Effects of NaCl and sultopride on striatal [3H]spiperone binding in neonatal, adult and senescent rats

Iw. J. L)evl. Neur~scie~~ce. Vol. 2. No. 5. pp. 421126, Pnntcd in Great Britain

073~5748184 $0.1.00+0.00 PergamonPressLtd. 0 1984 ISDN

1984.

EFFECTS OF NaCl AND SULTOPRIDE ON STRIATAL [“HISPIPERONE BINDING IN NEONATAL. ADULT AND SENESCENT RATS JUNKO MAKIHATA

Department

of Pharmacology,

and YASUYUKI NOMURA

Institute of Pharmaceutical Kasumi l-2-3. Minami-ku, (Ampted

Sciences. Hiroshima

29 Fehntary

Hiroshima University 734, Japan

School of Medicine.

1984)

Abstract-Effects of NaCl. (+)-and (-)-sultopride on striatal [3H]spiperone binding was investigated in 7-day. 70.day and 2-year-old rats. The amount of specific [‘Hlspiperone binding was the highest at 70 days and the value at adult stage was significantly (P
Aging.

NaCI, Sultopride,

[“H]Spiperone

binding,

Rat striatum.

It has been demonstrated that Naf is involved in the regulatory mechanism in radioligand binding to several neurotransmitter receptors, whose stimulation causes inhibitory influences on adenylate binding by NaCl appears to be due to procyclase activity. ‘.*Jx” The elevation of [“Hlspiperone tection of dopamine (DA) receptor complexes.‘7 In contrast, NaCl markedly enhances binding little has been known with affinity of substituted benzamide to DA;? receptors. y.‘3.‘5 However, regard to age-associated changes in effects of Na+ on stability of DA2 receptor complexes and on binding affinity of sultopride for DA2 receptors, although density of striatal [“Hlspiperone binding sites and DA-stimulated adenylate cyclase activity is changed during development and aging, in particular markedly low in the striatum of neonatal and senescent stages.4,7,” In the present study, effects of NaCl on the amount of [‘Hlspiperone binding and on inhibitory activities of (+)- and (-)-sultopride for [“Hlspiperone binding were investigated in striatal membranes of neonatal. adult and senescent rats. MATERIALS

AND METHODS

Wistar rats of both sexes were used. After birth all litters were culled to 10-12 pups/mother. Pups were maintained with their mother at 23°C in normal daylight conditions and were used at 7 days. Seventy-day and 2-year-old animals (approximately 23-24 months, retired breeders) were used as adult and senescent ones, respectively. After cervical decapitation of animals, the brains were rapidly removed and dissected on ice. The striatum was dissected out by the method of Nomura et al. “’ [“H]Spiperone binding assay was carried out by the method of Oki & Nomura. ” Briefly, isolated striatal tissues were homogenized on 50 mM Tris-HCl buffer (pH 7.7) with a Polytron@ (setting at 6) for 30 s and the homogenates were centrifuged twice at 50,000 g for 10 min with resuspension of the intermediary pellet in fresh buffer. The pellets were resuspended in 50 mM Tris-HCl buffer with 0.1% ascorbate and with or without 100 mM NaCl. A 0.7 ml portion of membrane suspension (approximately 0.4 mg protein) was transferred into 10 ml polyethylene tubes together with 0.1 ml of (+)- butaclamol solution (final concentration, 1 FM), (+)- or (-)-sultopride solution (final concentration, 10 nM-1 mM) or the buffer and 0.1 ml of [“Hlspiperone solution (final concentration, 0.5 nM or 0.02-l .O nM in case of Scatchard analysis). The reaction mixture was incubated for 15 min at 37°C. Five ml of ice-cold Tris-HCl buffer (pH 7.7) were then added, and the contents were filtered through a glass filter (Whatman GF/B) under reduced pressure. The filters were rinsed twice with an additional 5 ml of Tris-HCl buffer and then placed in liquid scintillator vials. Ten ml DN2:5-B 421

J. Makihata and Y. Nomura

322

of UnivergelG (Nakarai Chemicals Ltd.) were added and the radioactivity of bound [3H]spiperone determined with a Packard Tri-Carb liquid scintillation spectrometer (Model 3325). Specific (3H)spiperone binding was defined as that inhibited by 1 PM (+)-butaclamol. To estimate K,, and Bmax values, analysis of nonlinear upward curves of Scatchard plots was carried out on a Sharp MZ-2000 microcomputer system by the method described previously.” It was initially assumed that the curvature was due to the presence of only two components: one is the high-affinity component and the other is the low-affinity component. First, observed values were programmed into the computer and the points were plotted on the x- and !I-coordinate axes. Two independent. presumptive lines, actually two .I-- and F-intercepts, were then programmed into the computer and drawn in the same coordinate axes. On the basis of these two lines, the curvature. that is, the sum of the two components according to Rosenthal” were continuously plotted in the same figure, in which several observed values had been plotted previously. Observed values and a presumptive curvature were compared. Iterations of this procedure were performed at least 10 times and the best-fit lines were determined. and consequently the K,, and Bmax values were estimated. Estimation of protein was carried out by the Folin reagent method of Lowry et ul.’ ANOVA test was used for statistical difference of values at different ages. Student’s t-test was used for statistical analysis between two values. The drugs used were: [‘Hlspiperone (2.3 Ciimmol. Amersham). (+)-butaclamol hydrochloride (gift of Dr. J. Marshall, Aycrst Research Laboratories), (+)- and (-)-sultopride (Mitsui Pharmaceutical. Inc.). RESULTS effects

of NaCl

on striutal [3H]spiperone

binding in neonatal,

adult and senescent

ruts

Effects of Na ’ on striatal [“Hlspiperone binding was examined by incubating membranes with 0.5 nM [3H]spiperone and 100 mM NaCl for 15 min at 37°C. Specific [‘Hlspiperone binding wassignihcantly (P< 0.05) increased by the addition of NaCl into the binding medium at 70 days (Fig. 1). In both 7-day and 2-year-old animals. the amount of [‘Hlspiperone binding was significantly (P
Day

7

Year

?

0.1

0.:

0.2

Control

100 mM NaCl

Control

100 mM NaCl

Control

100 nM NaCl

Fig. I. Effects ot NaCl on the amount ofspecitic [‘H]spipcrone binding to striatal membranes in 7.day. 70. day and ?-year-old rats. Striatal membranes and 0.5 nM [3H]spiperone were incubated with or without 100 mM NaC‘I in 50 mM Tris-HCI buffer (pH 7.4) for I.5 min at 37°C. Each value represents the mean -c S.E. of three independent experiments. each of which was performed in triplicata Significance, ‘P
4ging of Na’

effects in striatal dopamine* receptors

423

[“Hlspiperone binding produced a nonlinear curve (Fig. 2). The data were compatible within the presence of two binding sites: the one is high affinity (Kd = 0.149 nM, Bmax = 0.180 pmol/mg protein) and the other is low affinity (& = 1.321 nM, Bmax = 0.475 pmol/mg protein) (Fig. 2A). NaCl(lO0 mM) caused a significant (P
(a) control

0.2 0 al L r

(b)

100

High

LOW

0.180+0.028

0.475+0.010

0.4

mM

0.8

0.6

NaCl

Ko(nM)

0.2 [3H]Spiperone

High

LOW

0.138+0.005

1.348+0.117

0.138+0.005

0.590+0.042*

0.4 bound

0.6

0.8

(pmol/mg)

Fig. 2, Effects of NaCl on high- and low-affinity [‘Hlspiperone binding in ?-day. 70-day and 2-year-old rats. Striatal membranes and 0.02-l .O nM [‘Hlspiperone were incubated with or without 100 mM NaCl for 15 min at 37°C. Nonlinear upward curves of Scatchard plots were separated into two distinct lines by the method of Rosenthal.‘” (A) Control (M); (B) 100 mM NaCl (M). The K,r and Bmax values were also shown. Significance, ‘kP
Effects ofNaC1 on inhibitory activity of (+)- and (-)-suftopride neonatal, adult and senescent rats

for striatal [“Hlspiperone binding in

Figure 3 shows effects of NaCl on concentration-inhibition curves of (+)- and (-)-sultopride on specific [“Hlspiperone binding to striatal membranes at 7 days, 70 days and 2 years. The uzsOvalues of (+)- and (-)-sultopride are shown in Table 1. In adult animals, (-)-sultopride is 34.4 fold more potent in inhibiting the binding compared to that of the (+)-enantiomer. Higher inhibitory effect of (-)-enantiomer than that of (+)-enantiomer was observed in neonatal and senescent animals as well. The q. values are not significantly different between animals at different stages. With regard to the effect of NaCl on the q. values of sultopride, a marked and significant (P< 0.001) reduction of the ICKY)of both (+)- and (-)-enantiomers was caused by 100 mM NaCl in adult rats. A reduction of the IC~()values of both enantiomers by 100 mM NaCl was also observed in neonatal and senescent rats. Hill coefficients of sultopride inhibition on [3H]spiperone binding were not affected by NaCl and not changed during development and aging. DISCUSSION In the present study, functional related changes were investigated.

involvement of Na+ in striatal DA receptor binding and its ageThe addition of 100 mM NaCl into the medium increased the

(+)-Sultopride f- )-Sultop’ride

1oOmM NaCl

22.3 rtO.8

8.1 CO.81 0.22 t 0.04/!

64.3 2.5

lc’so(PM)

on the inhibitory

0.13% 0.09t

26.2* -

Ratioof IC’~,, ..._____

Day 7

.-

activity of (+)-

1.26 0.78

1.10 0.75

nH ~.~. L 4.0 i0.f

9.9 I2.5ri 11.15 ‘-e0.031

62.0 1.8

~-”

Cl.lfJ? 0.(&t

34.4” _

Ratio of nscr

Day 70

Age

0.8’) 0.82

0.83 0.77

nH

for striatal ]“H]spiperone

~CVI( )LW

_____.-.

and (-)-sultopride

-

Year 2 -~-

and 2-year-old

10.3 -c 3.211 0.09 t O.Ml]/

Yl .o -+ 17.0$ 2.0 +- 0.3

O.ll? 0.0st

45.5” _

Ratio~ftc’.~,, IC.W(~M) __~_____^”

binding in 7-day, 7()&y

I .04 0.86

1.17 O.XY

nH

.-

rats

C-

I-sultooride

~cs(~ofsultopride in N&I

Ic~,,of

tcsrrof (+)-sultopride.

medium

IC~,~ofsultopride in control medium’ P
of ICgtr=

$ Significance, P Significance, ]] Significance,

i Ratio

* Ratlo of Icso =

The inhibitory activity of suhopride was expressed as the concentration of 50% inhibition (IC.W) which was obtained from the concentration-inhibitian curve of suhopride on [JH]spiperone binding (Fig. 3). Each ir‘s,, value shows the mean i S.E. of 3-l independent experiments, each of which was performed in triplicates. The Hill coefficient (nH) was determined from the Hill plot.

(~)-Sultopride (-)-Sultopride

Control

of N&t

Suhopride

I. Influence

Treatment

Table

Aging of Na’ effects in striatal dopaminez

-log [sultopride (MI]

425

receptors

-log [sultopride (M)]

-log [sultopride CM,]

Fig. 3. EffectsofNaCl on the inhibitory activity of(+)- and (-)-sultoprideforspecitic [-‘Hlspiperone binding in 7-day, 70-day and 2-year-old rats. NaCl (100 mM) and (+)- or (-)-sultopride (10 nM-1mM) were added to striatal membrane suspensions and the reaction mixtures were incubated with 0.5 nM [“Hlspiperone for IS min at 37°C. Each point represents the mean ? S.E. of three independent experiments, (A). (+)-sultopride in control: (A). (+)-sultopride in IO0 mM NaCl; (0). (0sultopride in control; (O), (-)-sultopridc in 100 mM NaCI.

amount of [“Hlspiperone binding on day 70. NaCl significantly increased the Bmax value of lowaffinity component of striatal [“Hlspiperone binding. An increase of the number of [“Hlspiperone binding sites has been suggested to be due to the protection of receptor complexes from degradation.” Since 100 mM NaCl significantly elevated the binding at all stages examined, Naf seems to protect DA receptor complexes from the degradation during incubation. The mechanism of protective effects of Nat is not known in detail but this is perhaps non-specific cation effects.” Lower amounts of [“Hlspiperone binding at 7 days than that at adult stage suggests an increase of DA receptors with age. In contrast, a significant reduction of the amount of [“Hlspiperone binding in 2year-old animals in comparison to that in 70-day-old ones is due to the reduction of the Bmax value of the binding with age but not due to the changes in the Kd value.“,‘.” (-)-Enantiomer of sultopride more potently inhibited [“Hlspiperone binding than (+)enantiomer at 7 days, 70 days and 2 years. Inhibitory activity of (-)-sultopride did not alter during aging and the ratio of ‘c50 of (+)- against the value of (-)-sultopride increased with age. It is posible. therefore, that DA receptors could recognize stereochemical structures of their ligands at 7 days and 2 years. It has been well established that substituted benzamides such as sulpiride and sultopride bind to DA2 receptors in a Na+-dependent manner.y.‘3,‘5,‘6 The results presented here indicate that 100 mM NaCl significantly enhanced inhibitory activity of (+)- and (-)-sultopride in adult rats. Since the increase of binding activity of sultopride to DA2 receptors by NaCl was observed in neonatal and senescent animals, the physiological mechanism of the involvement of Nat in benzamide binding already reach maturity at 7 days and still continue to work in striatal DA2 receptors at 2 years. The neurochemical mechanism by which Nat increases substituted benzamide binding to DA2 receptors has not been known in detail but the following are presumable: (1) holding the receptor in the preferred conformation for benzamide binding; (2) stabilizing benzamide binding to the receptors; (3) the involvement of the ionophore of Nat in benzamide binding sites. ” Findings that 50 p.M GTP does not alter ICKY)of sulpiride for [“HIspiperone bindingI suggest that no guanine nucleotide binding regulatory proteins are involved in Nat effects in benzamide affinity to DA, receptors. This well corresponds to the fact that GTP reduces agonist affinity but not antagonist one.3 In conclusion, Na+ can elevate the number of DA, receptors at 7 days, 70 days and 2 years and increasing effects of Na+ on benzamide binding affinity to DA? receptors continues through neonatal, adult and senescent stages in the rat striatum. authors are grateful for the kind advice of Prof. T. Segawa and for the gift of sultopride from Dr. A. Awaya, Mitsui Pharmaceuticals. Inc. This research was supported by Grant-in-Aid for Scientific Research (No. 58570925) from the Ministry of Education. Sciences and Culture. Japan.

Acknowledgemena-The

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426

J. Makihata

and Y. Nomura

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