,
tomical location of various specific 5-HT-containing cell groups with different inputs and outputs (Steinbusch, 1981; Bowker, Westlund, Sullivan and Coul ter, 1982). A second problem that accounts for some of the ambiguity in pharmacological studies of 5-HT and control of blood pressure is the presence of multiple subtypes of 5-HT receptors (Peroutka and Snyder, 1979; Leysen, 1985). Despite the methodological difficulties, there is need to investigate the role of 5-HT in cardiovascular control and the pathogenesis of hypertension. The aim of the present study was to examine the levels and metabolism of serotonin in rats made hypertensive by treatment with deoxycorticosterone acetate (DOCA) and sodium chloride (DOCA/ NaCl). A second goal was to investigate interactions between 5-HT and y-aminobutyric acid in hypertension, since GABA is known to be involved in cardiovascular regulation (Persson, 1980; DeFeudis, 1982; Gillis, Yamada, Dimicco, Williford, Segal, Hamosh and Norman, 1984) and GABA also alters the metabolism of 5-HT (Forchetti and Meek, 1981; Nishikawa and Scatton, 1985; Scatton, Serrano and Nishikawa, 1985). 417
R.
418
DAWSON
METHODS
Animals
Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, Massachusetts) were utilized in these experiments. Unilateral nephrectomy was performed in all rats when they were 4 weeks of age and a recovery period of 2 weeks was allowed. The 6 week-old rats were divided into two groups; one group was implanted subcutaneously with silastic tubing containing 200mg/kg of DOCA and sham surgery was performed on the second group. The rats treated with DOCA were immediately placed on 1.0% NaCl in the drinking water and the controls were given tap water. All rats were maintained on rat chow (Ralston Purina) and fluids ad libitum, in a colony room regulated for temperature and humidity, on a 12 hr light-dark cycle. Blood pressures were taken 48 hr prior to sacrifice by the tail cuff method. Studies with drugs
The metabolism of monoamines was indexed in rats treated with DOCA/NaCI and in control rats after 5 weeks of treatment (11 weeks old). PargylineHCI (100 mg/kg) or saline was administered intraperitoneally and rats treated with DOCA/NaCl and control rats were killed by decapitation 1 hr later. The accumulation of monoamines and decline in metabolites derived from monamine oxidase was assessed in regions of the brain. The effects of intraventricular (i.v.t.) administration of muscimol (1.0 pg/300 g body weight) on the monoamines and metabolites were examined in rats treated with DOCA/NaCl and control rats, 15 and 60 min after administration of drug. The details of the method of administration of muscimol have been previously described (Nagahama, Dawson and Oparil, 1985). Neurochemistry
All rats were killed by decapitation and the brains rapidly excised and dissected on ice. The following regions of the brain were analyzed; the medulla, pons, midbrain, hypothalamus, striatum and remaining telencephalon. Some studies evaluated monoamines and metabolites in the pons-medulla, hippocampus, forebrain (cortex rostra1 to a coronal cut at the level of the anterior commissure) and spinal cord. The
JR et al. regions of the brain were frozen in liquid nitrogen immediately after dissection and stored at -80°C until assay. Regions of the brain were weighed and frozen and then immediately homogenized in ice-cold 0.1 M perchloric acid which contained 1 mM glutathione, 50 mg/l disodium ethylenediamine tetraammonium (Na,EDTA) and an internal standard. The homogenate was centrifuged (10,000 x g for 10 min at 4°C) and the supernatant collected and filtered through 0.2 pm Nylon 66 filters. The unfiltered supernatant was directly injected for high performance liquid chromatographic separation and electrochemical detection, as previously described (Kontur, Dawson and Monjan, 1984). The analytical instrumentation consisted of a solvent delivery system (Waters M-45 or LDC Model 396 pump) and electrochemical detector (Bioanalytical Systems LC-4B or an ECD built in the laboratory). Monoamines and metabolites were separated using a 4.6 x 100 mm, 3 pm Cls column (Rainin Instrument Co., Inc., Woburn, Massachusetts). Statistics
Comparisons involving only treatment with DOCA/NaCl vs a control group were performed using the Student’s t-test. Studies involving treatments with both drug and DOCA/NaCI were analyzed using analysis of variance (ANOVA), followed by post hoc comparisons of treatment means and, where appropriate, by the least-significant-difference test (Dunnett, 1970). RESULTS
The blood pressure (BP), heart rate (HR) and body weight of the rats used in these studies are given in Table 1. Treatment with DOCA/NaCl increased the blood pressure and reduced body weight in all treated groups, compared to controls. Two weeks of treatment with DOCA/NaCl significantly elevated levels of 5-HT in the spinal cord, medulla and pons compared to controls (Fig. 1). The metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA) was elevated by treatment with DOCA/NaCl in all regions of the brain examined, except the spinal cord and forebrain (Fig. 2). No
Table I. Blood pressure, heart rate and body weight in rats given DOCA/NaCI control/water rats Gr0llp 1. II. 111. IV.
Weeks of treatment
BP
2 2 4 4 4 4 5 5
129+_3 l72+4* ll8?2 l78f7* ll3*2 188 f 5’ 12253 156?5*
CON/H,0 DOCA/NaCI CON/H,0 DOCA/NaCI CON/H,0 DOCA/NaCI CON/H,0 DOCA/NaCI
All values expressed *P < 0.05 (r-test) BP = blood pressure;
as K f
SE.
HR = heart rate.
HR 428+ 400 f 432 f 426 + 426 + 432k 416 + 444k
and
Body wt. 15 I6 20 25 13 I5 I2 I6
253+_6 231 ?6* 319 f IO 281 f 8’ 311 +6 278 + 8* 323 + 6 289 2 7*
5-HT and DOCA/NaCI hypertension
6810
419
r
.
Bi
113i
SPlNPLeom
OTHALAWS STRIATUM
FOREBRAIN
Fig. 1. Effects of 2 weeks of treatment with DOCA/NaCI on levels of 5-HT. Hatched bars represent the DOCA/NaCl-treated rats (n = 12) and the clear bars are controls (n = 12). (1135 pmol/g = 200 rig/g)) *P < 0.05 Control vs DOCA/NaCI (t-test).
other significant changes in monoamines or metabolites in the CNS were found in rats treated with DOCA/NaCI for 2 weeks, except for a significant (I’ < 0.001) increase in the content of norepinephrine (NE) in the pons (Table 2). Data for monoamines and metabolites for rats treated for 4 weeks with DOCA/NaCI are shown in Table 3. Levels of S-HIAA in rats treated with DOCA/NaCl were significantly (P < 0.05) elevated in the pons-medulla and hippocampus and increased in the hypothalamus, but this increase was not statistically significant. In contrast to the group treated for 2 weeks with DOCA/NaCI, 4 weeks of treatment with DOCA/NaCl did not elevate levels of 5-HT in the regions of the brain examined. Levels of NE in rats treated for 4 weeks with DOCA/NaCI were significantly (P < 0.01) elevated compared to controls in the pons-medulla. No other significant changes were present in the levels of monoamines or metabolites in the regions of the brain shown in Table
3. Monoamines and metabolites in the striatum were also unaltered by treatment with DOCA/NaCl (data not shown). Levels of monoamines and their accumulation after inhibition of monoamine oxidase with pargyline were measured in rats treated for 5 weeks with DOCA/NaCl. Analysis of variance demonstrated a significant (P < 0.001) effect of paryline on levels of monoamines and metabolites in all regions of the brain examined (Table 4). Rats treated with DOCA/NaCl for 5 weeks, when compared to controls, exhibited no significant alterations in either levels of 5-HT or 5-HIAA after injection of saline (Table 4). The increases in 5-HT and decreases in 5-HIAA after administration of pargyline in rats treated with DOCA/NaCl were similar to those of controls (Table 4). Levels of dopamine were significantly higher than in controls in the ponsmedulla and remaining forebrain structures and cortex of rats treated with DOCA/NaCl 1 hr after injec-
5230 1
9 *
4184
0
2 d E ,Q
3138
s z
2002
l
:, 1048
LaaPlNALmRo
lIli_ Mwvl_l_A
PONS
MIDBRAIN
PA-
STRIATUM
FOflEWWN
Fig. 2. Increase in levels of 5-HIAA produced by 2 weeks of treatment with DOCA/NaCl (hatched bars). (1046 pmol/g = 2OOng/g) *P i 0.05 Control vs DOCA/NaCI (r-test).
420
R. DAWSONJR et Table
al
2. Effects of two weeks of treatment with DOCA/NaCI metabolites in regions of brain
Brain region
NE
Spinal cord CON DOCA/NaCI Medulla CON DOCA/NaCI Pans CON
DOCA/NaCl
3416f 89*
on levels of monoamines
Dopamine
DOPAC
HVA
1507 k 83 1507i35
248 f I3 242 f 7
ND ND
ND ND
3529 f 242 3139 * 100
ND ND
ND ND
ND ND
2944 ? 71
ND ND
ND ND
ND ND
Midbrain CON
2873 f II2
770 k 46
DOCA/NaCI
2890f 100
810f 52
ND ND
ND ND
Hypothalamus CON
7359 k 231
3127 F 222
589 + 42
DOCA/NaCI
7743f 254
3414+ 261
589k 54
ND ND
42,636 + 4015 45,979 i_ 2866
3722 f I78 3787 k 208
2904 + I70 3217 f 258
6607 + 444 6496+418
856 f 54 797 i 36
ND ND
Striatum CON DOCA/NaCI Forebrain CON DOCA/NaCI
1194* 177 1218 & 130 1785? 89 1667 k 71
ND = not detectable. n = I2 per group. All values expressed as pmolig. *P < 0.001 DOCA vs CON (r-test). NE = norepinephrine; DOPAC = 3,4_dihydroxyphenylacetic
Table 3. Levels of monoamines
Pans-medulla CON/water DOCA/NaCl Hypothalamus CON/water DOCA/NaCI Hippocampus CON/water DOCA/NaCl
NE 4581 i 100 5060 f 106** 9918k644 I 1,455 * 686 2808 k I I2 2890 k 171
ND = not detectable. n = IO per group. Data are expressed as pmol/g f SE. 'P < 0.05CON YS DOCA/NaCl (t-test). **P < 0.01 CON vs DOCA/NaCI. NE = norepinephrine; DA = dopamine; hydroxytryptamine.
HVA = homovanillic
acid.
was found in a previous study that this was the time at which a maximum depressor response occurred (Naghama et al., 1985). A second experiment examined the neurochemical actions of muscimol, 60 min after administration, when significant effects of muscimol were present, but the blood pressure and epinephrine in plasma were not maximally affected (Naghama et al., 1985). Analysis of variance revealed significant (P < 0.05) effects of treatment with DOCA/NaCl on levels of 5-HIAA in the medulla, pons, midbrain and forebrain/cortex (Fig. 4). Individual comparisons showed that 15min after administration of vehicle (saline) no significant difference in levels of 5-HIAA were evident between DOCA/NaCl-treated and control rats except in the forebrain/cortex (Fig. 4). In contrast, after administration of muscimol the levels of 5-HIAA were significantly higher in several regions of the brain of rats treated with DOCA/NaCl than in controls (Fig. 4) despite an overall lack of effect of
tions of saline (Table 4). Accumulation of DA after pargyline was not different from controls in rats treated with DOCA/NaCI. Levels of NE were not different from controls after injections of saline, but rats treated with DOCA/NaCl did exhibit a significantly (P < 0.05) greater accumulation of NE in the pons-medulla and hypothalamus after treatment with pargyline than did controls (Fig 3). Tryptophan, the amino acid precursor of serotonin, was elevated by treatment with DOCA/NaCl in all regions of the brain examined (Table 5). The elevations were statistically significant in the ponsmedulla and hippocampus of rats treated for 4 weeks with DOCA/NaCl and the forebrain of rats treated for 2 weeks with DOCA/NaCl (Table 5). Muscimol, a GABA agonist, or saline vehicle was administered intraventricularly to rats treated with DOCA/NaC1(4 week treatment) or control rats. The rats were killed by decapitation 15 min after administration of drug or vehicle in one experiment, since it
Group
acid;
and
in rats treated DA
for 4 weeks with DOCA/NaCI 5-HIAA
5-HT
509 + 20 522 k I3
2636 + I10 3060 f 141;
4846 f 108 5096 f I I9
3375 f 157 3519 f 163
2186 k I78 2490 k 204
5153 * 159 5567 f 199
ND ND
1281 k I31 1820 li_ 204
2514 k I08 2650 f I31
5-HIAA = 5-hydroxyindoleacetic
acid;
S-H1 = 5-
.5-HT and DOCA/NaCl hypertension Table
4. Effects of treatment
Region
with pargyline DOCA/NaCl
(PAR) on levels of monoamines and metabolites (5 week treatment) and CON/water rats
Normetanephrine
of brain
Hypothalamus CON/water-SAL CON/water-PAR DOCA!NaCI-SAL DOCAINaCI-PAR Pans-&edulla CON/water-SAL CON/water-PAR DOCAiNaCl-SAL DOCA/NaCI-PAR Rest of brain CON/water-SAL CON/water-PAR DOCA/NaCI-SAL DOCA/NaCI-PAR
ND 551 i44 ND 671 f 71
Dopamine
DOPAC
3414 + 235 4185-t 118 3212 ~fr144 4446 + 157
571 i24 ND 535 * 18 ND
ND ND ND ND
379* I3 581?39 444 * 20** 640 + 33
ND ND ND ND
421
7814+ 10,230 + 8408 + 10,556 +
137 261 235’ 209
13593
tt:
ll229
c
c
1
SAL
PAR
Pons-medulla
SAL
PAR
Rest of brain
Fig. 3. Change in levels of NE 1hr after pargyline (PAR). Treatment with pargyline (100 mg/kg) resulted in a significantly (1182 pmol/g = 200 rig/g)) *P < 0.05 greater increase in levels of NE in D~A/NaCl-treaty rats (hatched bars) than controls in both the ins-m~ulla and hypothalamus. fn = 10 rats per group) (ANOVA folIowed by least
significance difference test).
treated
_+ 148 i: 289 & 261 * 221
3028 + I94 1151+7s 2819+ 173 1287&89
ND ND ND ND
3944 I 85 6793 + 267 4029 i: I36 7117~t329
1956 * 136 926 + 68 2076 rt 126 1030+68
719+30 ND 624 k 42 ND
2639 i: I14 4988k210 2741 I 119 5068 k I59
1083 + 146 649 F 68 1187k89 732k II0
5340 8700 5323 9301
with
5-HIAA
.5-HT
ND = not detectable. n = 9-10 per group. All values expressed as pmol/g wet weight k SE. P -e 0.001 for pargyline +P < 0.05 CON/water-SAL vs DOCA/NaCI-SAL (ANOVA followed by LSD). **P < 0.01 CON/water-SAL vs DOCA/NaCI-SAL (ANOVA followed by LSD). Abbreviations as in Tables 2 and 3.
lnuscimol on levels of 5-HIAA (i.e. no significant effect of treatment with muscimol by ANOVA). There were no significant interactions between DOCA and muscimol indicated by ANOVA, although in most regions of the brain they approached significance (P < 0.14.2 for all regions). Levels of 5-HT were not significantly altered by treatment with DOCA/NaCI alone, but there was a significant (P < 0.05) interaction between DOCA and tnuscimol in several regions of the brain as indicated by ANOVA. Individual comparisons of means demonstrated significantly higher levels of 5-HT in rats treated with DOCA/NaCl and given muscimol than in controls, in the spinal cord, medulla, pons and hypothalamus (Fig. 5). There were no differences between DOCA/NaCl-treated and control rats in levels of 5-HT in the striatum or forebrain/cortex of rats injected with either vehicle or muscinol (data not shown).
in rats
effect by ANOVA
Treatment with DOCA/NaCl had a significant effect on levels of S-HIAA in a11regions of the brain examined in the rats in the 60min study (Fig. 6). Individual comparisons showed that rats treated with DOCA/NaCl and given vehicle and decapitated 60min later had higher levels of 5-HIAA than controls, in all regions of the brain examined (Fig. 6). In the pons of rats treated with DOCA/NaCl and given muscimol, levels of 5-HIAA were higher than controls (Fig. 6). Analysis of variance demonstrated a significant (P < 0.01) effect of muscimol on levels of S-HIAA in all regions of the brain examined, except the midbrain and spinal cord. Muscimol signifi~ntly (P < 0.05) increased levels of 5-HIAA in control rats in all regions of the brain examined but only slightly increased levels of 5-HIAA in rats given DOCA/NaCl (Fig. 6). Levels of S-HT were not altered by treatment with either DOCA/NaCl or muscimol in any region of the brain examined, except the hypothalamus where muscimol significantly (P < 0.01) elevated levels of 5-HT in both control and rats treated with DOCA/NaCI (data not shown). significantly Administration of muscimol (P < 0.05) increased levels of DA in the pons and hypothalamus of rats treated with DOCA/NaCl and controls at both 15 and 60min after infusion (data not shown). Levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in the hypothalamus were also significantly (P < 0.05) elevated in rats treated with DOCA/NaCI and in control rats at both times after administration of muscimol (data not shown). DISCUSSION
The results of this study suggest that major changes occur in the metabolism of 5-HT during the development of hypertension induced by DOCA/NaCl. The increases in 5-HIAA appeared most pronounced with 2 weeks of treatment with DOCA/NaCl and were also present at 4 weeks but not with 5 weeks of treatment with DOCAINaCl. Levels of 5-HT were
R. DAWSONJR et
422
Table 5. Levels of trvptouhan
in control
4 weeks DOCA/NaCI Hippocampus Pans-medulla Cerebellum Midbrain Spinal cord 2 weeks DOCA/NaCI Forebrain
MEDULLA
PONS
rats
Control
25.5 + 1.9 (IO)* 30.0 + 2.0 (lo)* 30.9i2.7(11) 41. I i 2.7 (20) 36.3 k 2.5 (21)
17.9 + 15.0(10) 24.5 + l.Z(lO) 24.5+2.2(11) 34.9 jy 2.4(21) 31.3 + 1.8(22)
39.6 i 2.9 (12)**
25.2*
Data are expressed as lmol,‘g + SE. Numbers in parentheses represents the number *P < 0.05 DOCA YS Control (r-test). **P < 0.001 DOCA vs Control (r-test).
CORD
and DOCAiNaCI-treated
DOCA
Reeion of brain
SPINAL
al.
1.2(11)
of subjects.
MIDBRAIN
I-WFOTHALAMLtS
STRIATUM
FC%BRAIN/CTX
Fig. 4. Effects of treatment with DOCA/NaCl (hatched bars) and intraventricular administration of muscimol (MUS) on levels of 5-HIAA (n = 9912 rats per group) 15 min after infusion of muscinol. (1046 pmol/g = 200 rig/g)) *P < 0.05 Control/MUS vs DOCA/NaCI/MUS (ANOVA followed by least significance difference test).
1 9648
/
T
3513
1 7378 I P “,
6243
$ P ,P II II,
5103
3973
2833
SPINAL CORD
Fig. 5. Increases in levels of S-HT in rats treated with DOCA/NaCI (hatched bars) 15min after intraventricular administration of muscimol. (n = 9912 rats per group) (1135 pmol/g = 200 rig/g)) *P < 0.05 Control/MUS vs DOCA/NaCI/MUS (ANOVA followed by least significance difference test),
S-HT and DOCA/NaCl hypertension
423
1048
SPINAL CORD
KPJS
MEDULLA
MlDBRAlN
HYFOTHAIAWS
Fig. 6. Levels of S-HIAA in rats treated with DOCA/NaCI (hatched bars) and control rats 60 min after intraventricular infusion of muscimol or vehicle. (n = 9-12 rats per group) (1046 pmol/g = 200 rig/g)) *P <: 0.05 Control vs DOCA/NaCi (ANOVA followed by least significance difference test).
.
significantly elevated by 2 weeks of treatment with DOCA/NaCl, but were not elevated with 4 or 5 weeks of treatment with DOCA/NaCl. Thus, after 2 weeks of treatment with DOCA/NaCl, the alterations in the metabolism of 5-HT began to decline. The increase in metabolism of 5-HT appears generalized, in the sense that most regions of the brain examined appeared to exhibit increased levels of 5-HIAA in association with treatment with DOCA/NaCl. This generalized increased in metabolism of 5-HT may be due to the increased availability of the precursor, tr~tophan~ since levels of tryptophan in brain were increased by treatment with DOCA/NaCl. Whether the increase in levels of S-HIAA in rats treated with DOCA/NaCl represents increased release of 5-HT or increased intraneuronal metabolism of 5-HT is not evident from the current results. Previous studies that have examined the role of 5-HT in hypertension induced by DOCA/NaCl have been inconclusive. Treatment with the 5-HT neurotoxin, 5,6_dihydroxytryptamine, either lowered blood pressure (Finch, 1975) or had no effect (Myers, Reid and Lewis, 1974) on either the development or maintenance of induced by hypertension DOCA/NaCl. Pharmacological studies have shown treatment with DOCA/NaCl to potentiate the pressor responses of intraventricularly-administered 5-HT (Dalton, 1986) or report enhanced depressor responses to the administration of a blocker of the uptake of 5-HT combined with r_-%hydroxytryptophan (Fuiler, Holland, Yen, Bemis and Stamm, 1979). A recent study reported that chronic administration of dietary tryptophan reduced the intake of saline and prevented the rise in blood pressure associated with treatment with DOCA/NaCl (Fregly and Fater, 1986). No info~ation is available on the metabolism of serotonin during the development of hypertension induced by DOCA/NaCl. There is convincing evidence that the metabolism of 5-HT is increased in the early phase of hypertension induced by sinoaortic denervation (Chemerinski et al., 1980; Giarcovich-Martinez, Fernandez,
Chemerinski and Enero, 1983; Chalmers, 1975). Stores of serotonin (Felten et al., 1984; Saavedra, 1981; Wolfe and Kuhn, 1984a) and turnover (Smith et aZ., 1979; Koulu, Saavedra, Bjelogrlic, Niwa, Agren and Linnoila, 1986) are increased in spontaneously hypertensive rats (SHR). Intraventricular administration of 5,6_dihydroxytryptamine retards the development of hypertension in spontaneously hypertensive rats (Buckingham, Hamilton and Robson, 1976), however, depletion of 5-HT in brain with para-~horophenylalanine is associated with a rise in blood pressure in the spontaneously hypertensive rat (de Jong, Nijkamp and Bohus, 1975). Studies of the metabolism of serotonin or the effects of serotonin neurotoxins must be analyzed carefully since neurochemical, as well as other parameters, change as a function of age or duration of treatment in animal models of hypertension (Felten et al., 1984; Koulu et al., 1986; Giarcovich-Martinez et al., 1983). Administration of large doses of t-tryptophan to spontaneously hypertensive rats produce a doserelated decrease in blood pressure and increase the content of 5-HT in the central nervous system (Sved, van Itallie and Fernstrom, 1982; Wolf and Kuhn, 1984b). Echizen and Freed (1982) reported that infusion of L-5-hydroxytryptophan but not L-tryptophan over 24 hr decreased blood pressure in normotensive rats. The antihypertensive actions of L-tryptophan are complex and serotonin may (Sved, et al., 1982) or may not (Wolfe and Kuhn, 1984a) mediate the depressor response. The present results suggest that increases in the metabolism of 5-HT are associated with hypertension induced by DOCA/NaCl. This association of hypertension with altered metabolism of 5-HT is also evident in the spontaneously hypertensive rat and with hypertension induced by sinoaortic denervation. What is not clear is whether the changes seen in the metabolism of 5-HT are contributing to the pressor mechanism or if the changes in the metabolism of 5-HT are a response to the elevated blood pressure. Reflex and drug-induced elevations of blood pressure
424
R. DAWSONJR et a/.
are accompanied by increased concentrations of 5-HIAA in the extracellular fluid in the dorsal raphe and nucleus tractus solitarius (Freed, Echicen and Bhaskaran, 1985). Local application of S-HT in the nucleus tractus solitarius produces a neurogenic pressor response (Wolfe, Kuhn and Lovenberg, 1981). The fact that changes in the metabolism of .5-HT are most pronounced in the developmental phase of hypertension induced by DOCA/NaCl and in young spontaneously hypertensive rats (Smith et al., 1979; Koulu et al., 1986) suggest that increased metabolism of 5-HT is not important for the maintenance of hypertension. Thus, increased metabolism of 5-HT may be associated with the genesis of hypertension or metabolism of 5-HT may increase to attenuate a rise in blood pressure. Electrical stimulation of ascending serotonergic systems increases blood pressure, while electrical activation of bulbospinal 5-HT pathways can either increase or decrease blood pressure and sympathetic nervous activity (Wolfe et al., 1985). Therefore, 5-HT can be pressor or depressor, depending on the receptor population stimulated and the efferent circuitry activated. The effect of intravent~cularly administered muscimol on 5-HT and 5-HIAA in rats treated with DOCA/NaCl was complicated by the fact that levels of 5-HIAA were increased in rats treated with DOCA/NaCI compared to controls 60 min after injection of vehicle but not at 15 min after injection. This could have been the result of the stress associated with the intraventricular injection procedure or because these were different groups of rats. The metabolism of 5-HT appeared to be selectively altered in rats treated with DOCA/NaCl 15 min after administration of muscimol. This is in agreement with a previous study from this laboratory that demonstrated an enhanced depressor effect of muscimol in the DOCA/NaCl-induced hypertensive rat (Nagahama et al., 1985). Levels of 5-HIAA were increased in both rats treated with DOCA/NaCl and control rats 60 min after administration of muscimol, suggesting that the onset of action of muscimol was more rapid in rats treated with DOCA/NaCl than in controls. It is unclear whether the changes in 5-HIAA were associated with the depressor actions of muscimol or were a response to decreased blood pressure. Administration of 5-hydroxytryptophan attenuates the depressor actions of centrally administered GABA (Persson, 1980) and inhibitory responses of the sympathetic system, elicited by electrical stimulation of the medullary raphe nuclei, appear to be mediated by GABA (McCall and Humphrey, 1985). Thus, the bulbospinal 5-HT system appears to be under GABAergic control. Neurochemical evidence indicates that GABA can tonically inhibit the activity of 5-HT neuronal systems (Forchetti and Meek, 1981; Scatton et al., 1985). Chronic treatment with valproic acid, an inhibitor of the catabolism of GABA attenuates the development of hypertension induced by DOCA/NaCl Rotiroti, Palella, Losi, Ni-
stico and Caputi, 1982). The activity of glutamate decarboxylase is increased in the mesencephalon and GABA mimetics produce greater depressor responses in rats treated with DOCA/NaCl when compared to controls (Magri, Trimarchi, Quattrone, Squadrito, Di Giorgio, Costa and Caputi, 1984). Therefore, a disruption of interactions between S-HT and GABA in critical brainstem sites could contribute to the development of hypertension. The elevation in tryptophan in brain and consequent increases in levels of 5-HIAA seen in rats treated with DOCA/NaCI in this study could be the result of sympathetic activation and not a cause of the increased sympathetic activity seen in hypertension induced by DOCA/NaCl. Restraint stress increases levels of tryptophan and 5-HIAA in brain (Kennett and Joseph, 1981; Joseph and Kennett, 1983) and the increased uptake of tryptophan in brain can be attenuated by the inhibition of sympathetic activity with guanethidine (Franklin and Kelly, 1986). Thus, chronic sympathetic activation could increase levels of tryptophan in brain, although this appears to be unlikely in this study since the levels of 5-HIAA and 5-HT were not altered 5 weeks after treatment with DOCAiNaCl yet increased sympathetic activity was still presumed to be evident (de Champlain and van Ameringen, 1981). The adrenal steroids, hydrocortisone and corticosterone, alter key enzymes of tryptophan metabolism (Green, 1978). The possibility that DOCA could account for the changes in the metabolism of 5-HT or uptake of tryptophan, independent of any cardiovascular significance must also be considered. The increased levels of 5-HIAA and 5-HT in regions of the brain from rats treated with DOCA/NaCl, reported in the present study, are in contrast to the lack of effect of treatment with DOCA/NaCl on other monoamine systems in brain. An increase in levels of NE in the brainstem was found with both 2 and 4 weeks of treatment with DOCA/NaCl and acc~ulation of NE after pargyline was also increased in the brainstem of rats treated for 5 weeks with DOCA/NaCl. Alterations in NE in DOCA/NaCl-induced hypertension have been well characterized (Chalmers, 1975; de Champlain and van Ameringen, 1981) and participate in the increased sympathetic activity characteristic of hypertension induced by DOCA/NaCl. Few other changes in monoamines were found in the regions of the brain examined or the times sampled. Thus, it would appear that treatment with DOCA/NaCl provokes significant alterations in the metabolism of 5-HT which decline in magnitude during the established or maintenance phase of hypertension induced by DOCA/NaCl. Studies examining release of 5-HT, 5-HT receptors and the elaboration of interactions between 5-HT and GABA in controlling sympathetic outflow should yield important information in regards to the genesis of hypertension induced by DOCA/NaCl.
5-HT and DOCA/NaCI Acknowledgements-The authors wish to express their thanks to Victoria Redd Patterson for expert secretarial assistance in preparing this manuscript. This work was supported in pari by -NHLBI Grant-HL-25451, Hypertension Trainine Grant 5T32 HL07457 and a grant from the Division of-sponsored Research at the Uiiversity of Florida. REFERENCES
Baum T. and Shropshire A. T. (1975) Inhibition of efferent sympathetic nerve activity by 5-hydroxytryptophan and centrally administered 5-hydroxytryptamine. Neuropharmacology 14: 227-233. Bowker R. M.. Westlund K. N.. Sullivan M. C. and Coulter J. D. (198i) Organization of descending serotonergic projection to the spinal cord. Descending pathways to the spinal cord. Progress in Brain Research (Kuypers H. G. J. M. and Martin G. F., Eds), Vol. 57, pp. 239-265. Elsevier Biomedical Press, Amsterdam. Buckingham R. E., Hamilton T. C. and Robson D. (1976) Effect of intracerebroventricular 5,6-dihydroxytryptamine on blood pressure of spontaneously hypertensive rats. Eur. J. Pharmac. 36: 431437. Chalmers J. P. (1975) Brain amines and models of experimental hypertension. Circulation Res. 36: 469480. _ de Champlain J. and van Ameringen M. R. (1981) Evidence for the-participation of central noradrenergic mechanisms in DOCA-sodium hypertension in the rat. Central Nervous System Mechanisms in Hypertension (Buckley J. P. and Ferrario C. M., Eds), pp. 141~151. Raven Press, New York. Chemerinski E., Ramirez A. J. and Enero M. A. (1980) Sinoaortic denervation-induced changes in central serotonergic neurons. Eur. J. Pharmac. 64: 195-198. Dalton D. W. (1986) effects of centrallv . , The cardiovascular administered 5-hydroxytryptamine in the conscious normotensive and hypertensive rat. J. Autonomic Pharmac. 6: 67-75. DeFeudis F. V. (1982) Studies on GABA and cardiovascular function using in vivo and in vitro technics. Drug Dev. Res. 2: 507-517. De Jong W., Nijkamp F. P. and Bohus B. (1975) Role of noradrenaline and serotonin in the central control of blood pressure in normotensive and spontaneously hypertension rats. Archs int. Pharmacodyn. 213: 272-284. Dunnett C. W. (1970) Multiple comparison. Statistics in Endocrinoloev (McArthur J. W. and Colton T.. Eds). pp. 79%104.%T Press, Cambridge, MA. E;chizen H. and Freed C. R. (1982) Long-term infusion of L-5-hydroxytryptophan increases brain serotonin turnover and decreases blood pressure in normotensive rats. J. Pharmac. exp. Ther. 220: 579-584. Echizen H. and Freed C. R. (1984) Altered serotonin and norepinephrine metabolism in rat dorsal raphe nucleus after drug-induced hypertension. Life Sci. 34: 158 1-l 589. Felten S. Y., Weyhenmeyer J. A. and Felten D. L. (1984) Norepinephrine and serotonin in central autonomic nuclei in the spontaneously hypertensive rat and two normotensive control rats. Brain Res. Bull. 13: 437-441. Finch L. (1975) The cardiovascular effects of intraventricular 5,6_dihydroxytryptamine in conscious hypertensive rats. Clin. exp. Pharmac. Physiol. 2: 503-508. F:orchetti C. M. and Meek J. L. (1981) Evidence for a tonic GABAergic control of serotonin neurons in the median raphe nucleus. Brain Res. 206: 208-212. Franklin K. B. J. and Kelly S. J. (1986) Sympathetic control of tryptophan uptake and morphine analgesia in stressed rats. Eur. J. Pharmac. 126: 145-150. Freed C. R., Echizen H. and Bhaskaran D. (1985) V. Brain serotonin and blood pressure regulation: studies using in vivo electrochemistry and direct tissue assay. Lz> Sci. 37: 1783-1793. I,
hypertension
425
Fregly M. J. and Fater D. C. (1986) Prevention DOCAinduced hypertension in rats by chronic treatment with tryptophan: Clin. Exp. Pharmac. Physiol. 13: 767-776. Fuller R. W.. Holland D. R.. Yen T. T.. Bemis K. G. and Stamm N. b. (1979) Antihypertensive dffects of fluoxetine and L-5-hydroxtryptophan in rats. Life Sci. 25: 1237-1242. Giarcovich-Martinez S., Fernandez M., Chemerinski E. and Enero M. A. (1983) Central serotonergic activity after neurogenic hypertension. Eur. J. Pharmac. 86: 337-345. Gillis R. A., Yamada K. A., Dimicco J. A., Williford D. J., Segal S. A., Hamosh P. and Norman W. P. (1984) Central r-aminobutyric acid involvement in blood pressure control. Fed. Proc. 43: 32-38. Green A. R. (1978) The effects of dietary tryptophan and its peripheral metabolism on brain 5-hydroxytryptamine synthesis and function. Essays in Neurochemistry & Neuropharmacology (Youdim M. B. H., Lovenberg W., Sharman D. F. and Lagnado J. R., Eds), Vol. 3, pp. 104-127. Wiley, New York. Joseph M. H. and Kennett G. A. (1983) Stress-induced release of 5-HT in the hippocampus and its dependence on increased tryptophan availability: an in vivo electrochemical study. Brain Res. 270: 251-257. Kennett G. A. and Joseph M. H. (1981) The functional importance of increased brain tryptophan in the serotonergic response to restraint stress. Neuropharmacology 20: 3943. Kontur P., Dawson R. and Monjan A. (1984) Manipulation of mobile phase parameters for the HPLC separation of endogenous monoamines in rat brain tissue. J. Neurosci. Meth. 11: 5-18. Koulu M., Saavedra J. M., Bjelogrlic N., Niwa M., Agren H. and Linnoila M. (1986) Serotonin turnover in discrete hypothalamic nuclei and mesencephalic raphe nuclei of young and adult spontaneously hypertensive rats. Brain Res. 379: 257-263. Kuhn D. M., Wolf W. A. and Love&erg W. (1980) Review of the role of the central serotonergic neuronal system in blood pressure regulation. Hypertension 2: 243-255. Leysen J. E. (1985) Serotonergic binding sites. In: Serotonin and the Cardiovascular System (Vanhoutee P. M., Ed.), pp. 43-62. Raven Press, New York. M&i V., Trimarchi G. R., Quattrone G., Squadrito F., Di Gioreio R. M.. Costa G. and Canuti A. P. (1984) Increised cardidvascular responsiveness to GABkergic stimulation in DOCA-salt hypertensive rats. Res. Commun. them. Path. Pharmac. 45: 107-118. McCall R. B. and Humphrey S. J. (1982) Involvement of serotonin in the central regulation of blood pressure: evidence for a facilitating effect on sympathetic nerve activity. J. Pharmac. exp. Ther. 222: 94-102. McCall R. B. and Humphrey S. J. (1985) Evidence for GABA mediation of sympathetic inhibition evoked from midline medullary depressor sites. Brain Res. 339: 356360. Myers M. G., Reid J. L. and Lewis P. J. (1974) The effect of central serotonin depletion on DOCA-saline hypertension in the rat. Cardiovascular Res. 8: 806810. Nagahama S., Dawson R. and Oparil S. (1985) Enhanced depressor effect of muscimol in the DOCA/NaCl hypertensive rat: Evidence for altered GABAergic activity in brain. Proc. Sot. exp. Biol. Med. 180: 277-283. Nishikawa T. and Scatton 8. (1985) Inhibitory influence of GABA on central serotonergic transmission. Raphe nuclei as the neuroanatomical site of the GABAergic inhibition of cerebral serotonergic neurons. Brain Res. 331: 91-103. Peroutka S. J. and Snyder S. H. (1979) Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [‘HIlysergic acid diethylamide and [‘HIspiroperidol. Molec. Pharmac. 16: 687499. Persson B. (1980) Cardiovascular effects of intra-
426
R. DAWSONJR et al.
cerebroventricular GABA, glycine and muscimol in the rat. Naunyn-Schmiedebergs Arch. Pharmac. 313: 225-236. Pilowsky P. M., Kapoor V., Minson J. B., West M. J. and Chalmers J. P. (1986) Spinal cord serotonin release and raised blood pressure after brainstem kainic acid injection. Brain Res. 366: 354357. Rotiroti D.. Palella B., Losi E., Nistico G. and Caputi A. P. (1982) Evidence that a GABAergic mechanism influences the development of DOCA-salt hypertension in the rat. Eur. J. Pharmac. 83: 153-154. Saavedra J. M. (1981) Central biogenic amines and neuropeptides in genetic hypertension. Central Nervous System Mechanisms in Hypertension (Buckley J. P. and Ferrario C. M., Eds), pp. 129-139. Raven Press, New York. Scatton B., Serrano A. and Nishikawa T. (1985) GABA mimetics decrease extracellular concentrations of 5-HIAA (as measured by in uiuo voltammetry) in the dorsal raphe of the rat. Brain Res. 341: 372-376. Smith M. L., Browning R. A. and Myers J. H. (1979) In uiuo rate of serotonin synthesis in brain and spinal cord of young, spontaneously hypertensive rats. Eur. J. Pharmac. 53: 301-305.
Steinbusch H. W. M. (1981) Distribution of serotoninimmunoreactivity in the central nervous system of the rat-cell bodies and terminals. Neuroscience 6: 557-618. Sved A. F., Van Itallie C. M. and Fernstrom J. D. (1982) Studies on the antihypertensive action of L-tryptophan. J. Pharmac. exp. Ther. 221: 329-333. Vanhoutte P. M. (1985) Serotonin and the Cardiovascular Sysrem. Raven Press, New York. Wolf W. A., Kuhn D. M. and Lovenberg W. (1981) Blood pressure responses to local application of serotonergic agents in the nucleus tractus solitarii. Eur. J. Pharmac. 69: 29 1-299. Wolf W. A. and Kuhn D. M. (1984a) Antihypertensive effects of L-tryptophan are not mediated by brain serotonin. Brain Res. 295: 356359. Wolf W. A. and Kuhn D. M. (1984b) Effects of r.-tryptophan on blood pressure in normotensive and hypertensive rats. J. Pharmac. exp. Ther. 230: 324329. Wolf W. A., Kuhn D. M. and Love&erg W. (1985) Serotonin and central regulation of arterial blood pressure. In: Seroronin and the Cardiovascular System (Vanhoutte P. M., Ed.), pp. 63-74. Raven Press, New York.
Ot28-3908/88$3.00+ 0.00 Pergamon Press plc
CENTRAL SEROTONERGIC ALTERATIONS IN DEOXYCORTICOSTERONE ACETATE/NaCl (DOCA/NaCl)-INDUCED HYPERTENSION R. DAWSON JR,’ S. NAGAMHAMA’and S. OPARIL~ ‘Department of Pharmacodynamics, College of Pharmacy, University of Florida, J. Hillis Miller Health Center, Box J-487, Gainesville, Florida 32610, U.S.A. and ‘Hypertension Research Program, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, U.S.A. (Accepted 6 October 1987)
Summary-The present study investigated the metabolism of serotonin (5-HT) in rats made hypertensive by treatment with DOCA/NaCl. 5Hydroxytryptamine and its major metabolite, 5-hydroxyindoleacetic acid (5HIAA) were significantly elevated in a number of regions of the brain in rats treated for 2 weeks with DOCA/NaCl. Elevations in levels of 5-HIAA were present with 4 weeks of treatment with DOCA/NaCl but levels of S-HT were not altered. No changes in the metabolism of 5-HT were detectable with 5 weeks of treatment with DOCA/NaCl. Levels of tryptophan were also elevated in a number of regions of the brain by treatment with DOCA/NaCI. Alterations in norepinephrine (NE) in the brainstem were present with 2, 4 or 5 weeks of treatment with DOCA/NaCl. The neurochemical effects of muscimol, a GABA agonist, were also investigated in rats treated with DOCA/NaCI for 4 weeks. Significant increases in levels of 5-HIAA and 5-HT were present in rats treated with DOCA/NaCI but not in controls, 15 min after intraventricular administration of muscimol (1.0 pg/300 g body weight). Sixty min after muscimol, 5-HIAA was increased in both rats treated with DOCA/NaCl and control rats, but 5-HT was only increased in the hypothalamus. Treatment with DOCA/NaCl produced changes in the metabolism of 5-HT that may be important in the genesis of hypertension, but are not required for the maintenance of elevated arterial pressure. A disturbance of GABA-5-HT interactions between GABA and S-HT in brainstem sites may also contribute to the pathogenesis of hypertension induced by DOCA/NaCl. Key words: muscimol.
hypertension,
deoxycorticosterone
Serotonin (5-HT) in both the central and peripheral nervous systems appears to be involved in cardiovascular regulation (Kuhn, Wolfe and Lovenberg, 1980; Wolfe, Kuhn and Lovenberg, 1985; Vanhoutte, 1985; Chalmers, 1975). Alterations in levels and metabolism of 5-HT are present in genetic and experimentally-induced forms of hypertension (Chemerinski, Ramirez and Enero, 1980; Echizen and Freed, 1984; Saavedra, 1981; Smith, Browning and Myers, 1979; Felten, Weyhenmeyer and Felten, 1984). The exact nature or locus of the abnormalities in the neurochemistry of serotonin in the central nervous system, associated with hypertension have remained elusive. The role of 5-HT in normal cardiovascular control has been difficult to assess and the question of whether serotonergic efferent pathways are excitatory and/or inhibitory to the sympathetic system in not completely resolved (Baum and Shropshire, 1975; Wolfe et al., 1985; McCall and Humphrey, 1982; Pilowsky, Kapoor, Minson, West and Chalmers, 1986). Much of the difficulty in interpreting the role of 5-HT in cardiovascular control arises from the complexity of 5-HT neuronal pathways and the ana-
acetate,
serotonin,
5-hydroxyindoleacetic
acid,
tomical location of various specific 5-HT-containing cell groups with different inputs and outputs (Steinbusch, 1981; Bowker, Westlund, Sullivan and Coul ter, 1982). A second problem that accounts for some of the ambiguity in pharmacological studies of 5-HT and control of blood pressure is the presence of multiple subtypes of 5-HT receptors (Peroutka and Snyder, 1979; Leysen, 1985). Despite the methodological difficulties, there is need to investigate the role of 5-HT in cardiovascular control and the pathogenesis of hypertension. The aim of the present study was to examine the levels and metabolism of serotonin in rats made hypertensive by treatment with deoxycorticosterone acetate (DOCA) and sodium chloride (DOCA/ NaCl). A second goal was to investigate interactions between 5-HT and y-aminobutyric acid in hypertension, since GABA is known to be involved in cardiovascular regulation (Persson, 1980; DeFeudis, 1982; Gillis, Yamada, Dimicco, Williford, Segal, Hamosh and Norman, 1984) and GABA also alters the metabolism of 5-HT (Forchetti and Meek, 1981; Nishikawa and Scatton, 1985; Scatton, Serrano and Nishikawa, 1985). 417
R.
418
DAWSON
METHODS
Animals
Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, Massachusetts) were utilized in these experiments. Unilateral nephrectomy was performed in all rats when they were 4 weeks of age and a recovery period of 2 weeks was allowed. The 6 week-old rats were divided into two groups; one group was implanted subcutaneously with silastic tubing containing 200mg/kg of DOCA and sham surgery was performed on the second group. The rats treated with DOCA were immediately placed on 1.0% NaCl in the drinking water and the controls were given tap water. All rats were maintained on rat chow (Ralston Purina) and fluids ad libitum, in a colony room regulated for temperature and humidity, on a 12 hr light-dark cycle. Blood pressures were taken 48 hr prior to sacrifice by the tail cuff method. Studies with drugs
The metabolism of monoamines was indexed in rats treated with DOCA/NaCI and in control rats after 5 weeks of treatment (11 weeks old). PargylineHCI (100 mg/kg) or saline was administered intraperitoneally and rats treated with DOCA/NaCl and control rats were killed by decapitation 1 hr later. The accumulation of monoamines and decline in metabolites derived from monamine oxidase was assessed in regions of the brain. The effects of intraventricular (i.v.t.) administration of muscimol (1.0 pg/300 g body weight) on the monoamines and metabolites were examined in rats treated with DOCA/NaCl and control rats, 15 and 60 min after administration of drug. The details of the method of administration of muscimol have been previously described (Nagahama, Dawson and Oparil, 1985). Neurochemistry
All rats were killed by decapitation and the brains rapidly excised and dissected on ice. The following regions of the brain were analyzed; the medulla, pons, midbrain, hypothalamus, striatum and remaining telencephalon. Some studies evaluated monoamines and metabolites in the pons-medulla, hippocampus, forebrain (cortex rostra1 to a coronal cut at the level of the anterior commissure) and spinal cord. The
JR et al. regions of the brain were frozen in liquid nitrogen immediately after dissection and stored at -80°C until assay. Regions of the brain were weighed and frozen and then immediately homogenized in ice-cold 0.1 M perchloric acid which contained 1 mM glutathione, 50 mg/l disodium ethylenediamine tetraammonium (Na,EDTA) and an internal standard. The homogenate was centrifuged (10,000 x g for 10 min at 4°C) and the supernatant collected and filtered through 0.2 pm Nylon 66 filters. The unfiltered supernatant was directly injected for high performance liquid chromatographic separation and electrochemical detection, as previously described (Kontur, Dawson and Monjan, 1984). The analytical instrumentation consisted of a solvent delivery system (Waters M-45 or LDC Model 396 pump) and electrochemical detector (Bioanalytical Systems LC-4B or an ECD built in the laboratory). Monoamines and metabolites were separated using a 4.6 x 100 mm, 3 pm Cls column (Rainin Instrument Co., Inc., Woburn, Massachusetts). Statistics
Comparisons involving only treatment with DOCA/NaCl vs a control group were performed using the Student’s t-test. Studies involving treatments with both drug and DOCA/NaCI were analyzed using analysis of variance (ANOVA), followed by post hoc comparisons of treatment means and, where appropriate, by the least-significant-difference test (Dunnett, 1970). RESULTS
The blood pressure (BP), heart rate (HR) and body weight of the rats used in these studies are given in Table 1. Treatment with DOCA/NaCl increased the blood pressure and reduced body weight in all treated groups, compared to controls. Two weeks of treatment with DOCA/NaCl significantly elevated levels of 5-HT in the spinal cord, medulla and pons compared to controls (Fig. 1). The metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA) was elevated by treatment with DOCA/NaCl in all regions of the brain examined, except the spinal cord and forebrain (Fig. 2). No
Table I. Blood pressure, heart rate and body weight in rats given DOCA/NaCI control/water rats Gr0llp 1. II. 111. IV.
Weeks of treatment
BP
2 2 4 4 4 4 5 5
129+_3 l72+4* ll8?2 l78f7* ll3*2 188 f 5’ 12253 156?5*
CON/H,0 DOCA/NaCI CON/H,0 DOCA/NaCI CON/H,0 DOCA/NaCI CON/H,0 DOCA/NaCI
All values expressed *P < 0.05 (r-test) BP = blood pressure;
as K f
SE.
HR = heart rate.
HR 428+ 400 f 432 f 426 + 426 + 432k 416 + 444k
and
Body wt. 15 I6 20 25 13 I5 I2 I6
253+_6 231 ?6* 319 f IO 281 f 8’ 311 +6 278 + 8* 323 + 6 289 2 7*
5-HT and DOCA/NaCI hypertension
6810
419
r
.
Bi
113i
SPlNPLeom
OTHALAWS STRIATUM
FOREBRAIN
Fig. 1. Effects of 2 weeks of treatment with DOCA/NaCI on levels of 5-HT. Hatched bars represent the DOCA/NaCl-treated rats (n = 12) and the clear bars are controls (n = 12). (1135 pmol/g = 200 rig/g)) *P < 0.05 Control vs DOCA/NaCI (t-test).
other significant changes in monoamines or metabolites in the CNS were found in rats treated with DOCA/NaCI for 2 weeks, except for a significant (I’ < 0.001) increase in the content of norepinephrine (NE) in the pons (Table 2). Data for monoamines and metabolites for rats treated for 4 weeks with DOCA/NaCI are shown in Table 3. Levels of S-HIAA in rats treated with DOCA/NaCl were significantly (P < 0.05) elevated in the pons-medulla and hippocampus and increased in the hypothalamus, but this increase was not statistically significant. In contrast to the group treated for 2 weeks with DOCA/NaCI, 4 weeks of treatment with DOCA/NaCl did not elevate levels of 5-HT in the regions of the brain examined. Levels of NE in rats treated for 4 weeks with DOCA/NaCI were significantly (P < 0.01) elevated compared to controls in the pons-medulla. No other significant changes were present in the levels of monoamines or metabolites in the regions of the brain shown in Table
3. Monoamines and metabolites in the striatum were also unaltered by treatment with DOCA/NaCl (data not shown). Levels of monoamines and their accumulation after inhibition of monoamine oxidase with pargyline were measured in rats treated for 5 weeks with DOCA/NaCl. Analysis of variance demonstrated a significant (P < 0.001) effect of paryline on levels of monoamines and metabolites in all regions of the brain examined (Table 4). Rats treated with DOCA/NaCl for 5 weeks, when compared to controls, exhibited no significant alterations in either levels of 5-HT or 5-HIAA after injection of saline (Table 4). The increases in 5-HT and decreases in 5-HIAA after administration of pargyline in rats treated with DOCA/NaCl were similar to those of controls (Table 4). Levels of dopamine were significantly higher than in controls in the ponsmedulla and remaining forebrain structures and cortex of rats treated with DOCA/NaCl 1 hr after injec-
5230 1
9 *
4184
0
2 d E ,Q
3138
s z
2002
l
:, 1048
LaaPlNALmRo
lIli_ Mwvl_l_A
PONS
MIDBRAIN
PA-
STRIATUM
FOflEWWN
Fig. 2. Increase in levels of 5-HIAA produced by 2 weeks of treatment with DOCA/NaCl (hatched bars). (1046 pmol/g = 2OOng/g) *P i 0.05 Control vs DOCA/NaCI (r-test).
420
R. DAWSONJR et Table
al
2. Effects of two weeks of treatment with DOCA/NaCI metabolites in regions of brain
Brain region
NE
Spinal cord CON DOCA/NaCI Medulla CON DOCA/NaCI Pans CON
DOCA/NaCl
3416f 89*
on levels of monoamines
Dopamine
DOPAC
HVA
1507 k 83 1507i35
248 f I3 242 f 7
ND ND
ND ND
3529 f 242 3139 * 100
ND ND
ND ND
ND ND
2944 ? 71
ND ND
ND ND
ND ND
Midbrain CON
2873 f II2
770 k 46
DOCA/NaCI
2890f 100
810f 52
ND ND
ND ND
Hypothalamus CON
7359 k 231
3127 F 222
589 + 42
DOCA/NaCI
7743f 254
3414+ 261
589k 54
ND ND
42,636 + 4015 45,979 i_ 2866
3722 f I78 3787 k 208
2904 + I70 3217 f 258
6607 + 444 6496+418
856 f 54 797 i 36
ND ND
Striatum CON DOCA/NaCI Forebrain CON DOCA/NaCI
1194* 177 1218 & 130 1785? 89 1667 k 71
ND = not detectable. n = I2 per group. All values expressed as pmolig. *P < 0.001 DOCA vs CON (r-test). NE = norepinephrine; DOPAC = 3,4_dihydroxyphenylacetic
Table 3. Levels of monoamines
Pans-medulla CON/water DOCA/NaCl Hypothalamus CON/water DOCA/NaCI Hippocampus CON/water DOCA/NaCl
NE 4581 i 100 5060 f 106** 9918k644 I 1,455 * 686 2808 k I I2 2890 k 171
ND = not detectable. n = IO per group. Data are expressed as pmol/g f SE. 'P < 0.05CON YS DOCA/NaCl (t-test). **P < 0.01 CON vs DOCA/NaCI. NE = norepinephrine; DA = dopamine; hydroxytryptamine.
HVA = homovanillic
acid.
was found in a previous study that this was the time at which a maximum depressor response occurred (Naghama et al., 1985). A second experiment examined the neurochemical actions of muscimol, 60 min after administration, when significant effects of muscimol were present, but the blood pressure and epinephrine in plasma were not maximally affected (Naghama et al., 1985). Analysis of variance revealed significant (P < 0.05) effects of treatment with DOCA/NaCl on levels of 5-HIAA in the medulla, pons, midbrain and forebrain/cortex (Fig. 4). Individual comparisons showed that 15min after administration of vehicle (saline) no significant difference in levels of 5-HIAA were evident between DOCA/NaCl-treated and control rats except in the forebrain/cortex (Fig. 4). In contrast, after administration of muscimol the levels of 5-HIAA were significantly higher in several regions of the brain of rats treated with DOCA/NaCl than in controls (Fig. 4) despite an overall lack of effect of
tions of saline (Table 4). Accumulation of DA after pargyline was not different from controls in rats treated with DOCA/NaCI. Levels of NE were not different from controls after injections of saline, but rats treated with DOCA/NaCl did exhibit a significantly (P < 0.05) greater accumulation of NE in the pons-medulla and hypothalamus after treatment with pargyline than did controls (Fig 3). Tryptophan, the amino acid precursor of serotonin, was elevated by treatment with DOCA/NaCl in all regions of the brain examined (Table 5). The elevations were statistically significant in the ponsmedulla and hippocampus of rats treated for 4 weeks with DOCA/NaCl and the forebrain of rats treated for 2 weeks with DOCA/NaCl (Table 5). Muscimol, a GABA agonist, or saline vehicle was administered intraventricularly to rats treated with DOCA/NaC1(4 week treatment) or control rats. The rats were killed by decapitation 15 min after administration of drug or vehicle in one experiment, since it
Group
acid;
and
in rats treated DA
for 4 weeks with DOCA/NaCI 5-HIAA
5-HT
509 + 20 522 k I3
2636 + I10 3060 f 141;
4846 f 108 5096 f I I9
3375 f 157 3519 f 163
2186 k I78 2490 k 204
5153 * 159 5567 f 199
ND ND
1281 k I31 1820 li_ 204
2514 k I08 2650 f I31
5-HIAA = 5-hydroxyindoleacetic
acid;
S-H1 = 5-
.5-HT and DOCA/NaCl hypertension Table
4. Effects of treatment
Region
with pargyline DOCA/NaCl
(PAR) on levels of monoamines and metabolites (5 week treatment) and CON/water rats
Normetanephrine
of brain
Hypothalamus CON/water-SAL CON/water-PAR DOCA!NaCI-SAL DOCAINaCI-PAR Pans-&edulla CON/water-SAL CON/water-PAR DOCAiNaCl-SAL DOCA/NaCI-PAR Rest of brain CON/water-SAL CON/water-PAR DOCA/NaCI-SAL DOCA/NaCI-PAR
ND 551 i44 ND 671 f 71
Dopamine
DOPAC
3414 + 235 4185-t 118 3212 ~fr144 4446 + 157
571 i24 ND 535 * 18 ND
ND ND ND ND
379* I3 581?39 444 * 20** 640 + 33
ND ND ND ND
421
7814+ 10,230 + 8408 + 10,556 +
137 261 235’ 209
13593
tt:
ll229
c
c
1
SAL
PAR
Pons-medulla
SAL
PAR
Rest of brain
Fig. 3. Change in levels of NE 1hr after pargyline (PAR). Treatment with pargyline (100 mg/kg) resulted in a significantly (1182 pmol/g = 200 rig/g)) *P < 0.05 greater increase in levels of NE in D~A/NaCl-treaty rats (hatched bars) than controls in both the ins-m~ulla and hypothalamus. fn = 10 rats per group) (ANOVA folIowed by least
significance difference test).
treated
_+ 148 i: 289 & 261 * 221
3028 + I94 1151+7s 2819+ 173 1287&89
ND ND ND ND
3944 I 85 6793 + 267 4029 i: I36 7117~t329
1956 * 136 926 + 68 2076 rt 126 1030+68
719+30 ND 624 k 42 ND
2639 i: I14 4988k210 2741 I 119 5068 k I59
1083 + 146 649 F 68 1187k89 732k II0
5340 8700 5323 9301
with
5-HIAA
.5-HT
ND = not detectable. n = 9-10 per group. All values expressed as pmol/g wet weight k SE. P -e 0.001 for pargyline +P < 0.05 CON/water-SAL vs DOCA/NaCI-SAL (ANOVA followed by LSD). **P < 0.01 CON/water-SAL vs DOCA/NaCI-SAL (ANOVA followed by LSD). Abbreviations as in Tables 2 and 3.
lnuscimol on levels of 5-HIAA (i.e. no significant effect of treatment with muscimol by ANOVA). There were no significant interactions between DOCA and muscimol indicated by ANOVA, although in most regions of the brain they approached significance (P < 0.14.2 for all regions). Levels of 5-HT were not significantly altered by treatment with DOCA/NaCI alone, but there was a significant (P < 0.05) interaction between DOCA and tnuscimol in several regions of the brain as indicated by ANOVA. Individual comparisons of means demonstrated significantly higher levels of 5-HT in rats treated with DOCA/NaCl and given muscimol than in controls, in the spinal cord, medulla, pons and hypothalamus (Fig. 5). There were no differences between DOCA/NaCl-treated and control rats in levels of 5-HT in the striatum or forebrain/cortex of rats injected with either vehicle or muscinol (data not shown).
in rats
effect by ANOVA
Treatment with DOCA/NaCl had a significant effect on levels of S-HIAA in a11regions of the brain examined in the rats in the 60min study (Fig. 6). Individual comparisons showed that rats treated with DOCA/NaCl and given vehicle and decapitated 60min later had higher levels of 5-HIAA than controls, in all regions of the brain examined (Fig. 6). In the pons of rats treated with DOCA/NaCl and given muscimol, levels of 5-HIAA were higher than controls (Fig. 6). Analysis of variance demonstrated a significant (P < 0.01) effect of muscimol on levels of S-HIAA in all regions of the brain examined, except the midbrain and spinal cord. Muscimol signifi~ntly (P < 0.05) increased levels of 5-HIAA in control rats in all regions of the brain examined but only slightly increased levels of 5-HIAA in rats given DOCA/NaCl (Fig. 6). Levels of S-HT were not altered by treatment with either DOCA/NaCl or muscimol in any region of the brain examined, except the hypothalamus where muscimol significantly (P < 0.01) elevated levels of 5-HT in both control and rats treated with DOCA/NaCI (data not shown). significantly Administration of muscimol (P < 0.05) increased levels of DA in the pons and hypothalamus of rats treated with DOCA/NaCl and controls at both 15 and 60min after infusion (data not shown). Levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in the hypothalamus were also significantly (P < 0.05) elevated in rats treated with DOCA/NaCI and in control rats at both times after administration of muscimol (data not shown). DISCUSSION
The results of this study suggest that major changes occur in the metabolism of 5-HT during the development of hypertension induced by DOCA/NaCl. The increases in 5-HIAA appeared most pronounced with 2 weeks of treatment with DOCA/NaCl and were also present at 4 weeks but not with 5 weeks of treatment with DOCAINaCl. Levels of 5-HT were
R. DAWSONJR et
422
Table 5. Levels of trvptouhan
in control
4 weeks DOCA/NaCI Hippocampus Pans-medulla Cerebellum Midbrain Spinal cord 2 weeks DOCA/NaCI Forebrain
MEDULLA
PONS
rats
Control
25.5 + 1.9 (IO)* 30.0 + 2.0 (lo)* 30.9i2.7(11) 41. I i 2.7 (20) 36.3 k 2.5 (21)
17.9 + 15.0(10) 24.5 + l.Z(lO) 24.5+2.2(11) 34.9 jy 2.4(21) 31.3 + 1.8(22)
39.6 i 2.9 (12)**
25.2*
Data are expressed as lmol,‘g + SE. Numbers in parentheses represents the number *P < 0.05 DOCA YS Control (r-test). **P < 0.001 DOCA vs Control (r-test).
CORD
and DOCAiNaCI-treated
DOCA
Reeion of brain
SPINAL
al.
1.2(11)
of subjects.
MIDBRAIN
I-WFOTHALAMLtS
STRIATUM
FC%BRAIN/CTX
Fig. 4. Effects of treatment with DOCA/NaCl (hatched bars) and intraventricular administration of muscimol (MUS) on levels of 5-HIAA (n = 9912 rats per group) 15 min after infusion of muscinol. (1046 pmol/g = 200 rig/g)) *P < 0.05 Control/MUS vs DOCA/NaCI/MUS (ANOVA followed by least significance difference test).
1 9648
/
T
3513
1 7378 I P “,
6243
$ P ,P II II,
5103
3973
2833
SPINAL CORD
Fig. 5. Increases in levels of S-HT in rats treated with DOCA/NaCI (hatched bars) 15min after intraventricular administration of muscimol. (n = 9912 rats per group) (1135 pmol/g = 200 rig/g)) *P < 0.05 Control/MUS vs DOCA/NaCI/MUS (ANOVA followed by least significance difference test),
S-HT and DOCA/NaCl hypertension
423
1048
SPINAL CORD
KPJS
MEDULLA
MlDBRAlN
HYFOTHAIAWS
Fig. 6. Levels of S-HIAA in rats treated with DOCA/NaCI (hatched bars) and control rats 60 min after intraventricular infusion of muscimol or vehicle. (n = 9-12 rats per group) (1046 pmol/g = 200 rig/g)) *P <: 0.05 Control vs DOCA/NaCi (ANOVA followed by least significance difference test).
.
significantly elevated by 2 weeks of treatment with DOCA/NaCl, but were not elevated with 4 or 5 weeks of treatment with DOCA/NaCl. Thus, after 2 weeks of treatment with DOCA/NaCl, the alterations in the metabolism of 5-HT began to decline. The increase in metabolism of 5-HT appears generalized, in the sense that most regions of the brain examined appeared to exhibit increased levels of 5-HIAA in association with treatment with DOCA/NaCl. This generalized increased in metabolism of 5-HT may be due to the increased availability of the precursor, tr~tophan~ since levels of tryptophan in brain were increased by treatment with DOCA/NaCl. Whether the increase in levels of S-HIAA in rats treated with DOCA/NaCl represents increased release of 5-HT or increased intraneuronal metabolism of 5-HT is not evident from the current results. Previous studies that have examined the role of 5-HT in hypertension induced by DOCA/NaCl have been inconclusive. Treatment with the 5-HT neurotoxin, 5,6_dihydroxytryptamine, either lowered blood pressure (Finch, 1975) or had no effect (Myers, Reid and Lewis, 1974) on either the development or maintenance of induced by hypertension DOCA/NaCl. Pharmacological studies have shown treatment with DOCA/NaCl to potentiate the pressor responses of intraventricularly-administered 5-HT (Dalton, 1986) or report enhanced depressor responses to the administration of a blocker of the uptake of 5-HT combined with r_-%hydroxytryptophan (Fuiler, Holland, Yen, Bemis and Stamm, 1979). A recent study reported that chronic administration of dietary tryptophan reduced the intake of saline and prevented the rise in blood pressure associated with treatment with DOCA/NaCl (Fregly and Fater, 1986). No info~ation is available on the metabolism of serotonin during the development of hypertension induced by DOCA/NaCl. There is convincing evidence that the metabolism of 5-HT is increased in the early phase of hypertension induced by sinoaortic denervation (Chemerinski et al., 1980; Giarcovich-Martinez, Fernandez,
Chemerinski and Enero, 1983; Chalmers, 1975). Stores of serotonin (Felten et al., 1984; Saavedra, 1981; Wolfe and Kuhn, 1984a) and turnover (Smith et aZ., 1979; Koulu, Saavedra, Bjelogrlic, Niwa, Agren and Linnoila, 1986) are increased in spontaneously hypertensive rats (SHR). Intraventricular administration of 5,6_dihydroxytryptamine retards the development of hypertension in spontaneously hypertensive rats (Buckingham, Hamilton and Robson, 1976), however, depletion of 5-HT in brain with para-~horophenylalanine is associated with a rise in blood pressure in the spontaneously hypertensive rat (de Jong, Nijkamp and Bohus, 1975). Studies of the metabolism of serotonin or the effects of serotonin neurotoxins must be analyzed carefully since neurochemical, as well as other parameters, change as a function of age or duration of treatment in animal models of hypertension (Felten et al., 1984; Koulu et al., 1986; Giarcovich-Martinez et al., 1983). Administration of large doses of t-tryptophan to spontaneously hypertensive rats produce a doserelated decrease in blood pressure and increase the content of 5-HT in the central nervous system (Sved, van Itallie and Fernstrom, 1982; Wolf and Kuhn, 1984b). Echizen and Freed (1982) reported that infusion of L-5-hydroxytryptophan but not L-tryptophan over 24 hr decreased blood pressure in normotensive rats. The antihypertensive actions of L-tryptophan are complex and serotonin may (Sved, et al., 1982) or may not (Wolfe and Kuhn, 1984a) mediate the depressor response. The present results suggest that increases in the metabolism of 5-HT are associated with hypertension induced by DOCA/NaCl. This association of hypertension with altered metabolism of 5-HT is also evident in the spontaneously hypertensive rat and with hypertension induced by sinoaortic denervation. What is not clear is whether the changes seen in the metabolism of 5-HT are contributing to the pressor mechanism or if the changes in the metabolism of 5-HT are a response to the elevated blood pressure. Reflex and drug-induced elevations of blood pressure
424
R. DAWSONJR et a/.
are accompanied by increased concentrations of 5-HIAA in the extracellular fluid in the dorsal raphe and nucleus tractus solitarius (Freed, Echicen and Bhaskaran, 1985). Local application of S-HT in the nucleus tractus solitarius produces a neurogenic pressor response (Wolfe, Kuhn and Lovenberg, 1981). The fact that changes in the metabolism of .5-HT are most pronounced in the developmental phase of hypertension induced by DOCA/NaCl and in young spontaneously hypertensive rats (Smith et al., 1979; Koulu et al., 1986) suggest that increased metabolism of 5-HT is not important for the maintenance of hypertension. Thus, increased metabolism of 5-HT may be associated with the genesis of hypertension or metabolism of 5-HT may increase to attenuate a rise in blood pressure. Electrical stimulation of ascending serotonergic systems increases blood pressure, while electrical activation of bulbospinal 5-HT pathways can either increase or decrease blood pressure and sympathetic nervous activity (Wolfe et al., 1985). Therefore, 5-HT can be pressor or depressor, depending on the receptor population stimulated and the efferent circuitry activated. The effect of intravent~cularly administered muscimol on 5-HT and 5-HIAA in rats treated with DOCA/NaCl was complicated by the fact that levels of 5-HIAA were increased in rats treated with DOCA/NaCI compared to controls 60 min after injection of vehicle but not at 15 min after injection. This could have been the result of the stress associated with the intraventricular injection procedure or because these were different groups of rats. The metabolism of 5-HT appeared to be selectively altered in rats treated with DOCA/NaCl 15 min after administration of muscimol. This is in agreement with a previous study from this laboratory that demonstrated an enhanced depressor effect of muscimol in the DOCA/NaCl-induced hypertensive rat (Nagahama et al., 1985). Levels of 5-HIAA were increased in both rats treated with DOCA/NaCl and control rats 60 min after administration of muscimol, suggesting that the onset of action of muscimol was more rapid in rats treated with DOCA/NaCl than in controls. It is unclear whether the changes in 5-HIAA were associated with the depressor actions of muscimol or were a response to decreased blood pressure. Administration of 5-hydroxytryptophan attenuates the depressor actions of centrally administered GABA (Persson, 1980) and inhibitory responses of the sympathetic system, elicited by electrical stimulation of the medullary raphe nuclei, appear to be mediated by GABA (McCall and Humphrey, 1985). Thus, the bulbospinal 5-HT system appears to be under GABAergic control. Neurochemical evidence indicates that GABA can tonically inhibit the activity of 5-HT neuronal systems (Forchetti and Meek, 1981; Scatton et al., 1985). Chronic treatment with valproic acid, an inhibitor of the catabolism of GABA attenuates the development of hypertension induced by DOCA/NaCl Rotiroti, Palella, Losi, Ni-
stico and Caputi, 1982). The activity of glutamate decarboxylase is increased in the mesencephalon and GABA mimetics produce greater depressor responses in rats treated with DOCA/NaCl when compared to controls (Magri, Trimarchi, Quattrone, Squadrito, Di Giorgio, Costa and Caputi, 1984). Therefore, a disruption of interactions between S-HT and GABA in critical brainstem sites could contribute to the development of hypertension. The elevation in tryptophan in brain and consequent increases in levels of 5-HIAA seen in rats treated with DOCA/NaCI in this study could be the result of sympathetic activation and not a cause of the increased sympathetic activity seen in hypertension induced by DOCA/NaCl. Restraint stress increases levels of tryptophan and 5-HIAA in brain (Kennett and Joseph, 1981; Joseph and Kennett, 1983) and the increased uptake of tryptophan in brain can be attenuated by the inhibition of sympathetic activity with guanethidine (Franklin and Kelly, 1986). Thus, chronic sympathetic activation could increase levels of tryptophan in brain, although this appears to be unlikely in this study since the levels of 5-HIAA and 5-HT were not altered 5 weeks after treatment with DOCAiNaCl yet increased sympathetic activity was still presumed to be evident (de Champlain and van Ameringen, 1981). The adrenal steroids, hydrocortisone and corticosterone, alter key enzymes of tryptophan metabolism (Green, 1978). The possibility that DOCA could account for the changes in the metabolism of 5-HT or uptake of tryptophan, independent of any cardiovascular significance must also be considered. The increased levels of 5-HIAA and 5-HT in regions of the brain from rats treated with DOCA/NaCl, reported in the present study, are in contrast to the lack of effect of treatment with DOCA/NaCl on other monoamine systems in brain. An increase in levels of NE in the brainstem was found with both 2 and 4 weeks of treatment with DOCA/NaCl and acc~ulation of NE after pargyline was also increased in the brainstem of rats treated for 5 weeks with DOCA/NaCl. Alterations in NE in DOCA/NaCl-induced hypertension have been well characterized (Chalmers, 1975; de Champlain and van Ameringen, 1981) and participate in the increased sympathetic activity characteristic of hypertension induced by DOCA/NaCl. Few other changes in monoamines were found in the regions of the brain examined or the times sampled. Thus, it would appear that treatment with DOCA/NaCl provokes significant alterations in the metabolism of 5-HT which decline in magnitude during the established or maintenance phase of hypertension induced by DOCA/NaCl. Studies examining release of 5-HT, 5-HT receptors and the elaboration of interactions between 5-HT and GABA in controlling sympathetic outflow should yield important information in regards to the genesis of hypertension induced by DOCA/NaCl.
5-HT and DOCA/NaCI Acknowledgements-The authors wish to express their thanks to Victoria Redd Patterson for expert secretarial assistance in preparing this manuscript. This work was supported in pari by -NHLBI Grant-HL-25451, Hypertension Trainine Grant 5T32 HL07457 and a grant from the Division of-sponsored Research at the Uiiversity of Florida. REFERENCES
Baum T. and Shropshire A. T. (1975) Inhibition of efferent sympathetic nerve activity by 5-hydroxytryptophan and centrally administered 5-hydroxytryptamine. Neuropharmacology 14: 227-233. Bowker R. M.. Westlund K. N.. Sullivan M. C. and Coulter J. D. (198i) Organization of descending serotonergic projection to the spinal cord. Descending pathways to the spinal cord. Progress in Brain Research (Kuypers H. G. J. M. and Martin G. F., Eds), Vol. 57, pp. 239-265. Elsevier Biomedical Press, Amsterdam. Buckingham R. E., Hamilton T. C. and Robson D. (1976) Effect of intracerebroventricular 5,6-dihydroxytryptamine on blood pressure of spontaneously hypertensive rats. Eur. J. Pharmac. 36: 431437. Chalmers J. P. (1975) Brain amines and models of experimental hypertension. Circulation Res. 36: 469480. _ de Champlain J. and van Ameringen M. R. (1981) Evidence for the-participation of central noradrenergic mechanisms in DOCA-sodium hypertension in the rat. Central Nervous System Mechanisms in Hypertension (Buckley J. P. and Ferrario C. M., Eds), pp. 141~151. Raven Press, New York. Chemerinski E., Ramirez A. J. and Enero M. A. (1980) Sinoaortic denervation-induced changes in central serotonergic neurons. Eur. J. Pharmac. 64: 195-198. Dalton D. W. (1986) effects of centrallv . , The cardiovascular administered 5-hydroxytryptamine in the conscious normotensive and hypertensive rat. J. Autonomic Pharmac. 6: 67-75. DeFeudis F. V. (1982) Studies on GABA and cardiovascular function using in vivo and in vitro technics. Drug Dev. Res. 2: 507-517. De Jong W., Nijkamp F. P. and Bohus B. (1975) Role of noradrenaline and serotonin in the central control of blood pressure in normotensive and spontaneously hypertension rats. Archs int. Pharmacodyn. 213: 272-284. Dunnett C. W. (1970) Multiple comparison. Statistics in Endocrinoloev (McArthur J. W. and Colton T.. Eds). pp. 79%104.%T Press, Cambridge, MA. E;chizen H. and Freed C. R. (1982) Long-term infusion of L-5-hydroxytryptophan increases brain serotonin turnover and decreases blood pressure in normotensive rats. J. Pharmac. exp. Ther. 220: 579-584. Echizen H. and Freed C. R. (1984) Altered serotonin and norepinephrine metabolism in rat dorsal raphe nucleus after drug-induced hypertension. Life Sci. 34: 158 1-l 589. Felten S. Y., Weyhenmeyer J. A. and Felten D. L. (1984) Norepinephrine and serotonin in central autonomic nuclei in the spontaneously hypertensive rat and two normotensive control rats. Brain Res. Bull. 13: 437-441. Finch L. (1975) The cardiovascular effects of intraventricular 5,6_dihydroxytryptamine in conscious hypertensive rats. Clin. exp. Pharmac. Physiol. 2: 503-508. F:orchetti C. M. and Meek J. L. (1981) Evidence for a tonic GABAergic control of serotonin neurons in the median raphe nucleus. Brain Res. 206: 208-212. Franklin K. B. J. and Kelly S. J. (1986) Sympathetic control of tryptophan uptake and morphine analgesia in stressed rats. Eur. J. Pharmac. 126: 145-150. Freed C. R., Echizen H. and Bhaskaran D. (1985) V. Brain serotonin and blood pressure regulation: studies using in vivo electrochemistry and direct tissue assay. Lz> Sci. 37: 1783-1793. I,
hypertension
425
Fregly M. J. and Fater D. C. (1986) Prevention DOCAinduced hypertension in rats by chronic treatment with tryptophan: Clin. Exp. Pharmac. Physiol. 13: 767-776. Fuller R. W.. Holland D. R.. Yen T. T.. Bemis K. G. and Stamm N. b. (1979) Antihypertensive dffects of fluoxetine and L-5-hydroxtryptophan in rats. Life Sci. 25: 1237-1242. Giarcovich-Martinez S., Fernandez M., Chemerinski E. and Enero M. A. (1983) Central serotonergic activity after neurogenic hypertension. Eur. J. Pharmac. 86: 337-345. Gillis R. A., Yamada K. A., Dimicco J. A., Williford D. J., Segal S. A., Hamosh P. and Norman W. P. (1984) Central r-aminobutyric acid involvement in blood pressure control. Fed. Proc. 43: 32-38. Green A. R. (1978) The effects of dietary tryptophan and its peripheral metabolism on brain 5-hydroxytryptamine synthesis and function. Essays in Neurochemistry & Neuropharmacology (Youdim M. B. H., Lovenberg W., Sharman D. F. and Lagnado J. R., Eds), Vol. 3, pp. 104-127. Wiley, New York. Joseph M. H. and Kennett G. A. (1983) Stress-induced release of 5-HT in the hippocampus and its dependence on increased tryptophan availability: an in vivo electrochemical study. Brain Res. 270: 251-257. Kennett G. A. and Joseph M. H. (1981) The functional importance of increased brain tryptophan in the serotonergic response to restraint stress. Neuropharmacology 20: 3943. Kontur P., Dawson R. and Monjan A. (1984) Manipulation of mobile phase parameters for the HPLC separation of endogenous monoamines in rat brain tissue. J. Neurosci. Meth. 11: 5-18. Koulu M., Saavedra J. M., Bjelogrlic N., Niwa M., Agren H. and Linnoila M. (1986) Serotonin turnover in discrete hypothalamic nuclei and mesencephalic raphe nuclei of young and adult spontaneously hypertensive rats. Brain Res. 379: 257-263. Kuhn D. M., Wolf W. A. and Love&erg W. (1980) Review of the role of the central serotonergic neuronal system in blood pressure regulation. Hypertension 2: 243-255. Leysen J. E. (1985) Serotonergic binding sites. In: Serotonin and the Cardiovascular System (Vanhoutee P. M., Ed.), pp. 43-62. Raven Press, New York. M&i V., Trimarchi G. R., Quattrone G., Squadrito F., Di Gioreio R. M.. Costa G. and Canuti A. P. (1984) Increised cardidvascular responsiveness to GABkergic stimulation in DOCA-salt hypertensive rats. Res. Commun. them. Path. Pharmac. 45: 107-118. McCall R. B. and Humphrey S. J. (1982) Involvement of serotonin in the central regulation of blood pressure: evidence for a facilitating effect on sympathetic nerve activity. J. Pharmac. exp. Ther. 222: 94-102. McCall R. B. and Humphrey S. J. (1985) Evidence for GABA mediation of sympathetic inhibition evoked from midline medullary depressor sites. Brain Res. 339: 356360. Myers M. G., Reid J. L. and Lewis P. J. (1974) The effect of central serotonin depletion on DOCA-saline hypertension in the rat. Cardiovascular Res. 8: 806810. Nagahama S., Dawson R. and Oparil S. (1985) Enhanced depressor effect of muscimol in the DOCA/NaCl hypertensive rat: Evidence for altered GABAergic activity in brain. Proc. Sot. exp. Biol. Med. 180: 277-283. Nishikawa T. and Scatton 8. (1985) Inhibitory influence of GABA on central serotonergic transmission. Raphe nuclei as the neuroanatomical site of the GABAergic inhibition of cerebral serotonergic neurons. Brain Res. 331: 91-103. Peroutka S. J. and Snyder S. H. (1979) Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [‘HIlysergic acid diethylamide and [‘HIspiroperidol. Molec. Pharmac. 16: 687499. Persson B. (1980) Cardiovascular effects of intra-
426
R. DAWSONJR et al.
cerebroventricular GABA, glycine and muscimol in the rat. Naunyn-Schmiedebergs Arch. Pharmac. 313: 225-236. Pilowsky P. M., Kapoor V., Minson J. B., West M. J. and Chalmers J. P. (1986) Spinal cord serotonin release and raised blood pressure after brainstem kainic acid injection. Brain Res. 366: 354357. Rotiroti D.. Palella B., Losi E., Nistico G. and Caputi A. P. (1982) Evidence that a GABAergic mechanism influences the development of DOCA-salt hypertension in the rat. Eur. J. Pharmac. 83: 153-154. Saavedra J. M. (1981) Central biogenic amines and neuropeptides in genetic hypertension. Central Nervous System Mechanisms in Hypertension (Buckley J. P. and Ferrario C. M., Eds), pp. 129-139. Raven Press, New York. Scatton B., Serrano A. and Nishikawa T. (1985) GABA mimetics decrease extracellular concentrations of 5-HIAA (as measured by in uiuo voltammetry) in the dorsal raphe of the rat. Brain Res. 341: 372-376. Smith M. L., Browning R. A. and Myers J. H. (1979) In uiuo rate of serotonin synthesis in brain and spinal cord of young, spontaneously hypertensive rats. Eur. J. Pharmac. 53: 301-305.
Steinbusch H. W. M. (1981) Distribution of serotoninimmunoreactivity in the central nervous system of the rat-cell bodies and terminals. Neuroscience 6: 557-618. Sved A. F., Van Itallie C. M. and Fernstrom J. D. (1982) Studies on the antihypertensive action of L-tryptophan. J. Pharmac. exp. Ther. 221: 329-333. Vanhoutte P. M. (1985) Serotonin and the Cardiovascular Sysrem. Raven Press, New York. Wolf W. A., Kuhn D. M. and Lovenberg W. (1981) Blood pressure responses to local application of serotonergic agents in the nucleus tractus solitarii. Eur. J. Pharmac. 69: 29 1-299. Wolf W. A. and Kuhn D. M. (1984a) Antihypertensive effects of L-tryptophan are not mediated by brain serotonin. Brain Res. 295: 356359. Wolf W. A. and Kuhn D. M. (1984b) Effects of r.-tryptophan on blood pressure in normotensive and hypertensive rats. J. Pharmac. exp. Ther. 230: 324329. Wolf W. A., Kuhn D. M. and Love&erg W. (1985) Serotonin and central regulation of arterial blood pressure. In: Seroronin and the Cardiovascular System (Vanhoutte P. M., Ed.), pp. 63-74. Raven Press, New York.