Metabolic Brain Disease, Vol. 7, No. 4, 1992

A Simple Biological Way to Screen Dopaminergic Agonists F.A.M. de Azeredo 1' 2 and M.F. Ribeiro 1

Received November 16, 1992; accepted December 26 1992

The velocity of propagation of the/n vitro retinal model of spreading depression is very sensitive to changes in the ionic composition of the exlracellular medium and also to the the addition of different drugs. 10 txM of SKF 38393, a D1 agonist, increases the velocity of propagation of the wave while 10 p.M of Quinpirole, a D2 agonist, decreases it. Both changes are blockedby their specific antagonists, SCH 23390 and 1-sulpiriderespectively. This assay can biologically screen potential dopaminergic drugs indicating its physiological D1 and/or D2 preferred effect in the tissue for future analysis by other different methodolgies. KEY WORDS: spreading depression; dopaminergic agonists; DA 1 and DA 2 receptors.

INTRODUCTION Two categories of dopamine receptors were hypothesized by Kebabian and Calne (1979) based on biochemical parameters such as ligand binding and adenylate cyclase activity, Receptors associated with stimulation of adenylate cyclase were termed D 1 receptors where as who did not present any effect on adenylate cyclase activity, or even inhibit it, were named D2 receptors (Stoof, 1983; Stoof and Kebabian, 1984). Selective agonists and antagonists were then identified in pharmacological experiments allowing the definition of these different receptors in several tissues (Tsurata et al., 1981; Trabucchi et al., 1976; Kebabian et al., 1984). Several studies indicate the presence of both D1 and D2 dopamine receptors in the mammalian retina (Dubocovich and Weiner, 1985; Brann and Young, 1986). In the rat retina Qu et al., (1989) showed that D1 receptors were linked to activation of adenylate cyclase and D2 receptors were negatively coupled to the enzyme. Histochemically these receptors were found in both plexiform layers; D1 receptors were also described at the inner nuclear layer (Elena et al., 1989) and D2 receptors were also found on rods in bovine retina (Brann and Young, 1986). 1 Laboratorio de Biociencias, Instituto de Biologia, Universidade Federal Fluminense, Caixa Postal 100229, Niteroi, RJ, Brazil. 2 To whom correspondence should be addressed. 211 0885-7490/92/1200-0183506.50/09 1992PlenumPublishingCorporation

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In the in vitro preparation of the chick retina the phenomenon of spreading depression can be visually accompanied after mechanical stimulation of the tissue. The event in the brain shows abrupt changes in extracellular ion concentration and pH (Chesler, 1990; de Azeredo, 1991) and it is suggested that these fluxes are followed by an indiscriminate release of transmitters thereby increasing the ionic permeability of post synaptic membranes. This in vitro model has been already used to show temporal release of ACh and GABA (Rodrigues et al., 1988). Also dopamine was showed to be released from sviatum during SD and anoxia (Moghaddam et al., 1987). Although this transmitter has not been shown to be released from the retina during SD the tissue has a storage of dopamine in amacrine and interplexiform cells (Dowling, 1987). If the retina has the transmitter, the receptors and DA is released during SD it is feasible to expect its participation in the phenomenon. One property of SD which is very sensitive to changes in the extracellular environment is the velocity of propagation. In the present manuscript we show that selective agonists of D1 an D2 receptors affect the velocity of propagation of SD and the effect is also blocked by their specific antagonists allowing its use as a simple and inexpensive biological method for screening of putative dopaminergic drugs. METHODS

One week old chicks are used throughout the experiments. Chicks are decapitated and the eyeballs are removed and sectioned along the equator. The posterior hemispheres are placed in a petri dish containing physiological solution (PS) with the following composition in mM: 100 NaC1, 4 KCI, 1 MgSO4, 1 NaH2PO4, 30 NaHC13, 20 glucose and 1 CaCI2. This solution has a osmolarity of 280 miliosmols/1 and a pH equal to 8.2. All the experiments are carded out between 25 ~ and 28~ centrigrades. The eye cup is moved to a plastic block containing a rounded cavity connected by holes to plastic tubing in order to superfuse the tissue. The preparation is then covered with transparent coverslides and superfusion is started with medium with the above composition for 20 min with a flow of 0.6 ml/min. The retina is then mechanically stimulated every 20 min to elicit a wave of spreading depression in the chick retina. The velocity of propagation is measured based on the time elapsed between two parallel lines located at the ocular of the stereomicroscope equivalent to 2 mm in the preparation and expressed in mm/min. Initially one set of eye cups (up to 10) is routinely superfused with normal physiological solution and stimulated every 20 min during 200 min to determine the mean velocity of propagation of the wave at each time and simulate the period of time that the preparation will be subjected to the contact of solutions with different compositions. The standard deviation is calculated and the mean value plus and minus two standard deviations are normalized to show a zone where any calculated velocity of propagation will be considered not significantly different from the control values. During the experiment the first two stimulus, if reproducible, are used to calculate the control value of the velocity of propagation for this specific preparation. Each experiment is performed several times with the same sequence of superfusion and points in the figures represent the mean value. The data are normalized by the following formula:

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Table I . Velocity of Propagation of SD in mm/min in a Control Retina (mmlmin)

20

40

60

80

100

120

140

160

180

200

Velocity of Propagation (mm/min)

3.;95

3.16

3.25

3.16

3.16

3.43

3.53

3.43

334

3.43

Y=X ~ x ; conlrol value

X = + 1 if ratio > 1; X = -1 if ratio < 1

All drugs used in the experiments were biochemical grade. SKF 38393, SCH 23390, 1-sulpiride and quinpirole were supplied by RBI. Concentrations of drugs in the superfusion fluid were never over 200 ~M so as not to modify ionic activity of calcium and magnesium which can by themselves bias the velocity of propagation.

RESULTS

Mean Velocity of Propagation of SD in PS and Quality Control Charts The quality control chart is detemained based on the results obtained of velocity of propagation for retinas superfused in PS. We will use one preparation as an example. First of all we determine the velocity of propagation of SD in mm/min in a control retina stimulated ten times at a 20 rain interval (Table I). Then, we can calculate the mean velocity of propagation for this specific experiment and also the correspondent standard deviation which are 3.31 and 0.14, respectively. As expected, 95% of the control values will be located in a range between X + 2x SD and X 2 x SD, what will be 3.59 and 3.03, respectively. The data are normalized to allow us to compare experiments performed at different days. The mean is designated as +1 at the plot and the maximum and minimum values of the chart are calculated as described in Methods. The maximum value will be + 1.09 and the minimum equal to -1.10. Observe that negative values for -2 SD at the normalized chart are different from +2 SD. At our example we obtained -1.10 and + 1.09, respectively. We consider that any calculated velocity which lies between the maximum and the minimum values of the normalized chart (dashed region) will not be significantly different from control values.

Effect of Dopamine on the Velocity of Propagation of SD Dopamine was superfused through the retina under the presence of 200 I.tM of ascorbate to find at which concentration there was any significant change in the velocity of propagation, if any. At Fig. 1, we show a sequence of superfusion where a significant change

Fig. 1. Effect of dopamine on the velocity of propagation of SD in the retina eye cup preparation. The following sequence of superfusion was used in theses experiments: PS;PS;PS § 10 [,tM dopamine; PS + 10 ~tM dopamine, PS;PS;PS + 100 ~tM dopamine, PS + 100 ~tM dopamine; PS; PS. Dopamine addition was done in the presence of 200 gM ascorbate which does not affect the velocity of propagation of the wave by itself. Dots(,) that rest inside the dashed region are considered to not be significantly different from controls. Those located outside the dashed region are considered significantly different from controls. Each point represents the mean of three experiments.

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in the velocity of propagation was only observed at concentrations of dopamine close to 100 gM. At this concentration dopamine induced a 20% increase in the velocity of propagation of SD. Stimulation of the D1 Receptor with SKF 38393: Antagonism of SCH 23390 At Fig. 2 we show the following sequence of supeffusion: PS; PS; SKF; SKF; PS; PS; SKF; SKF; PS; PS. I0 pM of SKF, a specific D1 agonist (lower concentration did not show any effect at all) induced a 20 to 34% increase in the velocity of propagation. The effect was cancelled during superfusion with PS but the addition of SKF to the incubation medium increased again the velocity of propagation which came back to normal values after returning to superfuse the preparation with PS. In another sequence of superfusion, we kept SKF in the incubation medium and add SCH 23390, a specific D1 antagonist, at a concentration of 10 lxM (Fig. 3). Under these circumstances, the velocity of propagation returned to control values during the period where SCH was present in the medium. If the retina were superfused only with 10 lxM SCH 23390, no change in the velocity of propagation was observed showing no collateral effect of this drug on the velocity of propagation at the concentrations used in the experiments. The increase in the velocity of propagation of SD induced by SKF 38393 was dose-dependent. A two-fold increase in its concentration in the superfusion fluid can increase the velocity of propagation up to 56%. On the other hand, the reduction of the concentration to 5 l.tM did not change the velocity significantly. Stimulation of D2 Receptors with Quinpirole on the Velocity of Propagation of SD: Anatagonism of 1-sulpiride. The superfusion of the eye cup preparation with quinpirole (10 ~tM), a specific D2 agonist, showed a reduction of the velocity of propagation of SD. The following sequence of superfusion was used: PS; PS; Quinpirole; Quinpirole; PS;PS; Quinpirole, Quinpirole; PS; PS. The reduction in the velocity of propagation can reach 34% (Fig. 4 and Fig. 5) and always returned to control values when the preparation was superfused with PS. A continued superfusion of the preparation with quinpirole (10 ~tM) would decrease the velocity of propagation as expected. The effect was only interrupted by 1-sulpiride, a specific D2 antagonist, when added to the superfusion fluid. DISCUSSION The characterization of specific dopamine receptor agonists and antagonists is mainly based on receptor binding assays. These studies have suggested at least two dopamine receptors: D1 and D2. This methodology, however, requires specific equipment, knowledge of binding techniques and use of radioligands. Also, it tells us the affinity of the drugs to the receptor but it won't inform us ifa biological tissue will react the same way. The action in vivo will depend on the density of the receptors in the tissue and on the intensity on the cell response. In fact, some behavior studies have pointed to a dependence between D1 and D2 receptors (Carlson et al., 1987). Several substances classified as dopamine

Fig. 2. Effect of SKF 38393 on the velocity of propagation of SD in the retina eye cup preparation. The following sequence of superfusion was used in these experiments: PS; PS; PS + 10 Ix M SKF; PS +10 ~tM SKF; PS; PS; PS + 10 IxM SKF; PS + 10 IxM SKF; PS; PS. Each point represents the mean of four experiments.

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Fig. 3. Inhibition of the SKF 38393 effect on the velocity of propagation of SD induced by SCH 23390. The following sequence of superfusion was used in these experiments; PS; PS; PS + 10 laM SKF; PS + 10 gM SKF; PS + 10 gM SKF + 10 ktM SCH; PS + 10 gM SKF + 10 gM SCH; PS + 10btM SKF; PS + 10 paVlSKF; PS; PS. Each point represents the mean of five experiments.

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Fig. 4. Effect of Quinpirole on the velocity of propagation of SD in the retina eye cup preparation. The following sequence was used in these experiments: PS; PS; PS + 10 IxM Quinpirole; PS + 10 gM Quinpirole; PS; PS; PS + 10 ~tM Quinpirole; PS + 10 gM Quinpirole; PS; PS. Each point represents the mean of six experiments.

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was used in these experiments: PS; PS; PS + 10 ~tM Quinpirole; PS + 10 ~tM Quinpirole, PS + 10 ~M Quinpirole + 10 ~tM 1-sulpiride, PS + 10 ~tM Quinpirole + 10 OM 1-sulpiride; PS + 10 ~tM Quinpirole; PS + 10 p.M Quinpirole, PS; PS. Each point represents the mean of six

Fig. 5. Inhibition of the Quinpirole effect on the velocity of propagation of SD induced by 1-sulpiride. The following sequence of superfusion

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agonists/antagonists are now known as D1/D2 or D2/D1 depending on its preference (Seeman and Gregoriadis, 1987). There are several substances newly synthesized which show specific affinity for one of the receptors and were used in our experiments to test our biological preparation. With this preparation, we can suggest D1 or D2 specificity of a substance. D1 putative agonists shall increase the velocity of propagation of SD and the effect shall be cancelled by SCH 23390. D2 putative agonists shall reduce the velocity of propagation and the use of 1-sulpiride shall revert the effect. If any drug has a non-specific affinity to any of the dopamine receptors it is expected that there would be no significant change at the velocity of propagation as observed by the concomitant use of D1 and D2 agonists at the superfusion fluid (data not shown). Any significant change should only be observed at high concentrations as the one observed with the use of dopamine itself. Dopamine acts preferentially on D1 receptors at high concenlrations in our preparation, probably because of the competition between both receptors. On the other hand the characterization of an expected D1 or D2 antagonist is revealed the same way based on their efficacy to block to effect of the specific D1 (SKF 38393) or D2 (quinpirole) agonists. REFERENCES

Braun, M.R. and Young, W.S. IN (1986). Dopamine receptors are located on rods in bovine retina. Neurosci. Lett. 69: 221-226. Carlson, J.H., Bergstrom, D.A. and Walter, J.R. (1987). Stimulation of both D1 and D2 dopamine receptors appears necessary for full expression of postsynaptic effect of dopamine agonists; a neurophysiological study. Brain Res. 400: 205-218. Chesler, M. (1990). The regulation and modulation of the pH in the nervous system. Progr. NeurobioL 34:401-427. de Azeredo, F.A.M. (1991). Transient changes in energy metabolites and intracellular pH during spreading depression in the chick retina. Met. Brain Dis. 6: 75-82. Dowling, J.E. (1987). The Retina: an Approachable Part of the Brain. Harvard University Press, Cambridge, Mass. Dubocovich, M.L. and Weiner, N. (1985). Pharmacological differences between the D-2 autoreceptors and the D1 dopamine receptor in the rabbit retina. J. Pharmacol. Exp. Therap. 233: 747-754. Elena, P.P., Denis, P., Kosina-Boix, M. and Lapalus, P. (1989). Dopamine receptors in rabbit and rat eye: characterization and localization of D1 and D2 binding sites. Current Eye Res. 8: 73-83. Kebabian, J.W., Beaulieu, M. and Itoh, Y. (1984). Pharmacological and biochemical characterization of two categories of dopamine receptors. Can. J. Neurol. Sci. 11:114-117. Kebabian, J.W. and Caine D.B. (1979). Multiple receptors for dopamine. Nature 277: 93-96. Moghaddam, B., Schenk, J.O., Stewart, W.B. and Hansen, A.L (1987). Temporal relationship between neurolxansmitter release and ion flux during spreading depression and anoxia. Can. J. Physiol. Pharmacol. 65: 1105-1110. 0 u, Z.X., Fertel, R., Neff, N.H. and Hadjiconstantinou, M. (1989). Pharmacological characterization of rat retinal dopamine receptors. J. Pharmacol. Exp. Therap. 248: 621-625. Rodrigues, P.S., Guimaraes, A.P.O., de Azedredo, F.A.M. and Martins Ferreira, H. (1988). Involvement of GABA and ACh in retinal spreading depression: effect of low calcium - high magnesium solutions. Exp. Brain Res. 73: 659-664. Seeman, P. and Gregoriadis, D. (1987). Dopamine receptors in brain and periphery. Neurochem. Int. 10: 1-25.

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Stool, J.C. (1983). Dopamine receptors in the neostriatum: biochemical and pharmacological studies. In Kaiser, C., Kebabian, J.W., (eds.), Dopamine Receptors, American Chemical Society Press, Washington, pp. 117-145. Stoof, J.C., Kebabian, J.W. (1984). Two dopamine receptors: biochemistry, physiology and pharmacology. Life Sci. 35: 2281-2296. Trabucchi, M., Spano, P.F., Tono, C.W., Frattola, L. (1976). Effect of bromocripfine on central dopaminergic receptors. Life Sci. 19: 229-232. Tsurata, K., Frey, E.A., Grene, C.W., Cote, T.E. Eskay, R.L and Kebabian, J.W. (1981). Evidence that LY-141865 specifically stimulates the D2 dopamine receptor. Nature 292: 463-465.

A simple biological way to screen dopaminergic agonists.

The velocity of propagation of the in vitro retinal model of spreading depression is very sensitive to changes in the ionic composition of the extrace...
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