Somatostatin receptor elevation in rat striatum after diisopropylfluorophosphate administration

0361-9X30/92 $5.00 + .OO Copyright c 1992 Pergamon Press Ltd.

Brarn Rtwun~h Bukrrn, Vol. 28, pp. 5 13-5 18. 1992 Printed in the USA. All nghts reserved.

Somatostatin Receptor Elevation in Rat Striatum After Diisopropylfluorophosphate Administration I. ALVARO

AND E. ARILLA’

~Molmrlur Neuroendocrinology

Unit, Department of’BiochsnistrJs and Molecular Biolog?,, Medical School, C~niversity qf’Alcala. Madrid, Spain Received 2 July 199 1

I. AND E. ARILLA. Somuloslatin rrwptor clwation in ral .sirrutwi after Llii.roJprop~,Ifllrorol,/io.\p/fut~,dmrn~ rtruliorr BRAIN RES BULL 28(4) 513-518, 1992.-The acute and chronic administration of diisopropylfluorophosphate (DFP). an inhibitor of acetylcholinesterase or of atropine, a blocker of muscarinic cholinergic receptors. did not affect somatostatin-like Immunoreactivity (SLI) content in the striatum of rats. Acute and chronic DFP administration increased the number of specific “51-Tyr”-somatostatin (“51-Tyr”-SS) receptors in cells dissociated from the striatum without changing the affinity constant. Although the increase could be blocked by pretreatment with atropine. it was not due to a direct effect by DFP on somatostatin of DFP ( IV5 .2f) when added in (SS) receptors. because no rise in “’ I- T y r”-SS binding was produced by high concentrations vitro. The acute administration of atropine alone had no observable effect on the number of SS receptors. However. repeated binding in cells dissociated from the striatum. although atropine administration produced a significant decrease in the ‘Z51-Tvr”-SS , the afinity constant was unchanged. The results suggest that interactions between somatostatinergic and cholinergic receptors may be of importance in the rat striatum.

ALVARO.

Diisopropylfluorophosphate

Atropine

Somatostatin

IT is now generally accepted that the somatostatinergic and cholinergic systems appear to be particularly important in the control of motor behavior (14,32). An anatomical basis for the interaction between these systems has been provided by the observation that fields of somatostatinergic-containing (9,19,4 I) and cholinergic (4.35) nerve terminals overlap in the striatum and that the striatal muscarinic receptors are associated with SScontaining neurons (2). Likewise, Delfs et al. (8) showed the coexistence of acetylcholinesterase and SS-immunoreactivity in neuron culture from rat cerebrum. Although the effects of acetylcholine and atropine, a muscarinic receptor blocker. on the release of SS and of SS on the release of acetylcholine have been the subject of a great deal of investigation (26,33), the effects of the muscarinic cholinergic system on SLI levels and SS receptors in the striatum are not well known. The present study was undertaken to determine whether the cholinergic system may exert some of its effect by changing SLI levels and/or SS binding to dissociated cells from the striatum. a nucleus primarily involved in the control of movement (28). Accordingly. we measured SLI levels and ‘251-Tyr”-SS binding in the rat striatum following acute or chronic administration of DFP, a substance that inhibits acetylcholinesterase and prolongs cholinergic receptor stimulation from the acetylcholine released at the synapses (40). Since atropine blocks muscarinic cholinergic receptors and thus the acetylcholine can only bind to the nicotinic cholinergic receptor, the present study also examines the effect

receptors

Striatum

Rat

of atropine pretreatment to evaluate somatostatinergic system. MATERIALS

AND

the etfects of DFP on the

METHODS

Synthetic Tyr”-SS and SS-I4 were purchased from Universal Biologicals, Ltd. (Cambridge, UK); bacitracin, diisopropylfluorophosphate (DFP), atropine. and bovine serum albumin (BSA) (fraction V) from Sigma (St. Louis, MO): and carrier-free Na “‘1 (IMS 30: 100 mCi/ml) from the Radiochemical Centre (Amersham. UK). Tyr”-SS was radioiodinated by the chloramine-T method (16). The tracer was purified on a Sephadex G25 coarse column (I X IOO), which had been equilibrated with 0. I dl acetic acid containing bovine serum albumin 0.1% (wt/ wt). Specific tracer radioactivity was -400 Ci/g. The rabbit antibody used in the radioimmunoassay technique was purchased from the Radiochemical Centre (Amersham, UK). This antiserum was raised in rabbits against SS-I4 conjugated to BSA and is specific for SS, but because SS-I4 constitutes the C-terminal portions of both SS-25 and SS-28. the antiserum does not distinguish between these three forms. Esperirner~tul

Animals

Wistar rats (200-225 g) were housed in groups in a light- and temperature-controlled room (lights on from 07:OO to 19:OO h;

’ Requests for reprints should be addressed to E. Arilla, Molecular Neuroendocrinology Medical School. llniversity of Alcala, 2887 1 Alcala de Henares, Madrid. Spain.

Unit. Department

of Biochemistry

and Molecular

Biology,

514

ALVAKO

1

TABLE

EFFECTS OF ACUTE AND CHRONIC DIISOPROPYLFLUOROPHOSPHATE AND ATROPINE ON SOMATOSTATIN-LIKE IMMUNOREACTIVITY CONCENTRATION IN THE STRIATUM OF THE RAT Somatostatin-Like Immunoreactivity (ng/mg of Protein) Striatum

Control

Chronic

Acute

Treatment (olive oil)

DFP Control (saline) Atropine

5.23 4.99 3.53 4.26

+ 0.67 + 0.8 I + 0.45 +- 0.60

3.51 4.55 4.34 4.23

+ 0.27 i 0.20 f 0.36 -+ 0.80

Determinations were made in duplicate for each experiment. Results are expressed as means it S.E.M. of five separate experiments in each group.

No statistically significant differenceswere obtained when compared with the control

animals

of each group.

23°C) and given unlimited access to food and water. DFP dissolved in olive oil or atropine dissolved in saline were administered subcutaneously (s.c.) in volumes of 0.1 ml per 100 g body weight. Both acute and chronic treatments with DFP or atropine were carried out. In the acute treatment, a single DFP dose of 1 mg/kg was injected as described (38). In chronic treatment, DFP was injected at a dose of 1 mg/kg on day 1, 0.4 mg/kg on day 2, and 0.2 mg/kg on days 4- 10 as described (37). This schedule of DFP administration inhibits brain acetylcholinesterase activity by 80-90%, and thereby increases the stimulation of cholinergic receptors by retarding the hydrolysis of acetylcholine. Atropine treatment was carried out according to the following schemes as described (43). In acute treatment, a single atropine dose of 20 mg/kg was injected. In chronic treatment, a dose of 20 mg/kg atropine was administered daily for 14 days. In a third experimental group, a single atropine dose of 20 mg/kg was administered 1 h before a single DFP dose of I mg/kg. Also, in a fourth experimental group, atropine (20 mg/kg) was administered daily 1 h before the DFP for 10 days. In this group, the DFP was administered as described in the chronic treatment. Control animals for each group cited were injected with olive oil, saline, or saline plus olive oil, according to whether the corresponding experimental group was to be injected with DFP, atropine, or atropine + DFP. Animals were sacrificed by decapitation 6 h after the last injection as described (38). The brains were removed and the striatum rapidly dissected according to method of Glowinski and Iversen (15). In all experiments, tissues from control and treated rats were assayed in parallel.

Tissue Extraction and Somatostatin Radioimmunoassay The dissected striatum was rapidly homogenized for SLI measurements in 1 ml 1 Macetic acid using a Brinkmann Polytron (setting 5, 30 s) (Brinkmann Instruments, Westbury, NY). Extracts were boiled for 5 min in a water bath and then chilled in ice, and aliquots ( 100 ~1) were removed for protein estimation (22). Subsequently, homogenates were centrifuged at 15,OOOg for 15 min at 4’C, and the supernatant was stored at -70°C until assay. Just before assay, extracts were neutralized with 1 M NaOH. SS concentration was determined in tissue extracts by an RIA method previously described in detail (27). The lowest limit of sensitivity was 10 pg/ml. The possibility that substances present in the tissue extracts might interfere with the antibodyantigen binding, thus leading to erroneous results, was discarded

AND

.AKILL.~2

by performing serial dilutions of selected extracts in the assays and comparing the resulting changes in hormonal immunoreactivity with those in the diluted standards. In addition, known amounts of the authentic hormone were added to varying amounts of the extracts and serial dilutions were again assayed to determine if the added hormone could be reliably measured in the presence of tissue extracts. Incubation tubes, prepared in duplicate, contained 100 ~1 samples of unknown or standard solutions (O-500 pg cyclic SS tetradecapeptide) diluted in phosphate buffer (0.05 ,%fipH 7.5: containing 0.1% BSA and 0.25 M EDTA). 100 ~1 appropriately diluted anti-SS serum, 100 ~1 freshly prepared “51-Tyr”-SS diluted in buffer to give 5000 cpm (equivalent to 5-10 pg). and enough buffer to give a final volume of 0.8 ml. All reagents and assay tubes were kept chilled in ice before incubation for 48 h at 4°C. Separation of bound and free hormones was accomplished by the addition of 500 ~1 dextran-coated charcoal (OX wt/vol; dextran T-70. Pharmacia. Uppsala, Sweden; Norit-A 2% wt/vol charcoal, Serva, Feinbiochemica, Heidelberg, Germany). Dilution curves for striatum were parallel to the standard curve. Rats from different groups were included in each assay series. The intra- and interassay coefficients of variation were 6.9% and 8.0%). respectively. Binding Assay fhr Cell Preparations Cells from the striatum of adult Wistar rats (200-250 g) were dissociated by mechanical means as described previously (34). Briefly, brains were rapidly removed and placed on ice. After the blood vessels and pia mater were carefully removed, the striatum were minced into small chuncks (I mm’), and transferred to a 50 ml tube for suspension in 0.9% ice-cold saline solution. The tissue was mashed with the help of a I O-ml pipette and mixed using a Vortex for I min at maximum speed. The tissue suspension was first filtered through a Nitex Nylon-filter of 355 pm pore size, then through another with a pore size of 80 pm. The resulting cell suspension was centrifuged at IOOOg for 10 min and suspended in phosphate-buffered saline. Cell viability was always greater than 90% as measured by exclusion of @pan-blue dye. Cell protein was estimated using bovine serum albumin as a standard (22). Experimental conditions for SS binding were essentially as previously described (5). Briefly, dissociated cells from rat striaturn (1 mg protein/ml) were incubated in 0.5 ml of 50 mM trisHCI buffer (pH 7.5) containing 5 mM MgC&, 30 mM NaCI, 1% BSA, 0. I% bacitracin, and 100 pM ‘251-Tyr”-SS in the absence or presence of 0.01-10 nM unlabeled SS. After 60-min incubation at 25°C cell-bound peptide was isolated by centrifugation at 13,OOOgfor I .5 min, and the radioactivity determined in a Kontron gamma counter. Nonspecific binding was obtained from the amount of radioactivity bound in the presence of lo-’ M SS, and represents about 20%) of the binding observed in the absence of unlabeled peptide. This nonspecific component was substracted from the total bound radioactivity in order to obtain the corresponding specific binding. In all experiments, tissues from control and treated rats were assayed in parallel.

Evaluation o/‘Radiolabeled Peptide To determine the extent of tracer degradation during incubation, we measured the ability of preincubated peptide to bind to fresh cells as previously described (36). Briefly, ‘ZSI-Tyr”-SS ( IO0 PM) was incubated with dissociated cells from rat striatum (1 mg protein/ml) for 60 min at 25°C. After this preincubation, aliquots of the medium were added to fresh cells and incubated for an additional 60 min at 25°C. The fraction of the added radiolabeled peptide, which was specifically bound during the

DIISOPROPY

LFLUOROPHOSPHATE

AND

SOMATOSTATIN

RECEPTORS

515

Striotum Acute

0.14

0 10

8

Cm45 Bound, nM

0.090

FIG. I. Etlect of acutediisopropylfluorophosphate and atropine + diisopropylfluorophosphate on somatostatin binding to striatum dissociated cells. Left panel: cells ( I mg protein/ml) were incubated for 60 min at 25°C in the presence of 100 p,lf ‘251-Tyr”-SS and Increasing concentrations of native peptide. Points correspond to values for the animals in the control group pool (0): diisopropylfluorophosphate-treated group (0): and atropine + diisopropylfluorophosphate-treated group (A). The results express the value of a pool of the control medium groups. because the B,,, and kd values of the control groups were not affected by the saline or olive oil media. Each point is the mean of five replicate experiments. For the sake of clarity S.E.M. values are not represented. but were always below 104 of the mean values. Right panel: Scatchard analqs of the same data.

second incubation. was measured and expressed as a percentage of the binding that had been obtained in control experiments performed in the absence of cells during the preincubation period.

The LIGAND computer program (25) was used to analyze the binding data. The use of this program enabled models of receptors which best fit given sets of binding data to be selected. The same program was also used to present data in the form of Scatchard plots and to compute values for receptor affinity (kd) and density (B,,,) that best fit the sets of binding data for each rat. Values for these model-dependent parameters for each experimental group were expressed as mean ? S.E.M. The paired t test was applied to means derived from the best fit 01 data points for each individual animal. RESU1.X

The acute and chronic administration of either DFP or atropine to rats did not affect SLI content in striatum in comparison with the control group (Table I). The specific binding of ‘251-Tyr”-SS to dissociated cells from

in all rat groups bound “‘I-Tyr”-SS in a time-dependent fashion: an apparent equilibrium was observed between 50-120 min at 25°C (data not shown). All subsequent binding experiments were. therefore, conducted at 25°C for 60 min. Peptide degradation was determined to rule out the possibility of different SS degrading activities in all the preparations that might have affected the interpretation of the results. Dissociated cells from striatum showed a similar peptide degradation capacity, and the values varied bq no more than 10%~in all the experimental groups. Increasing concentrations of unlabeled SS competitively inhibited the specific binding of ‘*“I-Tyr”-SS to dissociated rat striatum cells in the preparations from both the control and experimental groups (Figs. I and 2. left panel). The specific binding of the tracer to dissociated cells from the striatum in the acute and chronic DFP administration groups was significantly higher than in the control animals. Scatchard analysis of these results indicates that acute and chronic DFP administration increased the number of SS receptors in the striatum without affecting the affinity constant (Figs. 1 and 2, right panels; Table 2). To assess whether DFP exerts a direct action on SS receptors. [IO- ‘1 M DFP was included in the incubation medium at the time of the binding assay with dissociated cells from striatum rat striatum

Stria turn

r

0

C Ironic

J

I

* 11

1

I

10

9

1

0

[SSI;IogM

0.045 Bound,nM

OD90

FIG. 2. Effect of chronic diisopropylfluorophosphate and atropine + diisopropylff uorophosphate on somatostatin binding to striatum-dissociated cells. Left panel: cells (1 mg protein/ml) were incubated for 60 min at 25°C in the presence of 100 pM ‘251-Tyr”-SS and increasing concentrations of native peptide. Points correspond to values for the animals in the control group pool (e); dii~propyIfluoropho~hate-~~~ group (0); and a&opine + ~i~propyl~uoroph~phate-tots group (A). The results express the value of a pool of the control medium groups, since the B- and kd vahzes of the control groups were not affected by the saline or olive oil media. Each point is the mean of fzve replicate experiments. For the sake of clarity S.E.M. values are not represented, but were always below 10%of the mean values. Right panel: Scatchard analysis of the same data.

of normal rats, The addition of DFP to the incubation medium changed neither the number nor the affinity of the SS receptors (data not shown). Pretreatment with atropine completely blocked the changes

in the number of SS receptors induced by acute or chronic DFP administration (Figs. 1 and 2, right panel; Table 2). The acute administration of atropine alone had no observable effect on the number of SS receptors (Table 3). However, without changing

TABLE 2 EFFECT OF ACUTE AND CHRONIC DIISOPROPYLFLUOROPHOSPHATE EQUILIBRIUM PARAMETERS OF SOMATOSTATIN

AND ATROPINE PLUS DiiSOPROPYL.FL.UOROPHOSPHATE BINDING TO STRIATUM DISSOClATED CELLS

Acute

Treatment Control (olive oil) DFP Control (saline + olive oil) Atropine + DFP

Chronic B (fmol/mg~~proteinf

f&U% (fmol/mg of protein) 45 95 57 58

t 4 & 4* + 7 +: 8

ON

0.52 0.11 0.70 0.57

If:0.15 + 0.12 -r-0.20 f 0. I7

46 79 63 61

&8 z!I2t + 9 lt7

0.67 0.54 0.49 0.58

4 0.12 + 0.12 z!z0.20 + 0.14

The maximum number of binding sites (B,) and dissociation constant (kd) were determined by least-squares linear regression. The values are means + S.E.M. of five separate experiments m each group. * Significant difference from control at p < 0.00 I. t Significant difference from control at p c 0.05.

DIISOPROPYLFLUOROPHOSPHATE

AND

SOMATOSTATIN

TABLE 3 EFFECT OF AClJTE AND CHRONIC ATROPINE ON EQUILIBRIUM PARAMETERS OF SOMATOSTATIN BINDING TO STRIATUM DISSOCIATED CELLS stnatum Bmar

Treatmmt

kd

(nM)

(fmol/mg of protein)

Acute Control (saline) Atropinc Chronic Control (saline) Atropine

51 fl 62t

0.42 + 0.18 0.72 t 0.10

IO

56 ? 6 29 2 9

0.72 + 0.22 0.51 i-o.15

(*)

The maximum number of binding sites (B,,,) and dissociation constant (kd) were determined by least-squares linear regression. The values are means i- S.E.M. of five separate experiments. * Significant difference from control at p < 0.02.

the affinity a significant

the striatum

constant.

repeated

atropine

administration

decrease in ‘251-Tyr”-SS binding (Table 3).

produced

in the cells from

DISCUSSION

These results showed a specific alteration in SS receptor density in the striatum following acute and chronic administration of DFP as well as following chronic atropine administration. The SLI content in the striatum, as well as the binding parameters of SS receptors in the control rats, were similar to those previously reported (12,30,39). The Scatchard analysis of the stoichiometric data suggested the existence of only one type of SS receptor. This finding agrees with some studies in rat brain membranes (6,12,39) but differs from other previously reported data (31.42). It is conceivable that the use of small SS analogs (31) or their labeling with very different isotopes (42) might explain this difference. The acute and chronic administration of DFP or atropine did not affect the content of SLI in the striatum. This lack of change in SLI content after atropine treatment is consistent with the studies of Epelbaum et al. (I I), Mufson et al. (24) and Fine et al. (l3), who did not find a change in SLI levels following cortical cholinergic denervation obtained by a selective lesion of the nucleus basalis, or by the studies of Pierotti and Simpson (29) on hippocampal SLI levels after cholinergic denervation of hippocampus. These previous findings, together with the present results suggest that the SLI levels in the brain areas studied are not influenced by cholinergic input. The mechanism by which DFP increases SS binding is unknown. However, the increase in ‘251-Tyr”-SS binding was not

RECEPTORS

due to a direct effect of DFP on SS receptors, because no change was detected in tracer binding following incubation of fresh dissociated brain cells with 10m5M DFP, a concentration that completely abolished acetylcholinesterase activity (10). Thus, the rise in the local concentration of acetylcholine released at the synapses. caused by the destruction of acetylcholinesterase. may have resulted in an overstimulation of choline@ receptors. This would have made it possible for cholinergic receptors to mediate, at least in part, the action of DFP because the increase in the number of SS receptors induced by DFP is prevented by pretreatment with the muscarinic cholinergic blocker. atropinc. In contrast to the DFP treatment, chronic atropine administration decreased the number of SS receptors in rat striatum. This decrease of SS receptors as a consequence of chronic atropine treatment suggests that insufficient cholinergic input via muscarinic receptors may modulate SS neuronal activity. It is of interest to note that a similar downregulation of SS receptors is seen in the cerebral cortex in Alzheimer’s disease (AD) (3). At present. cerebral cortical cholinergic deficiency appears to he the most severe and consistent neurochemical dehcit in AD ( 17). In addition. cholinergic basal forebrain lesions decrease the number of SS receptors in the frontal and occipital cortex ( I I). Stavinoha et al. (40) vvho measured the accumulation of acetylcholine in rat brain regions after administration of dichlorvos demonstrated that striatum had the highest rate of brain acetylcholine acumulation. The finding that both treatments alter receptor density but not affinity is consistent with the reported effects of both DFP and atropine on other receptor systems (1.7,21.38). Whether the change in the number of striatal SS receptors demonstrated in the present experiments is reflected in an altered physiological function has not been determined. However. it is possible that changes in motor behavior in the treated rats, may at least partly depend on changes in the number of receptors, because SS influences locomotor activity (32). At present, there is no direct evidence that the regulation of SS receptors by the cholinergic system has physiological significance. However, this mechanism may provide a means by which the brain environment could modulate SS receptor number and, therefore. SS sensitivity in a subset of SS-sensitive neurons. The fact that transsynaptic regulation of the level of a variety of intracellular peptides and proteins has already been documented ( I8,20.23) lends support to this possibility.

ACKNOWLEDGEMENTS

This study was supported by grants from the Comision Interministerial de Ciencia y Tecnologia (PB 87-0753) and the Fondo de Investigaciones Sanitarias de la Seguridad Social, of Spain (88/0903). We thank Carol F. Warren from the Alcala de Henares University Institute of Education Sciences for her editorial help.

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.AL,VARO AND ARI1.I A

25 Munson, P. J.; Rodbard, D. LIGAND: a versatile computerized

26

27

28. 29

30.

31. 32.

33. 34. 35.

36.

37. 38.

39. 40. 41.

42.

43.

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