Receptor Mechanisms Hype~ensive Rats

Received 3 1 July 1991

VAN DEN BUUSE, M., C. R. JONES AND J. WAGER. Brain dopai&e D-2 reeepror ~ec~~~~~ in s~n~~~~~~~ @per- tens&e ruts. BRAIN RES BULL zsfz) 289-297, 1992.-Brain dop~~~~c function was studied in s~n~~~usly hypertensive rats (SHR) with the selective dopamine D-2 antagonist sulpiride. Sulpiride dose-dependently inhibited locomotor activity of nor- motensive Wistar-Kyoto rats (WKY). SHR showed an increase in locomotor activity in response to low doses of sulpiride, whereas no effect was observed of higher doses. In a two-bottle salt-preference test, WKY showed increased preference for an 0.9% saline solution after treatment with sulpiride, whereas total fluid intake remained the same. In SHR, sulpiride influenced neither salt- preference nor total fluid intake. SHR and WKY with a unilateral lesion of the median forebrain bundle showed similar turning behaviour in response to treatment whh ~p~~oe. Pretreatment with 100 mg/kg suipiride virtually abolished atone- induced turning in WKY, but had little effect in SHR. Sulpiride dose-dependently increased seem prolactin concentrations in WKY and SHR. However, the increase was si~ni~c~tly greater in SHR. Dopamine D-2 receptor binding was measured with in vitro autoradiography, using [*z51+sulpiride as the ligand. Binding density was similar in the caudate nucleus and substantia nigm af SHR and WKY brain. Concentrations of the dopamine metabolites DOPAC and HVA, but not of dopamine itself, were signifi- cantly increased in frontal cortex, striatum and hypothalamus after treatment with 100 mgikg sulpiride. There were no significant differences between SHR and WKY in the increase in the DOPAClDA and HVA/DA ratio. These data show that SHR show differential changes in their response to central dopamine D-2 blockade when compared to WKY. Thus, in some tests @comotor a&&y after high doses, salt preference, turning behaviourj, SHR respond less to sufpiride. In contrast, in other aspects (locomo- tor activity after low doses, serum prolactin levefsf, SHR show increased functional responses to suipiride when compared to WKY. These differences are not the result of changes in postsynaptic receptor density, as dopamine D-2 receptor binding and the increase in dopamine turnover after D-2 receptor blockade were normal in SHR. It is concluded that SHR show increased func- tional in vivo central dopamine neurotransmission, possibly due to higher stress-induced central dopamine release and/or an altered coupling of dopamine D-2 receptors to (one of) their transduction mechanisms or second messenger systems. This could explain the relative lack of postsynaptic receptor blockade by sulpiride in this strain. However, presynaptic receptor density or sensitivity is upregulated in SHR to counteract the higher dopaminergic activity, which might explain tbe dis~~~b~to~ effect of LOW doses of sulpiride found in this strain.

Spontaneously hypertensive rat Dopamine Locomotor activity Salt preference D-2 receptors Turning behaviour Prolactin Metabolites

~~R~~S studies have suggested a role of central catechola- mine systems in the development of kQpx&miOn [for recent reviews, see (tl, 23, 39, 42)]. Thus changes in the central con- centrations and turnover of noradrenaline, adrenaline and dopa- mine in various brain regions of spontaneously hypertensive rats (SHR) have been described. Also the density of central catechol- amine receptors and the activity of catechol~e-synthesizing enzymes was shown to be altered in SHR.

Lesion studies have shown that forebrain dopamine depletion in young SHR inhibits the subsequent development of hyperten- sion (7, 37, 38, 40). This suggests that functional overactivity of brain dopamine systems is involved in the initiation of the rise in blood pressure in these animals, with the other changes in central catecholamine systems being a response to or inde- pendent of this process. However, it is unclear through which mechanism changes in the activity of central dopamine systems may play this role.

In the present study a number of functional tests were used to measure central dopaminergic activity in SHR. Previously, by

using an open-field tc?st, it was shown that SHR appear to be relatively insensitive to the inhibitory effects of dopamine D-2 receptor blockade on locomotor activity (36), a finding which would be in line with the proposed increased central dopamine neurotransmission in this strain (see above). Particularly, the se- lective dopamine D-2 receptor antagonist sulpiride (17, 21, 26, 28) dissociated SHR from their normotensive controls, the Wis- tar-Kyoto rats (WKY). In the present study, the effect of sulpir~ ide was compared between SHR and WKY in four in vivo tests which may invoive different central dopamine systems. More- over, we measured in vitro dopamine receptor binding and the increase in dopamine turnover in response to dopamine D-2 re- ceptor blockade. The differential changes in the effects of sulpir- ide in SHR sheds new light on the in vivo activity of central dopamine systems in this strain.

METHOD Artimals, Chemicals

Male SHR and WKY were obtained from IFFA Credo,

289

290 VAN DEN BLJUSE. JONES AND WAGNEK

L’Arbresle Cedex, France, and kept in the center in a quiet, ch- matized animal room during at least one week prior to the ex- periments. The animals had free access to pellet food and tap water.

In one group of SHR and WKY (280-320 g body weight, n=7). we measured mean arterial pressure (MAP) and heart rate. Two days before the measurements, the animals were op- erated under pentobarbital anesthesia. A polyethylene cannula was inserted in the left femoral artery, tunneled under the skin and exteriorized in the neck. Blood pressure was measured from conscious rats with a Statham P23Db transducer connected to a Grass model 7E polygraph. Grass tachographs determined heart rate from the pressure pulse. Basal MAP and heart rate, calcu- lated as the average of II-13 readings 5 min apart, was 168 ?Z 4 mmHg in SHR and 117+3 mmHg in WKY, and 378 rt 10 beatslmin in SHR and 349t 8 beatsimin in WKY, respectively.

Throughout the study, the commercially available injectable solution of sulpiride (Dogmatil, 100 mg/2 ml, Delagrange) was used. Where necessary, this solution was diluted with 0.9% sa- line to yield the appropriate dose. Injections of sulpiride were intraperitoneal (IP) in a volume of 2 ml/kg or, in case of the highest dose in the locomotor activity experiment. 2.5 ml/kg.

Experiment 1: Locomotor Activity

The effect of sulpiride on locomotor activity of SHR and WKY was tested with automated activity meters (Opto-Varimex mini infrared beam activity monitors and Bascount computer program, Columbus Ins~ments). Naive rats of 200-225 g were injected with 0, 0.2, 1, 5, 25 or 125 mg/kg sulpiride one hour before being placed in the activity cages. Locomotor activity was subsequently measured during one hour. Experiments were per- formed between 9:00 a.m. and 1:OO p.m.

experiment 2: Saline Preference

The preference of rats to drink from either an 0.9% saline solution or normal tap water was measured in a two-bottle choice test (IO). Thus, on day 0, SHR and WKY of 300-320 g were weighed and put in single cages with food available but no wa- ter. After 23 hours, two bottles were put on each cage. One bottle contained normal tap water, the other an 0.9% solution of NaCl in tap water, After one hour the bottles were removed and the rats were weighed. The amount of fluid taken by the ani- mals was determined by weighing the bottles before and after the one hour drinking period. The procedure was repeated on day 2, 3 and 4.

Data from day 1 and 2 were considered to represent habitua- tion to the experimental procedure and were not used in the present ex~~ments. Data from day 3 were taken as baseline value. Thus, water intake, saline intake, and total fluid intake were measured and calculated as intake per kg body weight. Sa- line preference was calculated as the percentage saline intake per kg body weight of total fluid intake per kg body weight. On day 4, the animals were injected with different doses of sulpiride (0, 1, 10 or 100 mg/kg) 30 min before the bottles were placed on the cage. Since data from day 4 were compared with those from day 3, each animal served as its own control. The data of two experiments with essentially identical results were pooled.

Experiment 3: Turning Behaviour

Turning behaviour (25,34) was induced by a unilateral lesion of the median forebrain bundle. SHR and WKY of 175-200 g were pretreated with 20 mg/kg desipramine IP, and 30 min later

anesthetized with Hypnorm (fentanyl and fluanison, Dupharr. 6-Hyd~xydop~ne was dissolved 4 mg/ml in 0.9% saline with 0.1% ascorbic acid. A volume of 2 pl of this sotution was m- jetted stereotaxically at 2 mm posterior of the bregma. I .S mm lateral of the midline and 8 mm ventral of the dura. The tooth- bar of the stereotact was set at +5. After the operation, the rats were housed individually.

Starting from one week after the operation. the rats were tested weekly for turning behaviour. Only rats which showed a constant level of at least 100 turns per hour in response to an injection of 0.5 mglkg d-amphetamine were used. Thus 6 SHR and 6 WKY were selected for further testing. Over three 60-min control sessions, these groups showed 174 Z? 23 and 160 2 17 turns respectively after injection of d-amphetamine.

Starting from four weeks after the operation, the animals were pretreated with different doses of sulpiride (0. I, LO or 100 mgikg) one hour before receiving d-amphetamine. Thus. after four sessions, all animals had received all doses of sulpiride.

Experiment 4: Serum Prolactin Concentrations

SHR and WKY of 200-225 g were injected with different doses of sulpiride (0, 0.1, 1, 10, or 100 mglkg) and decapitated in a separate room 45 min later. Trunk blood was collected in EDTA-containing tubes and allowed to clot for 2 hours in ice- water. The blood was then centrifuged for 25 min at 3000 t-pm. Alliquots of 150 pl serum were taken, frozen on dry ice and kept at -80°C. The concentration of prolactin in the serum samples was performed with a double-antibody radioimmunoas- say by Dr. F. Gamier, bole Nationale Veterinaire de Lyon. Rat prolactin RP-3 from the National Institute of Arthritis, Diabetes and Digestive Kidney Diseases was used as a standard, while the anti-rat-prolactin antibody and anti-rabbit precipitating anti” body were purchased from UCB-Bioproducts. Sensitivity of the assay was 5 ng/ml and the intraasay coefficient of variation be- low 10%.

~per~ment 5: Autor~~ograph~ of Dopam~t~e Receptors

SHR and WKY (200-250 g body weight) under ether anaes- thesia were perfused with heparinised saline and 0.1% parafotm- aldehyde. The brains were dissected and in a cryostat microtome 10 pm sagittal sections were cut. The sections were mounted onto gelatine-coated microscope slides. Labelling of dopamine receptors was carried out by incubating the tissue sections with 300 pM [ ‘2sI]sul~iride for D-2 receptors (2,171 or, for compari- son, 150 pM [‘* I]SCH 23982 for D-l receptors (6) in an incu- bation buffer containing 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl,, and 1 mM MgCl, (pH 7.4). The incubation buffer contained 10 pM serotonin to block binding (especially of [‘251]SCH 23982) to central serotonin receptors. These con- centrations of ligand yield a 16% occupancy of D-2 receptors (17) and 32% occupancy of D-l receptors (6). Adjacent sections were incubated in the same buffer in the presence of 1 pM cis- flupenthixol to determine nonspecific binding for dopamine D-l receptors and 10 FM apomotphine for D-2 receptors. Labelling of receptors was carried out for 60 min at room temperature and the incubations were ended by dipping the slides into ice-cold incubation buffer for two 2-min washes for D-2 and one 5-min wash for D-l receptors. After washing, the labelled tissues were exposed to LKB Ultrofilm for 3-5 days to generate autoradio- grams. Quantification against known standards was performed as previously described (14) using an MCID image analysis system.

Experiment 6: Dop~m~ne Turnover

The effect of different doses of sulpiride (0, 1. 10 or 100

BRAIN ~PAM~~ FUNCTION IN HYPERTENS~UN

mglkg) on the ratio of DOPAC/dopamine and HVA/dopamine was measured three hours after injection (32). SHR and WKY of 200-225 g were decapitated and their brains and whole pitu- itaries were rapidly removed. On a cold plate, the brains were dissected into frontal cortex, striatum, h~~~a~rn~s and me- dulla obfongata. AH tissue parts were frozen in liquid nitrogen and kept at - 80°C.

The tissue parts were weighed before homogenisation. The overall mean tissue weight (n = 32), for SHR and WKY respec- tively, was: frontal cortex 238 t: 5 vs. 237+7 mg; striatum 43 ? 2 vs. 43 C 1 mg; hypothalamus 35 + 1 vs. 35 k 1 mg; me- dulla oblongata 198?3 vs. 21O-C6 mg; pituitary 8.920.2 vs. 7.1 t0. I mg. The difference in pituitary wet weight between SHR and WKY was significant.

The tissue parts were homogenised in an appropriate vol- ume of 0.2 N HClO, containing Na,S,O,, EDTA and 50 kg/l a-methyl-DOPA as internal standard. The homogenates were centrifuged and the supematant was used. The concentrations of dopamine and its metabolites DOPAC and HVA, as well as of noradrenaline and serotonin and its metabootite S-HfAA, were measured by high-performance liquid chromatography with elec- trochemical detection (HPLC-ECD) according to the method of Wagner et al. (43). Briefly, 10-50 pl of supernatant was in- jected in a Beckman Ultrasphere ion-pair column (250x 4.6 mm, 5 pm particle size), protected with a guard column (70 x 2 mm) filled with CQPeII UDS. The temperature of the column was maintained at 2g,5”C. The eluent consisted of 0.1 M NaH,PO,-methanol (82.5:17.5, v/v) with 2.6 mM OSA, 0.f mM EDTA and 0.25 mM triethylamine and was brought to pH 3.39 with H&I,. Flow rate was 1 mumin. The electrochemical detector (Bioanalytical systems) was set at 0.68 V. Concentra- tions were calculated as rig/g or ngimg (pituitary) tissue wet weight.

Statistics

Data are expressed as mean2 standard error of the mean (SEM). Comparison between SHR and WKY and between dif- ferent treatment groups was done with analysis of variance ~AN~VA) and Duncan’s multiple range test. In case of the sa- line-preference and of the t~in~~haviour data, a repeated measures design was used to analyze within-animal effects. Dif- ferences between groups were considered significant at ~~0.05.

RESULTS

After being placed in the test cages, SHR as well as WKY displayed an initially high level of activity which decreased as the animals habituated to the novel environment. Saline-treated SHR were significantly more active man saline-treated WKY in me first 30-min period, but not the second 3Omin period (Table f and Fig. I).

Table 1 shows the effect of pretreatment with stdpiride on locomotor activity during the first period of 30 min; Fig. 1 shows the data for the second period of 30 min. In both time periods a significant overall strain difference and a significant overall effect of suipiride was observed. The strain-treatment in- teraction failed to reach significance, however. WKY treated with the highest dose of smpiride showed sign~~~~dy Lower ac- tivity scores than saline-treated WKY both in the fit and the second 30-mitt period. SHR treated with 1 mg/kg or with 5 mg/kg sulpiride showed significantly higher scores than saline- treated SHR in the second period of 30 min (Fig. 1).

1500 r * *

2 _/ t 1OOO - \I i! 2 *---0 T A

r$

5

Z/l l -i\, .

Y 500 ” 1 \

t 0-U SHR *

l -• WKY \

8

0

VEH 0.2 1 5 25 125

dose @w/kg)

FIG. 1 s The effect of sulpIfide on locomotor activity of SHR and WKY. Data are from the second Period of 30 min after the rats were placed in the test cage. The number of animals is 6 per group, except fur the sa- line-treated groups where n= 12. *&0.05 for difference with saline- treated rats of the same strain. SHR treated with 1, 5, 25 or 125 mgkg sulpiride showed sign~~c~~y higher activity scores than simifarIy treated WKY.

Experiment 2: Saline Preference

There was no significant difference between control SHR and WKY or between the various treatment groups witbin one strain for any of the parameters on the controi day (day 3).

Treatment with I mg/kg sulpiride tended to increase total fluid intake in SHR. Thus, whereas total fluid intake showed no significant difference between the various SHR groups on the control day (day J), the group treated with 1 mgikg (day 4) showed significantly higher fluid intake when compared to the vehicle-treated SHR. However, the difference between total fluid intake on the experimental day (day 4) and the control day (day 3) of the SHR-treated with 1 mg/kg su~ppiride failed to reach sta- tistical significance. No significant changes in any of the other parameters was observed in SHR (Table 2 and Fig. 2).

Comparison of data obtained on the control day and on the experimental day showed that treatment with 10 mg/kg sulpiride induced a significant increase in water intake in WKY but that

TABLE 1

THE EFFECT QF SULPIRIDE ON LOCOMOTOR ACTIVITY OF SHR AND WKY DURING THE FIRST 30 MIN AFTER

PLACEMENT IN THE ACTMTY CAGES

Treatment (m&kg) SHR WKY

Vehicle 1335 f 49” 930 ” 90 Sulpiride 0.2 1303 + 110 1045 c 118 Sulpiride 1 1417 k la0 1163 e 145 Sulpiride 5 1599 k 233” 1007 zk 159 Suipiride 25 1299 + 150” 692 + 116 Sulpiride I25 I183 i 1s7* 224 2 75;

*p<O.O5 for difference between SHR and WKY. TpCO.05 for difference with saline-treated controls. The number of

animals is 6 per group, except for the saline-treated groups where n = 12.

292 VAN DEN BUUSE, JONES AND WAGNER

SHR

WKY

*

un 10 100 mg/kg

0 control m sulpiride

FIG. 2. The effect of sulpiride on saline preference of SHR and WKY in a two-bottle choice test. Data are the percentage saline intake per body weight of total fluid intake per body weight *the between-group SEM. All animals were tested on the control day (day 3, see the Method sec- tion) and the treatment day (day 4). *p<O.O5 for within-animal compar- ison between the control day and the treatment day (repeated-measures ANOVA).

100 mg/kg significantly decreased this parameter (Table 2). No corresponding changes were found for saline intake or total fluid intake. Saline preference significantly increased in WKY after treatment with 100 mg/kg sulpiride (Fig. 2). The effect of 10

O-O SHR \

0-O WKY t”

VEH 1 10 100

dose b-w/kg)

FIG. 3. The effect of sulpiride on amphetamine-induced turning behav- iour of SHR and WKY with a unilateral lesion of the median forebrain bundle. Data are the total number of turns in one hour. The number of animals is 6 per group. *p<O.O5 for difference with saline-treated con- trols of the same strain. WKY treated with 100 mg/kg sulpiride showed a significantly lower number of turns than similarly treated SHR.

mg/kg sulpiride failed to reach statistical significance 0, = 0.078).

Experiment 3: Turning Behaviour

Sulpiride treatment induced a decrease in the number of am- phetamine-induced turns in both SHR and WKY, but the effect was much greater in WKY (Fig. 3). Thus 100 mg/kg sulpiride virtually abolished turning in WKY (- 81%). but not in SHR (-38%).

Experiment 4: Serum Prolactin Concentrations

Basal serum concentrations of prolactin were not different between SHR and WKY (Fig. 4). Treatment with sulpiride in-

TABLE 2

THE EFFECT OF SULPIRIDE ON WATER INTAKE, SALINE INTAKE AND

TOTAL FLUID INTAKE OF SHR AND WKY IN A TWO-BOTTLE CHOICE TEST

Treatment Group (n)

Water Saline Total Fluid

Day 3 Day 4 Day 3 Day 4 Day 3 Day 4

SHR Vehicle (19) 16 t 3 14 2 3 42 -t 5 462 4 57 f 4 601-3

Sul 1 (7) 17 * 4 19 ? 8 48 +- 6 55 + I1 65 ? 6 74 + 7t

Sul 10 (19) 15 * 3 14 * 3 43 2 3 44+ 4 57 ” 2 58 + 3

Sul 100 (14) 17 5 2 19 2 3 36 r 4 4ot 5 53 + 3 59 + 3

WKY Vehicle (19) 14 2 2 13 ‘- 2 38 ? 4 44L 4 52 t- 3 57 * 3

Sul 1 (7) 12 +- 1 12 ? 2 41 * 4 39L 4 54 2 4 51 + 3

Sul 10 (19) 14 rfr 3 22 * 4* 36 ? 5 36-c 5 51 ?I 3 57 t- 3

Sul 100 (13) 18 ? 3 11 * 3* 33 * 4 352 6 50 + 3 47 ? 6

Data were calculated as ml per kg body weight during one hour. *p<O.O5 for difference with corresponding value on control day (day 3). @<0.05 for difference with saline-treated controls of the same strain.

BRAIN DOPAMINE UNCTION IN HYPERTENSION 293

VEH 0.1 1 10 toa

dose hg/kd

FIG. 4. The effect of st@iride 0~ Serum prolactin ~~n&e~~at~~~s of SfiR and WKY 45 tin after injection. There are 6 rats per group except in the control groups where n= 8. *ptO.M for difference. with saline- treated controls of the same strain, Sulpiricie increased serum prolactin concentrations to a greater extent in SHR than in WKY.

creased serum prolactin ~~~~n~~~~s in both strains, but this effect was greater in SHR than in WKY (Fig. 3). Thus, in SHR, 1 mg/kg sulpiride increased serum prolactin concretion sig- nificantly to 241% of control values. Treatment with 10 mg/kg and 100 mg&g induced a similar effect (226% and 251%, re- spectively). in WRY, 1 mg/kg did not s~gn~~~~dy affect serum p~l~tin concentrations, but 10 mg&g and 100 mgikg caused a significant increase of 206 and 22f%, respectively.

Typical autaradioptns for dopmine D-1 and D-2 receptors are shown in Fig, 5. Highest binding density of both receptor subtypes was found in the nucleus caudatus, nucfeus accumbens, olfactory tuhercfe and substantia nigra pars compacta. In addi- tion, high D-l binding was observed in the substantia nigra pars reticu~ata and moderate D-l binding in cortical regions. Moder- ate D-2 binding was observed in the superior and inferior collic- uius, the molecular layer of the hippocampus and in hypothalamic regions (Fig. 5). No gross changes were observed between SHR and WRY sections in arty of these regiom. The mean receptor density values in nucleus caudatus and subst~tia n&a of SHR and WRY are shown in Table 3+ There were no significant dif- ferences in receptor densities between the two groups in either regiou.

Table 4 shows the basal ~ncen~t~~~s of dopamine and its metabolites MlpAC and HVA, as well as of noradrenaline and serotonin and its metabolite 5-H&4, in frontal cortex, striatum, b~o~~~us, medulla oblongata and pituitary of SHR and WKY. No significant differences in baseline brain monoamine concen~a~ons were observed, except for a iower HVA concen- tration in the frontal cortex and the medufta obfongata of SHR when compared to WRY. Afso the basal HV~do~ne ratio was significantly lower in frontal cortex and medulla oblongata of SHR. In the pnnitary, despite the higher wet weight (see the Method section), the concentration of dopamine and of DOPAC

were significantly lower in SHR than m WKY. However, the penis ~PAC~d~~~ine ratio did not differ between the two strains. The levels of HVA were too low to be detected reliably in the pituitary.

In the brain, only the highest dose af sulpiride (100 mglkg) caused an increase in the con~n~iions of DOPAC and HVA, but not of dopamine (not shown). Consequently, there were sig- nificant increases in the DOFAC/dopam~ne ratio and HVAtdopa- mine ratio (Fig. 6). The overali effect of sulpiride was s~g~i~cant in frontal cortex and striatum, but there was no difference be- tween the strains (significant effect of treatment only). In the hy~~~~us the overall effect of sulpiride on the DOPACldo- pamine ratio failed to reach significance @ =0.08), This was apparantIy caused by the lack of an effect of sulpiride in WKY, despite a significant increase in the DOPACldopamine ratio in SHR (Fig. 6). Sulpiride increased the h~~~~a~c HVA/dopa- mine ratio similarly in SHR and WKY. In the medulla oblon- gata there was only an effect of sulpiride on the HVA/dopamine ratio. Despite the fact that there was an overall strain difference in the HV~dop~~ne ratio in the medulla obiongata (see also above), the increase caused by sulpiride was simifar in the two strains (significant effect of strain and of treatment, but no strain X treatment interac~o~).

In the pituitary the effect of suiphide differed somewhat from that in the brain. Thus a significant overall increase of the ~PA~dop~~ne ratio was observed in response to sulpiride, with no overah difference between the strains. fn contrast to the brain, however (see above and Fig. 6). the effect of suipiride was maximal at the IO mgkg dose, whereas the 100 mgJkg dose did not signi~cantly change the DOPA~~dop~ine ratio.

The present results show that the in vivo response of SHR in four different functionaf. tests differs from that of WRY in two main aspects. First, the effect of high doses of sulpirido (IOO- 125 mglkg), as found in WRY in the locomotor activity experi- ment, the saline preference test and the turning bchaviour experimern, is essentially absent in SHR. Second, a greater ef- feet of lower doses of sulpiride (i-5 mg/kg) is present in SHR in some tests, notably on locomotor activity and on serum pro- la&n levels. importantly, however, SHR and WRY generally show similar increases in the DOPA~~~opamine ratio and HVAI dopamine ratio in response to dopamme D-2 receptor blockade with sulpiride. Also, the in vitro D-2 receptor binding did not show differences between the strain, To explain these results, it is suggested that central ~~rn~nergic neuro~smiss~on is en- hanced in SHR and, possibly as a consequence, presynaptic in- hibition of dopamine release is upregulated. This results in an animal with app~a~tly normal baseline responses (e.g., se the Locomotor Activity and Prolactin Levels section), but altered responses to drugs such as the dopamine D-2 antagonist sulpir- ide. Also, in experiments with dopamine D-2 receptor agonists, SHR have been shown to display aherations in dop~ine-adze ated responses in some tests, but not others (9. t2, 36).

Xt is generally accepted that sulpiride is a selective dopamine a-2 ~tagonist (17, 21, 26, 28). In the brain, dop~in~ D-2 re- ceptors are found in high density in do~~ine-inne~ated regions [(6,17), present results], both postsynaptically to dopamine neu- rons and p~s~ap~c~ly on dopaminergic ne~e tern&ah and c&f bodies (Zg, 44, 45) Presynaptic- or autoreceptors are acti- vated by lower concentrations of dopaminergic agonists than postsynaptic receptors, leading to an inhibition of dopamine re- lease and dopamine synthesis (45) In viva, low doses of dopa- mine agonists may thus inhibit locomotor activity (1,45). Under

294 VAN DEN BUUSE, JONES AND WAGNEK

DI WK’

02 SHF;

D2 WK’I .I..: _.

FIG. 5. Representative examples of autoradiograms from sagittal sections of SHR and WKY labelled with [‘*‘IlSCH23892 (top panel) and [‘*‘I]sulpiride (bottom panel). Left of each pair of sections is the appropriate series of calibrated grey scales. Tbe middle dia- gram outlines anatomical details: nucleus caudatus (NC), nucleus accumbens (NA), olfac- tory tubercle (OT) cortex (Co), hip~~pus (H), superior colliculus (SC), inferior coIiiculus (IC), substantia nigra pars compacta (SNc), substantia nigra pars reticula&+ (SNr), cerebellum (Ce) and medulla (M). No differences were observed in either D-2 or D- 1 binding density between SHR and WKY.

certain circumstances, low doses of dopamine D-2 antagonists striatal slices of SHR indeed an enhanced presynaptic inhibition have been shown to increase motor activity in normotensive rats of dopamine release was detected (15). In the prolactin test, (4,24). In the present experiments, SHR, but not WKY, showed SHR show a similarly enhanced response after low doses of an increase in locomotor activity after treatment with low doses sulpiride as in the locomotor activity test. Also, other authors of sulpiride. This supports the notion that presynaptic receptors have shown exaggerated changes in blood prolactin levels in hy- on dopamine nerve terminals in the brain are more sensitive in pertension (8, 22, 31). Whereas dopamine D-2 receptors are lo- these animals. In this respect, it is interesting to note that in cated postsynaptically on mammotrophs, it has been suggested

BRAIN DOPAMINE FUNCTION IN HYPERTENSION 295

TABLE 3 TABLE 4

DOPAMINE RECEPTOR LEVELS IN SHR AND WKY BRAINS LABELLED WITH [‘Z51]SULPIRIDE (D-2 RECEF’TORS) AND

[‘251]SCH23982 (D-l RECEPTORS)

BASAL CONCENTRATIONS OF DOPAMINE AND ITS METABOLITES DOPAC AND HVA, NORADRENALINE, AND SEROTONIN AND ITS

METABOLITE 5-HIAA IN BRAIN AND PITUITARY OF SHR AND WKY

Caudate-Putamen SHR WKY

[‘*51]Sulpiride

102 2 13 92 f 5

[‘ZSI]SCH23892 SHR WKY

Frontal Cortex 561 k 40 Dopamine 191 + 29 152 2 25 494 + 27 DOPAC 86 2 7 94 + 13

Substantia Nigra SHR WKY

19 _c 2 17 ? 2

HVA 58 k 4* 73 f 6 302 34 Noradrenaline 247 + 22 266 2 4 2 278 + 29 Serotonin 629 f 109 805 + 152

5-HIAA 364 f 31 437 2 29

Data are mean fmol/mg protein k S.E.M. of n = 7. No statistically significant differences were observed between SHR and WKY.

by a number of authors that these receptors respond to various dopaminergic drugs similarly as central presnyaptic D-2 dopa- mihe receptors (3,18). Thus brain presynaptic D-2 dopamine re- ceptors and pituitary postsynaptic D-2 dopamine receptors might constitute a subgroup of “inhibitory” D-2 dopamine receptors, which appears to be functionally upregulated in the SHR.

The lack of an effect on locomotor activity or saline prefer- ence at higher doses could also have been due to an increase in postsynaptic dopamine D-2 receptor density. In fact, in binding studies using striatal homogenates of SHR, some authors have shown increased binding of [3H]spiroperidol [for references, see (39)]. However, in our autoradiographic binding experiments with the more specific ligand [‘251]-sulpiride, we found no dif- ferences in brain dopamine D-2 binding density between SHR and WKY. Also, the generally similar increase in dopamine turnover in response to dopamine D-2 receptor blockade in SHR and WKY argues against changes in dopamine D-2 receptor density.

Another explanation for the different effect of the higher (postsynaptic) doses of sulpiride could be that endogenous dopa- mine release is markedly enhanced in these animals, thus shift- ing the antagonist dose-response curve to the right. This increase in brain dopamine release could induce a compensatory upregu- lation of presynaptic inhibition. SHR show behavioural and car- diovascular hyperreactivity to stress (35). Ikeda and co-workers (13) have indeed shown an enhanced in vivo release of dopa- mine in the striatum of SHR by voltammetry. In contrast, a slightly lower electrically stimulated dopamine release from stri- atal slices of SHR when compared to those of WKY was found by Linthorst et al. (15). Also, resting in vivo dopamine release was found to be lower in SHR than in WKY, and quinpirole caused a greater inhibition of dopamine release in SHR than in WKY (16). It could be argued that only stress-induced dopamine release is enhanced in SHR (13) and that the differences we ob- served between SHR and WKY especially in the locomotor test and perhaps also the saline-preference test, could at least in part be dependent on differences in this stress-induced dopamine release.

However, the amphetamine-induced turning might be ex- pected to involve a similar level of dopamine release in SHR and WKY. In previous experiments, we observed a similar re- sponse of SHR and WKY to the effect of amphetamine in an open-field test (36). Nevertheless, in the turning experiment, SHR responded less to sulpiride than WKY. Moreover, in the present experiments, the changes in DOPAC/dopamine and HVA/ dopamine turnover were generally similar in SHR and WKY (see Experiment 6). Thus an alternative mechanism than dopamine

Striatum Dopamine 6873 2 986 6035 2 529 DOPAC 2021 + 311 2040 f 110 HVA 838 5 73 836 2 71 Noradrenaline 87 2 18 108 f 21 Serotonin 889 2 172 698 + 55 5-HIAA 570 f 30 573 2 37

Hypothalamus Dopamine 315 f 33 323 + 50 DOPAC 160 f 27 172 -t 25 HVA 37 2 3 36 2 3 Noradrenaline 1568 f 188 1647 2 242 Serotonin 837 + 117 703 2 102 5-HIAA 838 -c 75 873 + 86

Medulla Oblongata Dopamine 48 -c 4 4Ok3 DOPAC 31 k 6 23 k 4 HVA 15 k 1* 23 r 2 Noradrenaline 478 k 28 393 r 35 Serotonin 895 2 81 788 k 55 5-HIAA 698 2 44 606 2 41

Pituitary Dopamine 0.22 -c 0.01* 0.41 f 0.02 DOPAC 0.029 2 0.005* 0.046 3t 0.003 Serotonin 0.47 -e 0.06 0.39 k 0.02 5-HIAA 0.10 f 0.02 0.08 + 0.01

*p<O.OS for difference between SHR and WKY. n= 8 for each group. Data are rig/g tissue wet weight for different brain regions and ng/mg for pituitary. The concentrations of HVA and noradrenaline were below the level of detection in the pituitary.

release might be altered in SHR. It is suggested that this alter- ation lies “behind” the postsynaptic receptor or involves changes in distinct dopamine D-2 receptor subtypes (as suggested also above). Thus it could involve changes in coupling or uncoupling of the dopamine D-2 receptor to some second messenger sys- tems (19,20) but not others, possibly through a genetic defect in G-protein-receptor coupling. For similar amounts of dopamine released an altered response of postsynaptic (and presynaptic) receptors is then observed. An interesting parallel to our find- ings has recently been described by Vaughn and co-workers (41). These authors found that subchronic treatment with the do- pamine agonist apomorphine sensitizes the rat to the effect of a challenge dose of dopamine without altering dopamine D-2 re- ceptor density or the change in dopamine turnover in response to a dopamine agonist. In our SHR model, this may reflect the higher stress-induced release of dopamine (subchronic agonist

296

Striatum Fr. Cortex

VAN

Hypothalamus

DEN BUUSE, JONES AND WAGNER

Medulla

Dose Sulpiride (mg/kg)

FIG. 6. The effect of sulpiride on the ratio of DOPAC/dopamine (top panels) and HVA/ dopamine (bottom panels) of different brain parts of SHR and WKY. For basal absolute concen~ations of dopamine, DOPAC and HVA, as well as of norad~n~ine, serotonin and S-HIAA, see Table 4. The number of animals was 8 per group. *p<O.O5 for difference with saline-treated controls of the same strain.

treatment), cawing the relative lack of an effect of a dopamine antagonist (functional postsynaptic upregulation of dopaminergic responses) without other changes in dopamine systems, as we have described in the present paper.

The other “postsynaptic” explanation for some of our results might imply that SHR display a defect in the levels of certain dopamine D-2-like receptor subtypes [(5,30), see also (29)J. Sulpiride has, in fact, been suggested to differentiate between different dopamine D-Z receptor subtypes in functional tests (24. 26, 27, 30). Such subtle changes could go unnotic~ in binding experiments.

In conclusion, by using a number of in vivo tests, we have

found differential changes in central dopamine D-2 receptor function in the SHR. Postsynaptic dopamine D-Z receptor func- tion is less responsive to an~gonist treatment in the in vivo tests. This is perhaps due to enhanced dopamine release under certain circumstances which has led to a decreased sensitivity to drugs. This decrease does not correlate with a change in receptor num- ber, but may be located “behind” the receptor. In contrast, pre- synaptic dopamine D-2 receptor tone is enhanced in SHR. The alterations in central dop~inergic transmission which result from these changes in receptor function could form part of the role which central dopamine, notably the nigrostriatal system (38,40), plays in the development of hypertension in the SHR.

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