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Reduction of Na+ enhances phosphoinositide hydrolysis and differentiates the stimulatory and inhibitory responses to quisqualate in rat brain slices
251
Brain Research, 536 (1990) 251-256 Elsevier BRES 16163
Reduction of Na ÷ enhances phosphoinositide hydrolysis and differentiates the stimulatory and inhibitory responses to quisqualate in rat brain slices Richard S. Jope, Xiaohua Li, George C. Ormandy, Ling Song and Mary B. Williams Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294 (U.S.A.) (Accepted 17 July 1990)
Key words: Inositol phosphate; Sodium-dependent; Quisqualate; Norepinephrine; Excitatory amino acid
The concentration of Na ÷ in the incubation medium significantly influenced phosphoinositide hydrolysis induced by some, but not all, agonists in rat cerebral cortical slices. Reductions of the Na ÷ concentration below 120 mM resulted in incremental increases in basal and norepinephrine-stimulated accumulation of [3H]inositol monophosphate in cortical slices that had been prelabelled with [3H]inositol, and maximal responses were obtained with 0 and 5 mM Na ÷. In contrast, the responses to carbachol and ibotenate were similar in medium containing 120 or 5 mM Na +. In medium with 120 mM Na t, quisqualate has two effects on phosphoinositide hydrolysis in cortical sfices, including a relatively weak stimulatory effect and an inhibitory modulation of the stimulation induced by norepinephrine. These two responses to quisqualate were differentially modulated by Nat; in 5 mM compared with 120 mM Na t the stimulatory response was greatly increased and the inhibitory effect was mostly eliminated. That these were two separate events was confirmed by the use of L-BOAA (fl-N-oxalyl-L-a,fl-diaminopropionic acid), which reproduces the inhibitory, but not the stimulatory effect of quisqualate on phosphoinositide hydrolysis. In 5 mM Na ÷, inhibition by L-BOAA of norepinephrine-stimulated phosphoinositide hydrolysis was completely eliminated. These results demonstrate that a physiological concentration of Na t maintains phosphoinositide hydrolysis at a submaximal level of sensitivity to some, but not all, agonists. The differential effects of Na + on the stimulatory and inhibitory effects of quisqualate further substantiate the suggestion that these are two separate processes and indicate that alterations of the Na t concentration may influence the effects of quisqualate, and other agonists, on phosphoinositide hydrolysis.
INTRODUCTION
13,17. For example, quisqualate stimulates phosphoinosi-
The hydrolysis of inositol-containing phospholipids is an important signal transduction mechanism in many tissues, including brain 6. Since excitatory amino acids, such as glutamate, apparently function as neurotransmitters at a large percentage of the synapses in the mammalian CNS 11, many investigators are studying the modulation of second messenger production associated with glutamate receptors. Several studies have demonstrated that phosphoinositide hydrolysis can be modulated by activation of specific excitatory amino acid receptor subtypes 1'13"17. Most of the widely used excitatory amino acid agonists have at some point been reported to stimulate phosphoinositide hydrolysis in brain tissue. The most consistently effective agonist is probably ibotenate, while quisqualate also often has been reported to activate this system TM. Several excitatory amino acid agonists also inhibit phosphoinositide hydrolysis induced by agonists of other neurotransmitters 1'
tide hydrolysis in rat cortical slices, but it also inhibits the stimulatory effect of norepinephrine s'13'lT'ls. Further studies indicated that the inhibitory, but not the stimulatory, response to quisqualate could be mimicked by L - B O A A (fl-N-oxalyl-L-Ct,fl-diaminopropionic acid) 14. However, the lack of effective and selective antagonists for these processes prevents an unequivocal identification of the receptors involved. As recently reviewed is, the effects on phosphoinositide hydrolysis of selective excitatory amino acid agonists and antagonists do not easily match the receptor subtype classifications that have been developed. Modifications of incubation buffers by reduction of the Na + concentration to low levels has been reported to enhance phosphoinositide hydrolysis in muscle 3'16. A brief report indicated that this condition increased basal and norepinephrine-stimulated phosphoinositide hydrolysis in rat cortical synaptoneurosomes 4. Therefore, in this study we measured the effect of altered Na +
Correspondence: R.S. Jope, Department of Psychiatry and Behavioral Neurobiology, Sparks Center 900, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
252 concentrations on phosphoinositide hydrolysis induced in response to a n u m b e r of agonists. Most notably, we report that reduced Na ÷ concentration enhances the
creases of approximately 7-fold occurring in media with 0 or 5 m M Na +. N o r e p i n e p h r i n e (100/~M) significantly stimulated the production of [3H]IP1 in 120 m M Na +, and
stimulatory, and blocks the inhibitory, response to
this response was increased incrementally as the Na ÷
quisqualate. Thus, this condition should provide a valu-
concentration was reduced. The inset in Fig. 1 shows the
able method to clarify the interactions of excitatory
difference in the [3H]IP x produced in media with reduced Na ÷ compared with that in 120 m M Na ÷, demonstrating
a m i n o acids with the phosphoinositide second messenger system. MATERIALS AND METHODS
Tissue preparation Male, Sprague-Dawley rats (200-250 g) were decapitated and the brains were rapidly dissected in ice-cold 0.32 M sucrose. Slices (0.3 mm x 0.3 mm) from the cerebral cortex, or hippocampus where indicated, were prepared with a Mcliwain tissue slicer and washed thoroughly with Krebs-bicarbonate-HEPES media (in mM: NaCI, 122; KCI, 5; MgCI2, 1.2; CaCI2, 1.3; KH2PO4, 1.2; NaHCO3, 3.6; glucose, 11; HEPES, 30; freshly bubbled with 95% 02/5% CO2; adjusted to pH 7.35). The slices were incubated in the same media at 37 °C for 60 min for regeneration, followed by several washes with fresh media.
that the enhanced response to n o r e p i n e p h r i n e is greater than, and i n d e p e n d e n t of, the increase in the basal rate of [3H]IP1 production and that these change s result in a reduced NE/basal ratio as the Na÷-concentration is reduced. Prazosin (10 /~M) completely blocked the response to norepinephrine in 120 and 5 m M Na ÷ (data not shown). Incubation with the sodium channel blocker tetrodotoxin (TTX) did not cause the same effects as did removal of Na ÷ from the m e d i u m indicating that Na ÷ influx
Assay of [3H]inositol phosphate production Preincubated slices were prelabelled by incubation at 37 °C for 1 h in fresh buffer containing0.5/tM myo-[2-3H]inositol(15 Ci/mmol) and 10 mM LiCI. After the incubation the slices were rapidly washed several times with media in which the NaCI was replaced with choline chloride to remove excess free [3H]inositol and extracellular Na ÷. Aliquots of [3H]inositol-prelabelled slices were incubated in a final volume of 500/~1 for 60 min or the indicated times in the presence of agonists or other agents. Where indicated, NaC1 was replaced with equal concentrations of choline chloride to generate the Na ÷ concentrations under study. For experiments with no sodium, NaHCO 3 was also replaced with KHCO3. The reaction was stopped by adding 1.7 ml of CHCI3/MeOH/12 N HC1 (1:2:0.01). Samples were transferred to extraction tubes and mixed with 1 ml of CHCI3 and 0.5 ml H20. The lipid phase was separated from the aqueous phase by centrifugation. Aqueous fractions were mixed with 0.5 ml of a 50% slurry of AG1-X8 resin, [3H]inositol phosphates were separated by the method of Berridge et al.2 as described previously9, and the radioactivity in each sample was measured. The lipid phase was dried overnight at room temperature and the radioactivity was measured. Results are expressed as the ratio of [3H]inositol phosphate/([3H]inositol + [3H]inositol phosphate + [3H]inositol phospholipid) to correct for variabilities in aliquots of brain slices.
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Materials Myo-[2-3H]inositol (15 Ci/mmol) was obtained from American Radiolabelled Chemical Inc. (St. Louis, MO). AG1-X8 anion exchange resin was from Bio-Rad (Richmond, CA). All other chemicals were of the highest grade available from commercial sources. RESULTS The accumulation of [3H]inositol monophosphate (IP1) was measured after a 60 min incubation of [3H]inositol-prelabelled cerebral cortical slices in media containing variable concentrations of Na ÷ (Fig. 1). In the absence of added agonist (basal), reduction of the Na ÷ concentration from 120 m M Na ÷ in controls resulted in e n h a n c e d accumulation of [3H]IP1, with maximal in-
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Fig. 1. Na÷-dependence of [3H]IPa production. Corticalslices were prelabelled with [3H]inositol for 60 rain and washed, as described in the Materials and Methods section. Slices were incubated for 60 rain in media with the indicated concentrations of Na + in the absence (Basal) or presence of 100/~M norepinephrine (NE). Values are means + S.E.M. from 4 experiments measured in triplicate. The inset shows the values for (O) basal and (0) norepincphrinestimulated [3H]IP1 production after subtraction of the values obtained with 120 mM Na +. These calculated values show that reduced Na + independently enhanced the response with each condition.
253 through voltage-dependent channels is not responsible for the depressed phosphoinositide hydrolysis observed in 120 mM Na ÷. Basal and norepinephrine-stimulated [3H]IP1 production were unaffected by inclusion of "I'TX in the medium, while the response to carbachol was significantly reduced by TTX concentrations of 0.3/~M and greater (Fig. 2). Fig. 3 shows the agonist-selectivity of the influence of Na ÷ on phosphoinositide hydrolysis. Phosphoinositide hydrolysis stimulated by norepinephrine was enhanced in low Na + and this increase was more than additive with the increased [3H]IP~ produced under basal conditions. In contrast, the responses to carbachol and ibotenate were only slightly enhanced in low Na ÷ and these increases appeared to be additive with the larger basal response, indicating that phosphoinositide hydrolysis induced by these two agents was not sensitive to the Na ÷ concentration. The most impressive Na+-sensitivity was observed with quisqualate which produced a large increase in [3H]IP1 accumulation in media with low Na ÷. We recently reported that glutamate, arachidonate, and quisqualate significantly inhibited norepinephrinestimulated phosphoinositide hydrolysis in rat cerebral cortical slicess'm. Fig. 3 shows that glutamate (0.5 mM) reduced [3H]IPI produced in response to norepinephrine by approximately 50% and that this degree of inhibition was not altered in media with low Na ÷. Arachidonate (0.2 mM) inhibited the response to norepinephrine by approximately 40% in 120 mM Na ÷ and this inhibition was only slightly reduced (to approximately 25%) in 5 mM Na ÷. In contrast, whereas quisqualate (0.5 mM) inhibited the response to norepinephrine by 60% in 120 mM Na ÷, there was no inhibition by quisqualate evident in 5 mM Na ÷. T r x did not affect the inhibitory effects of glutamate, arachidonate, or quisqualate. To examine if the apparent absence of an inhibitory
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Fig. 3. Effects of reduced Na + or tctrodotoxin on the modulation of phosphoinositide hydrolysis. Cortical slices were prelabelled with [3H]inositol for 60 rain and washed, as described in the Materials and Methods section. Slices were incubated for 60 rain in normal media containing 120 m M Na + (control; open bars), media with 5 x 10-7 M tetrodotoxin (T[~C; hatched bars), or media with 5 m M Na + (low Na+; solid bars). The production of [3H]IPt was measured after incubation with no added agonist (Basal), 100/~M norepi-
neplirine (HE), 500 #M quisqualate (QA), NE plus QA, NE plus 500/zM glutamate (Glu), NE plus 200/tM arachidonic acid (AA), 2 mM carbachol (CARB), or 0.1 or 1.0 mM ibotenate (IBO). Values are means + S.E.M. from 3 experiments (except n = 2 for IBO) measured in triplicate.
effect of quisqualate in low Na + was actually due to its being masked by enhanced quisqualate-stimulated phosphoinositide hydrolysis, we employed another excitatory amino acid agonist, L-BOAA. As reported previously 14, L-BOAA mimicks the inhibition caused by quisqualate but does not itself induce [3H]IP1 production in 120 mM Na ÷ (Fig. 4). In 5 mM Na ÷, inhibition by L-BOAA of the
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Fig. 4. Low Na ÷ blocked L-BOAA-induced inhibition of norepinephrine-stimulated [3H]IP1 production. Cortical slices that had been prelabelled with [3H]inositol and washed were incubated for 60 rain with no addition (BSL), 100/~M norepinephrine (NE; hatched bars), 0.3 or 1 mM L-BOAA, or L-BOAA and norepinephrine (hatched bars), in media containing 5 mM Na+ (Low Na +) or 120 mM Na + (Normal Na+). Values are means + S.E.M. of 3 experiments measured in triplicate.
254
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Fig. 5. Effect of Na ÷ on [3H]IP1 produced in response to quisqualate. Cortical slices that had been prelabelled with [3n]inositol and washed were incubated for 60 min with no agonist (BSL) or with concentrations of quisqualate (QA) varying from 10 -7 to 10 -3 M. Hatched bars indicate [SH]IP1 produced in response to quisqualate after subtraction of the corresponding basal value. Values are means + S.E.M. of 3 experiments measured in triplicate.
response to norepinephrine was totally blocked, while L - B O A A (1 mM) alone produced only a small rise in [3H]IP 1 which was much below that caused by quisqualate (Fig. 3). These results reinforce the conclusion that L - B O A A is more selective than is quisqualate in causing inhibition of norepinephrine-stimulated phosphoinositide hydrolysis without activating this process. Furthermore, the data indicate that the two responses to quisqualate are differentially affected by low Na t , supporting the
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Fig. 6. Effect of Na + on phosphoinositide hydrolysis in hippocampal slices. Hippocampal slices that had been prelabeUed with [3Hlinositol and washed were incubated for 60 min in the absence of added agonist (basal, B, open bars), with 100 ~M quisqualate (Q, hatched bars), or with 100/zM norepinephrinc (N, solid bars) in medium containing 120 mM Na + (Normal Na ÷) or 5 mM Na + (Low Na+). [3H]IPx (left ordinate), [3H]IP2, and [3H]IP3 (right ordinate) were measured as described in the Materials and Methods section. Values are means + S.E.M. of 3 experiments measured in triplicate.
hypothesis that they are mediated by different receptors or mechanisms. The enhancement by low Na + of quisqualate-stimulated [3H]IPx production was examined further by measuring the concentration-dependence of this response. In the presence of 5 mM Na + there was a remarkable enhancement of the potency of quisqualate so that 10 7 M quisqualate significantly elevated [3H]IP1 (Fig. 5). This is about a 100-fold lower concentration than is required to activate phosphoinositide hydrolysis in 120 mM Na +. Also, the maximal response to quisqualate was almost 6-fold higher in 5 mM Na + than in 120 mM Na +. These large effects of Na t on the quisqualate response contrast with the lack of Nat-sensitivity of ibotenate-stimulated phosphoinositide hydrolysis. Additionally, the inhibition caused by the higher concentrations of quisqualate in 120 mM Na ÷ are greatly attenuated in 5 mM Na ÷. Phosphoinositide hydrolysis in hippocampal slices was also influenced by the Na + concentration. Fig. 6 shows that compared with 120 mM Na ÷, in 5 mM Na ÷ [3H]IP1 production was increased under basal conditions and in response to norepinephrine and quisqualate. Also shown in Fig. 6 are the findings that in 5 mM compared with 120 mM Na + there were much greater accumulations of [3H]IP2 and slightly increased accumulations of [3H]IP3 in response to quisqualate or norepinephrine. DISCUSSION The results of this investigation have demonstrated that the concentration of Na t has a strong modulatory influence on phosphoinositide hydrolysis in rat brain slices and that this modulation is selective for only some of the agonists which activate this system. Most notable was the Nat-sensitivity of the responses to quisqualate. Quisqualate can directly stimulate phosphoinositide hydrolysis and it also inhibits norepinephrine-stimulated phosphoinositide hydrolysis s'13,17as. Reduction of the Na + concentration increased both the efficacy and the potency of the stimulatory response to quisqualate. Thus, low Na + converted what was a small quisqualate response requiring relatively high concentrations of quisqualate in normal Na ÷ to a strong response which was greater than that of ibotenate and was evident even at a very low quisqualate concentration. The suboptimal stimulation by quisqualate of phosphoinositide hydrolysis observed in high concentrations of Na ÷ suggests that Na ÷ limits this interaction in many situations and that under appropriate conditions quisqualate can activate phosphoinositide metabolism to a much greater extent than was recognized previously. In contrast to the enhancement by low Na t of the stimulatory response to quisqualate, low Na t eliminated quisqualate-induced inhibition of norepi-
255 nephrine-stimulated phosphoinositide hydrolysis. These two opposing effects of quisqualate precluded clear delineation of whether the inhibitory modulation was blocked by low Na + or if it was masked by the enhanced stimulation produced by quisqualate in low Na +. LB O A A mimicks the inhibitory, but not the stimulatory, response to quisqualate 14. The finding that low Na + also blocked the inhibition induced by L-BOAA indicates that the two responses to quisqualate are separate processes and they are differentially influenced by the concentration of Na +. Thus, low Na + enhanced the stimulation of phosphoinositide hydrolysis induced by quisqualate and blocked its inhibitory effect on the response to norepinephrine. These results indicate that if the Na + concentration is altered it will have an important influence on the interactions between quisqualate and phosphoinositide metabolism. The results with quisqualate also suggest that responses measured in 120 mM Na + are due to a summation of the stimulatory and inhibitory responses to quisqualate and that lowered Na + serves to separate these two processes so the stimulatory response is more evident at lower quiqualate concentrations while the inhibitory effect is only observed at 1 mM quisqualate. Others have also reported that omission of Na + from the incubation medium reduced inhibition by N-methylD-aspartate of carbachol-stimulated phosphoinositide hydrolysis in hippocampal slices 1'~2. Thus, physiological concentrations of Na + may be a common requirement for one mechanism by which excitatory amino acids inhibit agonist-stimulated phosphoinositide hydrolysis and the selective interactions among excitatory amino acid agonists and other neurotransmitter systems may be dependent upon the receptor distributions. Glutamate also inhibited norepinephrine-stimulated phosphoinositide hydrolysis but, unlike quisqualate, this effect of glutamate was not blocked by reduced Na +. This may be because glutamate induces inhibition by a different mechanism, such as by activation of phospholipase A 2 and subsequent inhibition of phosphoinositide hydrolysis by arachidonate 1°, or because the effect of Na + is only evident with the more structurally restricted 'excitatory amino acid analogs. The major observed effects of lowered Na + concentration on phosphoinositide metabolism, including increased basal hydrolysis, increased stimulatory responses to norepinephrine and quisqualate, and blocked inhibitory effects of quisqualate and L-BOAA may be due to multiple mechanisms. The binding characteristics of a number of neurotransmitter and hormone receptors are REFERENCES 1 Baudry, M., Evans, J. and Lynch, G., Excitatory amino acids
influenced by the Na + concentration. For example, Na + significantly reduced the quisqualate-sensitive [3H]glutamate binding in a soluble preparation from rat adrenal 19. Other investigators have suggested that enhanced phosphoinositide hydrolysis occurring in low Na + may be due to Na+/Ca 2+ exchange resulting in increased intracellular Ca 2+ (refs. 4, 16). This may be especially relevant to the increased basal rate of phosphoinositide hydrolysis in low Na + since increased intracellular Ca 2+ caused by depolarizing concentrations of K + also increases phosphoinositide hydrolysis. However, high K + has little effect on phosphoinositide hydrolysis induced by norepinephrine or quisqualate, suggesting that an additional mechanism is required to explain the enhanced responses with these agonists. Furthermore, the response to carbachol is greatly potentiated by high K + but is unaffected by low Na +, indicating that increased intracellular Ca 2+ may not be responsible for the enhanced responses to agonists in low Na +. Simply reducing the Ca 2+ in the media cannot be used to test the Ca2+-dependency of the effect of low Na + because the agonist responses are greatly impaired in the absence of Ca 2+ in brain slices. The present results may be due in part to modulation by Na + of G-proteins mediating phosphoinositide hydrolysis, as G-proteins associated with cyclic AMP production have previously been shown to be influenced by Na +. The inhibitory G-protein, Gi, associated with the cyclic AMP system has previously been reported to be Na+-dependent, so that physiological concentrations of Na + are required for optimal activity of neurotransmitters or hormones which inhibit adenylate cyclase7'15. Complex tissue- and brain region-dependent effects of Na + have been reported in adenylate cyclase studies and precise mechanisms of action of Na + are not clarified5. Nevertheless, it is interesting to speculate that an inhibitory G-protein may be associated with some phosphoinositide systems in the brain, such as the inhibition of norepinephrine-stimulated phosphoinositide hydrolysis by quisqualate, and that such an inhibitory G-protein may be inactive in low Na + in analogy with the cyclic AMP system. As suggested by Duman et al. 5 Na + flux associated with neuronal activity may modulate second messenger activity by actions on G-proteins. Acknowledgements. The authors thank Dot McAdory for preparing the manuscript. This study was supported by USPHS grant NS-26165 and by the U.S. Army Medical Research and Development Command under contract No. DAMD-17-89-C-9037. Opinions, interpretations and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.
inhibit stimulation of phosphatidylinositol metabolism by aminergic agonists in hippocampus, Nature, 319 (1986) 329-331. 2 Berridge, R., Downes, C.P. and Hanley, M.R., Lithium
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amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands, Biochem. J., 206 (1982) 587-595. Best, L. and Bolton, T.B., Depolarization of guinea-pig visceral smooth muscle causes hydrolysis of inositol phospholipids, Naunyn-Schmiedeberg's Arch. Pharmacol., 333 (1986) 78-82. Chandler, L.J. and Crews, ET., Calcium and sodium dependency of phosphoinositide hydrolysis in rat cerebral cortical synaptoneurosomes, Soc. Neurosci. Abstr., 14 (1988) 84. Duman, R.S., TerwiUiger,R.Z., Nestler, E.J. and Tallman, J.E, Sodium and potassium regulation of guanine nucleotide-stimulated adenylate cyclase in brain, Biochem. Pharmacol., 38 (1989) 1909-1914. Fisher, S.K. and Agranoff, B.W., Receptor activation and inositol lipid hydrolysis in neural tissues, J. Neurochem., 48 (1987) 999-1017. Jakobs, K.H. and Wieland, T., Evidence for receptor-regulated phosphotransfer reactions involved in activation of the adenylate cyclase inhibitory G protein in human platelet membranes, Eur. J. Biochem., 183 (1989) 115-121. Jope, R.S. and Li, X., Inhibition of inositol phospholipid synthesis and norepinephrine-stimulated hydrolysis in rat brain slices by excitatory amino acids, Biochem. Pharmacol., 38 (1989) 589-596. Jope, R.S., Casebolt, T.L. and Johnson, G.V.W., Modulation of carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral cortex, Neurochem. Res., 12 (1987) 693-700. Li, X., Song, L. and Jope, R.S., Modulation of phosphoinositide metabolism in rat brain slices by excitatory amino acids, arachidonic acid, and GABA, Neurochem. Res., 15 (1990) 725-738. Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and
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distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365-402. Morrisett, R.A., Chow, C.C. Sakaguchi, T., Shin, C. and McNamara, J.O., Inhibition of muscarinic-coupled phosphoinositide hydrolysis by N-methyl-D-aspartate is dependent on depolarization via channel activation, J. Neurochem., 54 (1990) 1517-1525. Nicoletti, F., Iadarola, M.J., Wroblewski, J.T. and Costa, E., Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: developmental changes and interaction with al-adrenoceptors, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 1931-1935. Ormandy, G.C. and Jope, R.S., Inhibition of phosphoinositide hydrolysis by the novel neurotoxin ~-N-oxalyl-L-a,fl-diaminopropionic acid, Brain Research, 510 (1990) 53-57. Rodbell, M., The role of hormone receptors and GTP-regulatory proteins in membrane transduction, Nature, 284 (1980) 17-21. Sasaguri, T. and Watson, S.P., Lowering of the extracellular Na + concentration enhances high-K+-induced formation of inositol phosphate in the guinea-pig ileum, Biochem. J., 252 (1988) 883-888. Schmidt, B.H., Weiss, S., Sebben, M., Kemp, D.E., Bockaert, J. and Sladeczek, E, Dual action of excitatory amino acids on the metabolism of inositol phosphates in striatal neurons, Mol. Pharmacol., 32 (1987) 364-368. Sladeczek, E, R6casens, M. and Bockaert, J., A new mechanism for glutamate receptor action: phosphoinositide hydrolysis, Trends Neurosci., 11 (1988) 545-549. Yoneda, Y. and Ogita, K., Characterization of quisqualatesensitive [3H]glutamate binding activity solubilized from rat adrenal, Neurochem. Int., 15 (1989) 137-143.
Brain Research, 536 (1990) 251-256 Elsevier BRES 16163
Reduction of Na ÷ enhances phosphoinositide hydrolysis and differentiates the stimulatory and inhibitory responses to quisqualate in rat brain slices Richard S. Jope, Xiaohua Li, George C. Ormandy, Ling Song and Mary B. Williams Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294 (U.S.A.) (Accepted 17 July 1990)
Key words: Inositol phosphate; Sodium-dependent; Quisqualate; Norepinephrine; Excitatory amino acid
The concentration of Na ÷ in the incubation medium significantly influenced phosphoinositide hydrolysis induced by some, but not all, agonists in rat cerebral cortical slices. Reductions of the Na ÷ concentration below 120 mM resulted in incremental increases in basal and norepinephrine-stimulated accumulation of [3H]inositol monophosphate in cortical slices that had been prelabelled with [3H]inositol, and maximal responses were obtained with 0 and 5 mM Na ÷. In contrast, the responses to carbachol and ibotenate were similar in medium containing 120 or 5 mM Na +. In medium with 120 mM Na t, quisqualate has two effects on phosphoinositide hydrolysis in cortical sfices, including a relatively weak stimulatory effect and an inhibitory modulation of the stimulation induced by norepinephrine. These two responses to quisqualate were differentially modulated by Nat; in 5 mM compared with 120 mM Na t the stimulatory response was greatly increased and the inhibitory effect was mostly eliminated. That these were two separate events was confirmed by the use of L-BOAA (fl-N-oxalyl-L-a,fl-diaminopropionic acid), which reproduces the inhibitory, but not the stimulatory effect of quisqualate on phosphoinositide hydrolysis. In 5 mM Na ÷, inhibition by L-BOAA of norepinephrine-stimulated phosphoinositide hydrolysis was completely eliminated. These results demonstrate that a physiological concentration of Na t maintains phosphoinositide hydrolysis at a submaximal level of sensitivity to some, but not all, agonists. The differential effects of Na + on the stimulatory and inhibitory effects of quisqualate further substantiate the suggestion that these are two separate processes and indicate that alterations of the Na t concentration may influence the effects of quisqualate, and other agonists, on phosphoinositide hydrolysis.
INTRODUCTION
13,17. For example, quisqualate stimulates phosphoinosi-
The hydrolysis of inositol-containing phospholipids is an important signal transduction mechanism in many tissues, including brain 6. Since excitatory amino acids, such as glutamate, apparently function as neurotransmitters at a large percentage of the synapses in the mammalian CNS 11, many investigators are studying the modulation of second messenger production associated with glutamate receptors. Several studies have demonstrated that phosphoinositide hydrolysis can be modulated by activation of specific excitatory amino acid receptor subtypes 1'13"17. Most of the widely used excitatory amino acid agonists have at some point been reported to stimulate phosphoinositide hydrolysis in brain tissue. The most consistently effective agonist is probably ibotenate, while quisqualate also often has been reported to activate this system TM. Several excitatory amino acid agonists also inhibit phosphoinositide hydrolysis induced by agonists of other neurotransmitters 1'
tide hydrolysis in rat cortical slices, but it also inhibits the stimulatory effect of norepinephrine s'13'lT'ls. Further studies indicated that the inhibitory, but not the stimulatory, response to quisqualate could be mimicked by L - B O A A (fl-N-oxalyl-L-Ct,fl-diaminopropionic acid) 14. However, the lack of effective and selective antagonists for these processes prevents an unequivocal identification of the receptors involved. As recently reviewed is, the effects on phosphoinositide hydrolysis of selective excitatory amino acid agonists and antagonists do not easily match the receptor subtype classifications that have been developed. Modifications of incubation buffers by reduction of the Na + concentration to low levels has been reported to enhance phosphoinositide hydrolysis in muscle 3'16. A brief report indicated that this condition increased basal and norepinephrine-stimulated phosphoinositide hydrolysis in rat cortical synaptoneurosomes 4. Therefore, in this study we measured the effect of altered Na +
Correspondence: R.S. Jope, Department of Psychiatry and Behavioral Neurobiology, Sparks Center 900, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
252 concentrations on phosphoinositide hydrolysis induced in response to a n u m b e r of agonists. Most notably, we report that reduced Na ÷ concentration enhances the
creases of approximately 7-fold occurring in media with 0 or 5 m M Na +. N o r e p i n e p h r i n e (100/~M) significantly stimulated the production of [3H]IP1 in 120 m M Na +, and
stimulatory, and blocks the inhibitory, response to
this response was increased incrementally as the Na ÷
quisqualate. Thus, this condition should provide a valu-
concentration was reduced. The inset in Fig. 1 shows the
able method to clarify the interactions of excitatory
difference in the [3H]IP x produced in media with reduced Na ÷ compared with that in 120 m M Na ÷, demonstrating
a m i n o acids with the phosphoinositide second messenger system. MATERIALS AND METHODS
Tissue preparation Male, Sprague-Dawley rats (200-250 g) were decapitated and the brains were rapidly dissected in ice-cold 0.32 M sucrose. Slices (0.3 mm x 0.3 mm) from the cerebral cortex, or hippocampus where indicated, were prepared with a Mcliwain tissue slicer and washed thoroughly with Krebs-bicarbonate-HEPES media (in mM: NaCI, 122; KCI, 5; MgCI2, 1.2; CaCI2, 1.3; KH2PO4, 1.2; NaHCO3, 3.6; glucose, 11; HEPES, 30; freshly bubbled with 95% 02/5% CO2; adjusted to pH 7.35). The slices were incubated in the same media at 37 °C for 60 min for regeneration, followed by several washes with fresh media.
that the enhanced response to n o r e p i n e p h r i n e is greater than, and i n d e p e n d e n t of, the increase in the basal rate of [3H]IP1 production and that these change s result in a reduced NE/basal ratio as the Na÷-concentration is reduced. Prazosin (10 /~M) completely blocked the response to norepinephrine in 120 and 5 m M Na ÷ (data not shown). Incubation with the sodium channel blocker tetrodotoxin (TTX) did not cause the same effects as did removal of Na ÷ from the m e d i u m indicating that Na ÷ influx
Assay of [3H]inositol phosphate production Preincubated slices were prelabelled by incubation at 37 °C for 1 h in fresh buffer containing0.5/tM myo-[2-3H]inositol(15 Ci/mmol) and 10 mM LiCI. After the incubation the slices were rapidly washed several times with media in which the NaCI was replaced with choline chloride to remove excess free [3H]inositol and extracellular Na ÷. Aliquots of [3H]inositol-prelabelled slices were incubated in a final volume of 500/~1 for 60 min or the indicated times in the presence of agonists or other agents. Where indicated, NaC1 was replaced with equal concentrations of choline chloride to generate the Na ÷ concentrations under study. For experiments with no sodium, NaHCO 3 was also replaced with KHCO3. The reaction was stopped by adding 1.7 ml of CHCI3/MeOH/12 N HC1 (1:2:0.01). Samples were transferred to extraction tubes and mixed with 1 ml of CHCI3 and 0.5 ml H20. The lipid phase was separated from the aqueous phase by centrifugation. Aqueous fractions were mixed with 0.5 ml of a 50% slurry of AG1-X8 resin, [3H]inositol phosphates were separated by the method of Berridge et al.2 as described previously9, and the radioactivity in each sample was measured. The lipid phase was dried overnight at room temperature and the radioactivity was measured. Results are expressed as the ratio of [3H]inositol phosphate/([3H]inositol + [3H]inositol phosphate + [3H]inositol phospholipid) to correct for variabilities in aliquots of brain slices.
12
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Materials Myo-[2-3H]inositol (15 Ci/mmol) was obtained from American Radiolabelled Chemical Inc. (St. Louis, MO). AG1-X8 anion exchange resin was from Bio-Rad (Richmond, CA). All other chemicals were of the highest grade available from commercial sources. RESULTS The accumulation of [3H]inositol monophosphate (IP1) was measured after a 60 min incubation of [3H]inositol-prelabelled cerebral cortical slices in media containing variable concentrations of Na ÷ (Fig. 1). In the absence of added agonist (basal), reduction of the Na ÷ concentration from 120 m M Na ÷ in controls resulted in e n h a n c e d accumulation of [3H]IP1, with maximal in-
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Fig. 1. Na÷-dependence of [3H]IPa production. Corticalslices were prelabelled with [3H]inositol for 60 rain and washed, as described in the Materials and Methods section. Slices were incubated for 60 rain in media with the indicated concentrations of Na + in the absence (Basal) or presence of 100/~M norepinephrine (NE). Values are means + S.E.M. from 4 experiments measured in triplicate. The inset shows the values for (O) basal and (0) norepincphrinestimulated [3H]IP1 production after subtraction of the values obtained with 120 mM Na +. These calculated values show that reduced Na + independently enhanced the response with each condition.
253 through voltage-dependent channels is not responsible for the depressed phosphoinositide hydrolysis observed in 120 mM Na ÷. Basal and norepinephrine-stimulated [3H]IP1 production were unaffected by inclusion of "I'TX in the medium, while the response to carbachol was significantly reduced by TTX concentrations of 0.3/~M and greater (Fig. 2). Fig. 3 shows the agonist-selectivity of the influence of Na ÷ on phosphoinositide hydrolysis. Phosphoinositide hydrolysis stimulated by norepinephrine was enhanced in low Na + and this increase was more than additive with the increased [3H]IP~ produced under basal conditions. In contrast, the responses to carbachol and ibotenate were only slightly enhanced in low Na ÷ and these increases appeared to be additive with the larger basal response, indicating that phosphoinositide hydrolysis induced by these two agents was not sensitive to the Na ÷ concentration. The most impressive Na+-sensitivity was observed with quisqualate which produced a large increase in [3H]IP1 accumulation in media with low Na ÷. We recently reported that glutamate, arachidonate, and quisqualate significantly inhibited norepinephrinestimulated phosphoinositide hydrolysis in rat cerebral cortical slicess'm. Fig. 3 shows that glutamate (0.5 mM) reduced [3H]IPI produced in response to norepinephrine by approximately 50% and that this degree of inhibition was not altered in media with low Na ÷. Arachidonate (0.2 mM) inhibited the response to norepinephrine by approximately 40% in 120 mM Na ÷ and this inhibition was only slightly reduced (to approximately 25%) in 5 mM Na ÷. In contrast, whereas quisqualate (0.5 mM) inhibited the response to norepinephrine by 60% in 120 mM Na ÷, there was no inhibition by quisqualate evident in 5 mM Na ÷. T r x did not affect the inhibitory effects of glutamate, arachidonate, or quisqualate. To examine if the apparent absence of an inhibitory
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Fig. 3. Effects of reduced Na + or tctrodotoxin on the modulation of phosphoinositide hydrolysis. Cortical slices were prelabelled with [3H]inositol for 60 rain and washed, as described in the Materials and Methods section. Slices were incubated for 60 rain in normal media containing 120 m M Na + (control; open bars), media with 5 x 10-7 M tetrodotoxin (T[~C; hatched bars), or media with 5 m M Na + (low Na+; solid bars). The production of [3H]IPt was measured after incubation with no added agonist (Basal), 100/~M norepi-
neplirine (HE), 500 #M quisqualate (QA), NE plus QA, NE plus 500/zM glutamate (Glu), NE plus 200/tM arachidonic acid (AA), 2 mM carbachol (CARB), or 0.1 or 1.0 mM ibotenate (IBO). Values are means + S.E.M. from 3 experiments (except n = 2 for IBO) measured in triplicate.
effect of quisqualate in low Na + was actually due to its being masked by enhanced quisqualate-stimulated phosphoinositide hydrolysis, we employed another excitatory amino acid agonist, L-BOAA. As reported previously 14, L-BOAA mimicks the inhibition caused by quisqualate but does not itself induce [3H]IP1 production in 120 mM Na ÷ (Fig. 4). In 5 mM Na ÷, inhibition by L-BOAA of the
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Fig. 2. Effects of tetrodotoxin on [3H]IP1production. Cortical slices that had been prelabelled with [3H]inositol and washed were incubated for 60 min with 100/~M norepinephrine (&) or 2 mM carbachol (0) in the presence of varying concentrations of tetrodotoxin. Values are means _+S.E.M. from 3 experiments measured in triplicate. *P < 0.05 compared with no tetrodotoxin.
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Fig. 4. Low Na ÷ blocked L-BOAA-induced inhibition of norepinephrine-stimulated [3H]IP1 production. Cortical slices that had been prelabelled with [3H]inositol and washed were incubated for 60 rain with no addition (BSL), 100/~M norepinephrine (NE; hatched bars), 0.3 or 1 mM L-BOAA, or L-BOAA and norepinephrine (hatched bars), in media containing 5 mM Na+ (Low Na +) or 120 mM Na + (Normal Na+). Values are means + S.E.M. of 3 experiments measured in triplicate.
254
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Fig. 5. Effect of Na ÷ on [3H]IP1 produced in response to quisqualate. Cortical slices that had been prelabelled with [3n]inositol and washed were incubated for 60 min with no agonist (BSL) or with concentrations of quisqualate (QA) varying from 10 -7 to 10 -3 M. Hatched bars indicate [SH]IP1 produced in response to quisqualate after subtraction of the corresponding basal value. Values are means + S.E.M. of 3 experiments measured in triplicate.
response to norepinephrine was totally blocked, while L - B O A A (1 mM) alone produced only a small rise in [3H]IP 1 which was much below that caused by quisqualate (Fig. 3). These results reinforce the conclusion that L - B O A A is more selective than is quisqualate in causing inhibition of norepinephrine-stimulated phosphoinositide hydrolysis without activating this process. Furthermore, the data indicate that the two responses to quisqualate are differentially affected by low Na t , supporting the
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Fig. 6. Effect of Na + on phosphoinositide hydrolysis in hippocampal slices. Hippocampal slices that had been prelabeUed with [3Hlinositol and washed were incubated for 60 min in the absence of added agonist (basal, B, open bars), with 100 ~M quisqualate (Q, hatched bars), or with 100/zM norepinephrinc (N, solid bars) in medium containing 120 mM Na + (Normal Na ÷) or 5 mM Na + (Low Na+). [3H]IPx (left ordinate), [3H]IP2, and [3H]IP3 (right ordinate) were measured as described in the Materials and Methods section. Values are means + S.E.M. of 3 experiments measured in triplicate.
hypothesis that they are mediated by different receptors or mechanisms. The enhancement by low Na + of quisqualate-stimulated [3H]IPx production was examined further by measuring the concentration-dependence of this response. In the presence of 5 mM Na + there was a remarkable enhancement of the potency of quisqualate so that 10 7 M quisqualate significantly elevated [3H]IP1 (Fig. 5). This is about a 100-fold lower concentration than is required to activate phosphoinositide hydrolysis in 120 mM Na +. Also, the maximal response to quisqualate was almost 6-fold higher in 5 mM Na + than in 120 mM Na +. These large effects of Na t on the quisqualate response contrast with the lack of Nat-sensitivity of ibotenate-stimulated phosphoinositide hydrolysis. Additionally, the inhibition caused by the higher concentrations of quisqualate in 120 mM Na ÷ are greatly attenuated in 5 mM Na ÷. Phosphoinositide hydrolysis in hippocampal slices was also influenced by the Na + concentration. Fig. 6 shows that compared with 120 mM Na ÷, in 5 mM Na ÷ [3H]IP1 production was increased under basal conditions and in response to norepinephrine and quisqualate. Also shown in Fig. 6 are the findings that in 5 mM compared with 120 mM Na + there were much greater accumulations of [3H]IP2 and slightly increased accumulations of [3H]IP3 in response to quisqualate or norepinephrine. DISCUSSION The results of this investigation have demonstrated that the concentration of Na t has a strong modulatory influence on phosphoinositide hydrolysis in rat brain slices and that this modulation is selective for only some of the agonists which activate this system. Most notable was the Nat-sensitivity of the responses to quisqualate. Quisqualate can directly stimulate phosphoinositide hydrolysis and it also inhibits norepinephrine-stimulated phosphoinositide hydrolysis s'13,17as. Reduction of the Na + concentration increased both the efficacy and the potency of the stimulatory response to quisqualate. Thus, low Na + converted what was a small quisqualate response requiring relatively high concentrations of quisqualate in normal Na ÷ to a strong response which was greater than that of ibotenate and was evident even at a very low quisqualate concentration. The suboptimal stimulation by quisqualate of phosphoinositide hydrolysis observed in high concentrations of Na ÷ suggests that Na ÷ limits this interaction in many situations and that under appropriate conditions quisqualate can activate phosphoinositide metabolism to a much greater extent than was recognized previously. In contrast to the enhancement by low Na t of the stimulatory response to quisqualate, low Na t eliminated quisqualate-induced inhibition of norepi-
255 nephrine-stimulated phosphoinositide hydrolysis. These two opposing effects of quisqualate precluded clear delineation of whether the inhibitory modulation was blocked by low Na + or if it was masked by the enhanced stimulation produced by quisqualate in low Na +. LB O A A mimicks the inhibitory, but not the stimulatory, response to quisqualate 14. The finding that low Na + also blocked the inhibition induced by L-BOAA indicates that the two responses to quisqualate are separate processes and they are differentially influenced by the concentration of Na +. Thus, low Na + enhanced the stimulation of phosphoinositide hydrolysis induced by quisqualate and blocked its inhibitory effect on the response to norepinephrine. These results indicate that if the Na + concentration is altered it will have an important influence on the interactions between quisqualate and phosphoinositide metabolism. The results with quisqualate also suggest that responses measured in 120 mM Na + are due to a summation of the stimulatory and inhibitory responses to quisqualate and that lowered Na + serves to separate these two processes so the stimulatory response is more evident at lower quiqualate concentrations while the inhibitory effect is only observed at 1 mM quisqualate. Others have also reported that omission of Na + from the incubation medium reduced inhibition by N-methylD-aspartate of carbachol-stimulated phosphoinositide hydrolysis in hippocampal slices 1'~2. Thus, physiological concentrations of Na + may be a common requirement for one mechanism by which excitatory amino acids inhibit agonist-stimulated phosphoinositide hydrolysis and the selective interactions among excitatory amino acid agonists and other neurotransmitter systems may be dependent upon the receptor distributions. Glutamate also inhibited norepinephrine-stimulated phosphoinositide hydrolysis but, unlike quisqualate, this effect of glutamate was not blocked by reduced Na +. This may be because glutamate induces inhibition by a different mechanism, such as by activation of phospholipase A 2 and subsequent inhibition of phosphoinositide hydrolysis by arachidonate 1°, or because the effect of Na + is only evident with the more structurally restricted 'excitatory amino acid analogs. The major observed effects of lowered Na + concentration on phosphoinositide metabolism, including increased basal hydrolysis, increased stimulatory responses to norepinephrine and quisqualate, and blocked inhibitory effects of quisqualate and L-BOAA may be due to multiple mechanisms. The binding characteristics of a number of neurotransmitter and hormone receptors are REFERENCES 1 Baudry, M., Evans, J. and Lynch, G., Excitatory amino acids
influenced by the Na + concentration. For example, Na + significantly reduced the quisqualate-sensitive [3H]glutamate binding in a soluble preparation from rat adrenal 19. Other investigators have suggested that enhanced phosphoinositide hydrolysis occurring in low Na + may be due to Na+/Ca 2+ exchange resulting in increased intracellular Ca 2+ (refs. 4, 16). This may be especially relevant to the increased basal rate of phosphoinositide hydrolysis in low Na + since increased intracellular Ca 2+ caused by depolarizing concentrations of K + also increases phosphoinositide hydrolysis. However, high K + has little effect on phosphoinositide hydrolysis induced by norepinephrine or quisqualate, suggesting that an additional mechanism is required to explain the enhanced responses with these agonists. Furthermore, the response to carbachol is greatly potentiated by high K + but is unaffected by low Na +, indicating that increased intracellular Ca 2+ may not be responsible for the enhanced responses to agonists in low Na +. Simply reducing the Ca 2+ in the media cannot be used to test the Ca2+-dependency of the effect of low Na + because the agonist responses are greatly impaired in the absence of Ca 2+ in brain slices. The present results may be due in part to modulation by Na + of G-proteins mediating phosphoinositide hydrolysis, as G-proteins associated with cyclic AMP production have previously been shown to be influenced by Na +. The inhibitory G-protein, Gi, associated with the cyclic AMP system has previously been reported to be Na+-dependent, so that physiological concentrations of Na + are required for optimal activity of neurotransmitters or hormones which inhibit adenylate cyclase7'15. Complex tissue- and brain region-dependent effects of Na + have been reported in adenylate cyclase studies and precise mechanisms of action of Na + are not clarified5. Nevertheless, it is interesting to speculate that an inhibitory G-protein may be associated with some phosphoinositide systems in the brain, such as the inhibition of norepinephrine-stimulated phosphoinositide hydrolysis by quisqualate, and that such an inhibitory G-protein may be inactive in low Na + in analogy with the cyclic AMP system. As suggested by Duman et al. 5 Na + flux associated with neuronal activity may modulate second messenger activity by actions on G-proteins. Acknowledgements. The authors thank Dot McAdory for preparing the manuscript. This study was supported by USPHS grant NS-26165 and by the U.S. Army Medical Research and Development Command under contract No. DAMD-17-89-C-9037. Opinions, interpretations and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.
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11
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