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Developmental aspects of muscarinic-induced inositol polyphosphate accumulation in rat cerebral cortex
European Journal of Pharmacology - Molecular Pharmacology Section, 172 (1989) 425-434
425
Elsevier EJPMOL 90041
Developmental aspects of muscarinic-induced inositol polyphosphate accumulation in rat cerebral cortex T h o m a s A. R o o n e y a n d Stefan R. N a h o r s k i Department of Pharmacology and Therapeutics, University of Leicester, Medical Sciences Building, University Road, Leicester LEI 9HN, U.K.
Received 12 April 1989, revisedMS received11 July 1989, accepted 28 July 1989
The ability of carbachol to stimulate phosphoinositide hydrolysis in developing brain was examined by assaying [3H]inositol phosphates in the presence and absence of lithium. Lithium (5 mM) enhanced carbachol-stimulated [3H]inositol monophosphate and [ 3H]inositol bisphosphate accumulations at every age tested but the enhancement of both [JH]inositol phosphates was greater at 7 days than at 40 days. A marked, time-dependent inhibition of [3H]inositol trisphosphate and [JH]inositol tetrakisphosphates accumulations, i.e. 29-33 and 76-79%, respectively, was produced by lithium at every age tested. Lithium also inhibited both [3H]inositol-l,3,4-trisphosphate and [3H]inositol1,4,5-trisphosphate by 29-38%. There were no developmental differences in the ECs0 values for lithium-induced potentiations of [JH]inositol mono- and bisphosphate accumulations (i.e. 0.4-0.6 and 4-6 mM, respectively). Similarly, negligible changes in the ECs0 values for carbachol-induced [ 3H]inositol mono- and bisphosphate accumulations were observed in the presence or absence of lithium at every age tested. Models of receptor coupling and the sensitivity of inositol polyphosphate dephosphorylation to lithium block during development are considered. Muscarinic receptors; Inositol polyphosphates; (Development)
1. Introduction It is now clear that a large variety of neurotransmitter receptors, including muscarinic receptors, are linked, via a guanine nucleotide-sensitive mechanism, to a phospholipase C-mediated hydrolysis of phosphoinositides in various regions of the central nervous system (Abdel-Latif, 1986; Downes, 1986; Nahorski et al., 1986; Rooney and Nahorski, 1986). Such a phosphodiesteratic cleavage produces the two putative second messengers inositol-l,4,5-trisphosphate (InsP3) and diacylglycerol (Berridge and Irvine, 1984). The former
Correspondence to: Dr. T.A. Rooney, Department of Pathology and Cell Biology,Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19102, U.S.A.
metabolite can mobilise intracellular bound calcium in many cells, including those of neuronal origin (Ueda et al., 1986), probably by a stereoselective interaction with a receptor recently reported in cerebellum (Willcocks et al., 1987). Recent evidence indicates that InsP3 can undergo either dephosphorylation to inositol-l,4-bisphosphate (InsP2) (Storey et al., 1984), or phosphorylation to inositol-l,3,4,5-tetrakisphosphate (InsP4) (Hawkins et ai., 1986; Irvine et al., 1986) and that this inositol phosphate may regulate Ca 2÷ homeostasis at the level of the plasma membrane (Irvine and Moor, 1986). Thus, the proportion of InsP 3 undergoing phosphorylation or dephosphorylation and its regulation would seem to be crucial to the functional significance of several cell surface receptors in a variety of different situations. The immature brain with its developing
0922-4106/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)
426
innervation and receptor density provides an opportunity to investigate the relationship between receptor activation and phosphoinositide responsiveness. In a previous report we have shown that by assaying total [3H]inositol phosphates in the presence of lithium, carbachol produces supramaximal phosphoinositide responses despite very low density of muscarinic receptors within the first week of development which decline to adult values at > 21 days when receptor density matures (Rooney and Nahorski, 1987). In the present study we have extended these striking observations by investigating inositol polyphosphate responses in the developing rat cortex.
2. Materials and methods
The methods used were essentially those previously described by Brown et al. (1984), with modifications (Batty et al., 1985; Batty and Nahorski, 1985). Male Sprague-Dawley rats of various ages (7, 14, 21 and 40 days) were killed by cervical dislocation, decapitated, the brains removed and the cerebral cortex dissected (Glowinski and Iversen, 1966) on ice. The cerebral cortex was crosschopped into 350 × 350 /~m slices and incubated for 60 rain in a modified Krebs-bicarbonate buffer, equilibrated previously with 95% 02-5% CO2, pH 7.4, at 37 °C in a shaking water bath. Samples (50 /~1) of packed slices were then incubated for 60 min with 1.0 /~M [3H]inositol either in the presence or absence of appropriate lithium concentration before labelled slices were exposed to agonist stimulation. Incubations were terminated by addition of 300 #1 of 7% (v/v) perchloric acid, tissue sedimented by centrifugation and the supernatants neutralised by addition of a freshly prepared 1 : 1 (v/v) mixture of Freon/tri-n-octylamine, as previously described (Downes et al., 1986). Inositol phosphates were separated on columns of Dowex-1 anion-exchange resin (200-400 mesh; formate form) or by gradient-elution high performance liquid chromatography (HPLC) as previously described (Batty and Nahorski, 1985).
2.1. Drugs [2-3H]Myo-inositol (15 Ci/mmol) was obtained from New England Nuclear (Boston, MA, U.S.A.). Tri-n-octylamine was obtained from Aldrich Chemical Co. Ltd. (Dorset, England). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). or Fisons p.l.c. (Loughborough, England).
2.2. Analysis of data The concentration of agonist producing halfmaximal stimulation (ECs0) was obtained by subjecting the concentration-effect curve to a four parameter logistic model using computer-assisted iterative curve-fitting (ALLFIT) (De Lean et al., 1978).
3. Results
3.1. Developmental profiles for carbachol-induced inositol polyphosphate accumulation The relative proportions of each individual inositol phosphate accumulating under stimulation by carbachol was investigated. The measurements of individual [3H]inositol phosphates were performed in the absence and presence of lithium over a 30 min stimulation period. It is clear from fig. 1 that lithium enhances carbachol-stimulated [ 3H]inositol monophosphate ([ 3H]InsP1) and [ 3H]inositol bisphosphate ([3HllnsP2) accumulations at every age tested. Moreover, lithium has a marked inhibitory effect on [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4) accumulations (i.e. by 36-46% and 6779% respectively) at every age. Lithium also produces a greater enhancement of [3H]InsP1 and [3H]InsP2 at the earlier ages (7 and 14 days) than in the more mature brain (21 and 40 days). Lithium enhances [3H]InsP1 accumulation by 9.1-, 6.6-, 4.5- and 4.3-fold at 7, 14, 21 and 40 days respectively. Lithium also enhances [3H]InsP2 accumulations by 2.6-, 2.5-, 2.3- and 2.1-fold at 7, 14, 21 and 40 days respectively. There are also significant accumulations of each of the [3H]inositol phos-
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Fig. 1. Measurements of individual [3H]inositol phosphates at various stages (age in days) of postnatal ontogeny in rat cerebral cortex. The effects of carbachol (1 mM) at 30 rain in the presence of 5 mM LiC1 are shown. The striped bars show the responses obtained in the presence of LiCI and the open bars show the responses in the absence of LiC1. Samples were incubated with [3H]inositol (5 t~Ci/tube) in the presence or absence of 5 mM lithium for 60 min and then stimulated with carbachol. The results are expressed as cpm/2 ml fraction of each batch sample. Each bar in the histogram represents the mean _+S.E.M, of three to six experiments, each performed in triplicate.
phates in the absence of lithium following carbachol stimulation. These measurements indicate that the stimulated levels of [3H]InsP3 and particularly [3H]InsP4 in the absence of lithium are significantly greater at the earlier ages (7 and 14 days) than those obtained at 21 and 40 days. There also seems to be a slightly greater accumulation of [3H]InsP2 in the absence of lithium at the earlier ages, but any differences in the levels of [3H]InsP1 are less obvious. Under these conditions, lithium enhanced basal [3H]InsPl, [3H]InsP2 and [3H]InsP3 accumulations at each age but had a negligible effect on basal [3H]InsP4 accumulation.
3.2. Dose-response curves for carbachol-stimulated inositol polyphosphate accumulations during developrnent In a previous report we have shown that doseresponse curves for carbachol obtained by measuring total [3H]inositol phosphates (>90% [3H] InsP1) show no variability in ECs0 during development (Rooney and Nahorski, 1987). These observations were further investigated in the present studies by performing dose-response curves to carbachol for each [3H]inositol phosphate. Thus, it was possible to observe whether agonist-stimu-
428
lated accumulations of total [3H]inositol phosphates faithfully reflect receptor activation and whether any variability in the coupling of the muscarinic-induced phosphoinositide responses could be detected by measuring inositol phosphate products which accumulate upstream from total [3H]inositol phosphate accumulations. To do this, dose-response curves for each [3H]inositol phosphate were obtained under carbachol stimulation both in the absence and presence of lithium. These dose-response curves were performed at two time points. Consequently, dose-response curves to 120"
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Fig. 2. Dose-response curves to carbachol for inositol polyphosphate accumulation in the immature (7 days) cerebral cortex. Responses obtained in the presence (shaded circles) and absence (shaded triangles) of 5 mM LiC1 are shown. Slices were incubated with [3H]inositol (5 vCi/tube) in the presence or absence of 5 mM lithium and then incubated for 5 min (A) or 30 rain (B) in the presence of carbachol. The 100% values in terms of cpm/2 ml fraction are shown as maximal values. Each dose-response curve represents the mean+S.E.M, of three experiments, each performed in triplicate. Agonist concentrations above 1 mM did not significantly further increase the production of inositol phosphates.
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- Log Carb Log Carb - Log Carb Fig. 3. Dose-response curves to carbachol for inositol polyphosphate accumulation in cerebral cortex (40 days). Responses were obtained in the presence (shaded circles) and absence (shaded triangles) of 5 mM LiCI. Incubations were performed at 5 min (A) and 30 rain (B) in the presence of carbachol. Slices were incubated with [3H]inositol (5/~Ci/tube) for 60 rain either in the presence or absence of 5 mM lithium before stimulation by agonist. The 100% values in terms of cpm/2 ml fraction are shown as maximal values. Each dose-response curve represents the mean 5-S.E.M. of three experiments, each performed in triplicate. Agonist concentrations above 1 mM did not significantly further increase the production of inositol phosphates. - Log Carb
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absence and presence of lithium (figs. 2B and 3B). Perhaps the most important results to note are that the ECs0 values for carbachol in p r o m o t i n g the accumulation of each of the [3H]inositol phosphates in the presence and absence of lithium in b o t h 7- and 40-day-old rats are almost identical (table 1). The enhancements p r o d u c e d by lithium on [3H]InsP1 and [3H]InsP2, as well as the inhibitory effects on [3H]InsP3 and [3H]InsP4, shown in the dose-response curves at b o t h ages, are also compatible with the effects observed in fig. 1 using
a single maximal dose of carbachol. The dose-response curves to carbachol performed in the absence and presence of lithium in b o t h 7- and 40-day-old rats do show some differences at a 5 min stimulation period. Firstly, although lithium still enhances [3H]InsP] and [3H]InsP2 accumulation more in the 7- than in the 40-day-old animal, it does so less m a r k e d l y than at the 30 min stimulation period (figs. 2A and 3A). Secondly, lithium does not seem to be able to inhibit [3H]InsP3 and [3H]InsP4 accumulations, in either the 7 day or
430 TABLE 1 Postnatal ontogeny of ECs0 values for carbachol-induced polyphosphate accumulation. The values represent the means + S.E.M. of two to three experiments. Cerebral cortical slices were labelled with [3H]inositol (5 # O / t u b e ) for 60 min either in the presence or absence of 5 m M LiC1. Samples were further incubated for 5 or 30 rain after addition of carbachol or Krebs medium. Samples were extracted and triplicates combined as described in Materials and methods. The ECs0 values for carbachol-induced polyphosphate accumulation were calculated as described in Materials and methods. Carbachol incubation
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the 40 day animals, at the 5 rain stimulation period. However, the ECs0 values for carbachol-induced accumulation of each of the [3H]inositol phosphates are very similar to those obtained at the 30 rain stimulation period (table 1).
3.3. Measurement of [3H]lnsP3 isomers during development In order to investigate the effects of lithium on [3H]InsP3 in more detail, measurements of [3H]Ins-l,3,4-P3 and [3H]Ins-l,4,5-P3 were made. Table 2 shows the effect of carbachol on these isomers in both 7- and 40-day-old rats. It is clear from this table that lithium inhibits the stimulated accumulation of both [3H]Ins-l,3,4-P3 and [3H]Ins-l,4,5-P3 by 29-38% at both ages with a 30 min stimulation period. No effect on the levels of the [3H]InsP3 isomers was observed at the 5 rain stimulation period. Carbachol produced a 5- and 3-fold stimulation of [3H]Ins-l,4,5-P3 accumulation at both time points in the absence of lithium in 7- and 40-day-old rats, respectively. In the presence of lithium, carbachol produced a 6- and 3-fold stimulation of [3H]Ins-l,4,5-P3 at both ages with a 5 min stimulation period. Carbachol also produced a 17- and ll-fold enhancement of [3H]Ins-l,3,4-P3 levels at both ages with a 5 min stimulation. It is difficult to determine the percentage stimulation produced by carbachol on [3H]Ins-l,3,4-P3 in the absence of lithium due to the undetectable control levels under such conditions. Lithium also pro-
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duced a slight enhancement of both [3H]Ins-l,3,4P3 and [3H]Ins-l,4,5-P3 basal levels under all conditions examined.
TABLE 2 Effects of 5 m M LiC1 on control and carbachol-stimulated (1 mM) accumulations of [3HllnsP3 isomers. Cerebral cortical slices were labelled with [3H]inositol ( 5 / ~ C i / t u b e ) for 60 min either in the presence or absence of 5 m M LiC1. Samples were further incubated for 5 or 30 min after addition of carbachol or Krebs medium. Samples were extracted and triplicates combined as described in Materials and methods and subsequently analysed by H P L C for [3HllnsP3. In order to perform this study in triplicate, tissue was obtained by pooling cerebral tissue from 7-8 animals of both ages. The data represent combined triplicates so as to facilitate the detection of both inositol phosphate isomers at both ages. Similar results have been obtained on at least one other occasion. Incubation
7-day-old rat Control (5 min) Carbachol (5 min) Control (30 rain) Carbachol (30 rain)
Inositol phosphates (cpm) Ins-l,3,4-P 3
Ins-l,4,5-P 3
+Li +
-Li +
+Li +
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34 573 72 357
547 562
573 3 380 694 2150
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47 497 50 348
476 507
507 1448 666 1024
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40-day-old rat Control (5 min) Carbachol (5 min) Control (30 min) Carbachol (30 min)
431
3.4. Effects of lithium on carbachol-stimulated inositol polyphosphate accumulation during development Having firmly established in the previous sections that the supramaximal responses produced by carbachol at early ages cannot be explained by a more efficiently coupled muscarinic receptor in these animals, the possibility of an altered sensitivity to lithium during development was examined by performing dose-response curves to lithium for each [3H]inositol phosphate. These dose-response curves were performed in both 7- and 40-day-old rats at a 30 rain Stimulation period. Lithium produced a dose-related potentiation of [3H]InsP1 in both 7- and 40-day-old rats (fig. 4). In the 7-dayold rat lithium produced a 10-11-fold enhancement with a maximal and half-maximal effect at 3-5 and 0.6 mM, respectively. In the 40-day-old rat, lithium produced a 5-fold enhancement with maximal and half-maximal effects at 3-5 and 0.4 mM, respectively. Similarly, lithium produced a significant, though less potent, enhancement of [3H]InsP2 by 5- and 2-fold at a concentration of 15-30 mM in both 7- and 40-day-old rats respectively. The half-maximal InsP2 response to lithium was produced at a concentration of 4-6 mM at both ages. In contrast to the potentiating effects on [3H]-
InsPa and [3H]InsP2, lithium produced a dose-related inhibition of both [3H]InsP3 and [3H]InsP4 in 7- and 40-day-old rats. The effect on [3H]InsP3 was evident between 0.1-0.3 mM and was halfmaximal by 0.4-0.5 mM at both ages. The inhibition was maximal by 3-5 mM and represented a 29-33% decrease of both ages. The effect on [3H]InsP4 was evident between 0.1-0.3 mM and was half-maximal by 0.3-0.4 mM at both ages. This represented a 76-79% decrease at both ages and was maximal by 3-5 mM.
4. Discussion
The results presented in this study indicate, that upon activation, the muscarinic receptor in young rats produces supramaximal polyphosphoinositide responses compared to adult. However, the muscarinic receptor does not appear to be more efficiently coupled to phosphoinositide hydrolysis as the ECs0 values for carbachol-induced inositol polyphosphate accumulation are similar to those obtained in adults. These results are consistent with previous measurements of total [3H]InsPs which also show supramaximal responses to carbachol with no shift in its dose-response curve in rat cerebral cortex of young rats (Balduini et al., 1987; Rooney and Nahorski, 1987).
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Fig. 4. Dose-related effects of lithium on carbachol-stimulated inositol polyphosphate accumulations in cerebral cortex from 7- and 40-day-old rats. Slices were incubated with [3H]inositol (5/~Ci/tube) in the presence of appropriate lithium concentration for 60 min. Samples were then incubated with carbachol (1 mM) for a further 30 rain. The results are expressed as cpm/2 ml fraction of each batch sample. Data obtained in the absence of lithium (not shown) was not significantly different to that observed at 10-4 M lithium. Results represent the means_+S.E.M. of three experiments, each performed in triplicate.
432 Furthermore, the relative intrinsic activity of the muscarinic partial agonist, arecoline, has been shown to remain constant throughout development (Rooney and Nahorski, 1987). It should be noted, however, that the present data do not agree with a recent report by Heacock et al. (1987) who suggest an enhanced coupling (exhibited as a lower ECs0 value for agonists) of the muscarinic receptor to cortical phosphoinositide metabolism in the young rat. The reasons for these differences are not known, though it is noteworthy that the low ECs0 value for carbachol reported in young rats by these authors is identical to that obtained at all ages in the present study. The present studies attempt to elucidate the basis of this markedly elevated muscarinic receptor-stimulated inositol phosphate production. The developmental changes in muscarinic receptor-induced inositol polyphosphate accumulation could relate to a greater ability of lithium to enhance [ 3H]InsPl and [ 3H]insP2 accumulation in young animals. However, this greater enhancing property of lithium in younger animals does not seem to be due to an increased sensitivity to lithium at these ages. Lithium has the same ECs0 value in 7- and 40-day-old rats for both [3H]InsPl and [3H]InsP2 accumulations (i.e. 0.4-0.6 and 4-6 raM, respectively). These results are consistent with previous reports in brain (Berridge et al., 1982; Brown et al., 1984; Batty and Nahorski, 1985) as well as in hepatocytes and pituitary GH 3 cells (Drummond et al., 1984; Thomas et al., 1984). It is therefore clear that the respective phosphatases regulating InsP 1 and InsP2 dephosphorylation, although sensitive to lithium during development, do not change with regard to their sensitivity for lithium block. The greater enhancement produced by lithium, predominantly on [3H]InsP1 accumulation in younger animals is also accompanied by a dose-dependent inhibition of [ 3H]insP 3 and [ 3H]insP4 accumulation at the more prolonged stimulation times. This inhibition of [3H]InsP3 and [3H]InsP4 accumulation is also evident in 40-day-old rats where the lithium enhancement of [3H]InsP1 accumulation is less than in 7-day-old rats. Lithium produces a 30-40% inhibition of [3H]InsP3 accumulation and a 67-79% inhibition of [3H]InsP4 accumulation in both 7- and
40-day-old rats. The differential enhancing properties of lithium on [3H]InsP1 could be explained if the various isomers of this inositol phosphate which could potentially be formed from both [3H]Ins-l,4,5-P3 and Ins-l,3,4-P 3 dephosphorylation were produced, in part, via lithium-insensitive dephosphorylation pathways (see Majerus et al., 1988). Thus, it is possible there is a change in the relative activity of these phosphatases as the animals mature. Such possibilities would result in the lower [3H]InsP1 accumulation in the presence of lithium observed with no shift in the dose-response curve to carbachol. As an examination of the isomeric forms of [3H]InsP1 produced at the various stages of development was not performed in the present studies it is impossible to tell which isomers are potentiated by lithium or indeed formed at the various stages of development. These questions are the focus of current investigations in this laboratory. Although lithium has been shown to potentiate carbachol-induced [3H]InsP1 and [3H]InsP2 accumulations, it seems to have an inhibitory effect on both stimulated [3H]InsP3 and [3H]InsP4 levels. A similar inhibitory effect on [3H]InsP3 accumulation was originally reported by Batty and Nahorski, 1985, although it was subsequently shown that a large part of the inhibition was due to a reduction in the levels of [3H]InsP4 which represented a contaminant of the original [3H]InsP3 fraction (Batty and Nahorski, 1987). It is also important to note that the effect of lithium on both [3H]InsP3 and [3H]InsP4 accumulations seems to be time-dependent. Thus, lithium inhibits both InsP3 and InsP4 at the 30 min but not the 5 min stimulation period. The effect of lithium is also dependent on the length of preincubation period with lithium. For example, we previously found no effect on [3 H]insP 3 accumulation if agonist and lithium were added together (Batty and Nahorski, 1987) rather than having lithium present during the labelling period with [3H]inositol before agonist stimulation. As yet, the reasons for lithium's inhibitory effects on both [3H]InsP3 and [3H]InsP4 accumulations are unknown although some possibilities have been discussed (Batty and Nahorski, 1987). For example, the effects could be explained by a depletion of a receptor-associated phospholipid
433
pool, although this seems unlikely as labelled inositol is present in the incubation buffer throughout the period of agonist and lithium treatments. Moreover, lithium produces its inhibitory effect on [3H]InsP3 accumulations at a concentration where its potentiation of [3H]InsP1 accumulations is still linear. If it is assumed that all of the InsP~ measured is derived from InsP 3, as is suggested by the similar ECs0 values for both inositol phosphates upon agonist activation, then it is unlikely that the inhibitory effects of lithium on InsP3 and InsP4 can be explained by a decrease in the pool of receptor-associated PtdlnsP 2. Another possibility, consistent with the observed lag period associated with the onset of lithium's inhibitory action on both InsP 3 and InsP4, is that the effects could be mediated via protein kinase C activation by an elevation of diacylglycerol following observed lithium treatment (Drummond and Raeburn, 1984; Downes and Stone, 1986). Such an activation could have negative feedback effects on the various phosphoinositide kinases and phosphatases or directly at the level of the receptor. For example, phorbol esters have been shown to inhibit muscarinic receptorinduced inositol polyphosphate accumulation (Orellana et al., 1985) and to increase the hydrolysis of Ins-l,4,5-P 3 by an activation of a 5-phosphatase (Molina y Vedia and Lapetina, 1986). Since it is likely that the same 5-phosphatase is involved in dephosphorylating both Ins-1,4,5-~ and Ins-l,3,4,5-P4 then this may, in part, explain the inhibitory effect produced by lithium on both of these inositol phosphates. Such an indirect effect of lithium is further supported by the fact that the Ins-l,4,5-P 3 5-phosphatase has been shown to be insensitive to any direct effects of lithium (Downes et al., 1982; Seyfred et al., 1984; Connolly et al., 1985). There are some anomalies in the effects of lithium on both InsP 3 and InsP4. Lithium inhibits InsP 4 accumulation by 67-79% but only inhibits InsP 3 levels by 36-46%. A more detailed analysis of InsP3 accumulations shows that Ins-l,3,4-P 3 and Ins-l,4,5-P3 are inhibited to the same degree by lithium. Thus, a 67-79% inhibition of InsP4 only results in a 30-38% inhibition of Ins-l,3,4-P 3. As Ins-l,3,4-P 3 has been shown to be formed
solely from a 5-phosphatase attack on Ins-l,3,4,5P4 (Batty et al., 1985; Hansen et al., 1986; Hawkins et al., 1986) the disproportionate decreases in InsP4 and Ins-l,3,4-p~ produced by lithium could be explained if lithium also enhanced Ins-l,3,4-P 3 accumulation. This possibility has been reported by Burgess et al. (1985), and indeed lithium has been shown to elevate total InsP 3 levels in hepatocytes and G H 3 cells (Drummond et al., 1984; Thomas et al., 1984). In conclusion, it is clear from these results that the supramaximal responses produced in young rats cannot be explained by an enhanced coupling of the muscarinic receptor or by a differential sensitivity to lithium. However, the extent to which the various pathways of inositol polyphosphate dephosphorylation are used a n d / o r blocked by lithium may vary during development. Moreover, the time-dependent inhibitory effects of lithium on both InsP 3 and lnsP 4 require further investigation in order to obtain a greater understanding of how lithium perturbs the phosphoinositide signalling system in brain. Future studies should be focussed on a more detailed analysis of the effects of lithium on the separated isomeric forms of inositol phosphates produced during development.
Acknowledgements The authors would like to thank Dr. I.H. Batty for running of HPLC samples and Jenny Bell for help in the preparation of the manuscript.
References Abdel-Latif, A.A., 1986, Calcium mobilising receptors, polyphosphoinositides and the generation of second messengers, Pharmacol. Rev. 38, 227. Balduini, W., S.D. Murphy and L.G. Costa, 1987, Developmental changes in muscarinic receptor-stimulated phosphoinositide metabolism in rat brain, J. Pharmacol. Exp. Ther. 241,421. Batty, I.H. and S.R. Nahorski, 1985, Differential effects of lithium on muscarinic receptor stimulation of inositol phosphates in rat cerebral cortex slices, J. Neurochem. 45, 1514. Batty, I.H. and S.R. Nahorski, 1987, Lithium inhibits muscarinic receptor-stimulated inositol tetrakisphosphate accumulation in rat cerebral cortex, Biochem. J. 247, 797.
434 Batty, I.H., S.R. Nahorski and R.F. Irvine, 1985, Rapid formation of inositol 1,3,4,5-tetrakisphosphate following muscarinic receptor stimulation of rat cerebral cortical slices, Biochem. J. 232, 211. Berridge, M.J., C.P. Downes and M.R. Hanley, 1982, Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands, Biochem. J. 206, 587. Berridge, M.J. and R.F. Irvine, 1984, Inositol trisphosphate, a novel second messenger in signal transduction, Nature 302, 315. Brown, E., D.A. Kendall and S.R. Nahorski, 1984, Inositol phospholipid hydrolysis in rat cerebral cortical slices. I. Receptor characterisation, J. Neurochem. 42, 1379. Burgess, G.M., J.S. McKinney, R.F. Irvine and J.W. Putney, 1985, Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca 2+-mobilising hormone-activated cells, Biochem. J. 232, 211. Connolly, T.M., T.E. Bross and P.W. Majerus, 1985, Isolation of a phosphomonoesterase from human platelets that specifically hydrolyses the 5-phosphate of inositol 1,4,5-trisphosphate, J. Biol. Chem. 260, 7868. De Lean, A., P.J. Munson and D. Rodbard, 1978, Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay and physiological dose-response curves, Am. J. Physiol. 235, E97. Downes, C.P., 1986, Agonist-stimulated phosphatidylinositol 4,5-bisphosphate metabolism in the nervous system, Neurochem. Int. 11,394. Downes, C.P., P.T. Hawkins and R.F. Irvine, 1986, Inositol 1,3,4,5-tetrakisphosphate and not phosphatidylinositol 3,4bisphosphate is the probable precursor of inositol 1,3,4-trisphosphate in agonist stimulated parotid gland, Biochem. J. 238, 501. Downes, C.P., M.C. Mussat and R.H. Michell, 1982, The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane, Biochem. J. 203, 169. Downes, C.P. and M.A. Stone, 1986, Lithium-induced reduction in intracellular inositol supply in cholinergically stimulated parotid gland, Biochem. J. 234, 199. Drummond, A.H., M. Bushfield and C.H. Macphee, 1984, Thyrotropin-releasing hormone-stimulated [3H]inositol metabolism in GH 3 pituitary tumor cells. Studies with lithium, Mol. Pharmacol. 25, 210. Drummond, A.H. and C.A. Raeburn, 1984, The interaction of lithium with thyrotropin-releasing hormone-stimulated lipid metabolism in GH 3 cells, Biochem. J. 224, 129. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain. I. The disposition of 3Hnorepinephrine, 3H-dopamine and 3H-dopa in various regions of the brain, J. Neurochem. 13, 655. Hansen, C.A., S. Mah and J.R. Williamson, 1986, Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver, J. Biol. Chem. 261, 8100. Hawkins, P.T., L. Stephens and C.P. Downes, 1986, Rapid
formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in rat parotid glands may both result indirectly from phosphatidylinositol 4,5-bisphosphate, Biochem. J. 238, 507. Heacock, A.M., S.K. Fisher and B.W. Agranoff, 1987, Enhanced coupling of neonatal muscarinic receptors in rat brain to phosphoinositide turnover, J. Neurochem. 48, 1904. Irvine, R.F., A.J. Letcher, J.P. Heslop and M.J. Berridge, 1986, The inositol tris/tetrakisphosphate pathway-demonstration of Ins(I,4,5) 3-kinase activity in animal tissues, Nature 320, 631. Irvine, R.F. and R.M. Moor, 1986, Micro-injection of inositol 1,3,4,5-tetrakisphosphate activates sea urchin eggs by a mechanism dependent on external Ca 2+, Biochem. J. 240, 917. Majerus, P.W., J.M. Connolly, V.S. Bansal, R.C. Inhorn, T.S. Ross and D.L. Lips, 1988, Inositol phosphate synthesis and degradation, J. Biol. Chem. 263, 3051. Molina y Vedia, L.M. and E.G. Lapetina, 1986, Phorbol 12,13-dibutyrate and 1-oleyl-2-acetyldiacylglycerol stimulate inositol trisphosphate dephosphorylation in human platelets, J. Biol. Chem. 261, 10493. Nahorski, S.R., D.A. Kendall and I.H. Batty, 1986, Receptors and phosphoinositide metabolism in the central nervous system, Biochem. Pharmacol. 35, 2447. Orellana, S.A., P.A. Solski and J.H. Brown, 1985, Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilisation in cultured astrocytoma cells, J. Biol. Chem. 260, 5236. Rooney, T.A. and S.R. Nahorski, 1986, Regional characterisation of agonist and depolarisation-induced phosphoinositide hydrolysis in rat brain, J. Pharmacol. Exp. Ther. 239, 873. Rooney, T.A. and S.R. Nahorski, 1987, Postnatal ontogeny of agonist and depolarisation-induced phosphoinositide hydrolysis in rat cerebral cortex, J. Pharmacol. Exp. Ther. 243, 333. Seyfred, M.A., L.E. Farrell and W.W. Wells, 1984, Characterisation of D-myo-inositol 1,4,5-trisphosphate phosphatase in rat liver plasma membranes, J. Biol. Chem. 259, 13204. Storey, D.J., S.B. Shears, C.J. Kirk and R.H. Michell, 1984, Stepwise enzymatic dephosphorylation of inositol 1,4,5-trisphosphate to inositol in liver, Nature 312, 374. Thomas, A.P., J. Alexander and J.R. Williamson, 1984, Relationship between inositol polyphosphate production and the increase of cytosolic free Ca 2 + induced by vasopressin in isolated hepatocytes, J. Biol. Chem. 259, 5574. Ueda, T., C. Sheaui-Huei, M.W. Noel and D.L. Gill, 1986, Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the NIE115 neuronal cell line, J. Biol. Chem. 261, 3184. Willcocks, A.L., A.M. Cooke, B.V.L. Potter and S.R. Nahorski, 1987, Stereospecific recognition sites for [3H]inositol(1,4,5)trisphosphate in particulate preparations of rat cerebellum, Biochem. Biophys. Res. Commun. 146, 1071.
425
Elsevier EJPMOL 90041
Developmental aspects of muscarinic-induced inositol polyphosphate accumulation in rat cerebral cortex T h o m a s A. R o o n e y a n d Stefan R. N a h o r s k i Department of Pharmacology and Therapeutics, University of Leicester, Medical Sciences Building, University Road, Leicester LEI 9HN, U.K.
Received 12 April 1989, revisedMS received11 July 1989, accepted 28 July 1989
The ability of carbachol to stimulate phosphoinositide hydrolysis in developing brain was examined by assaying [3H]inositol phosphates in the presence and absence of lithium. Lithium (5 mM) enhanced carbachol-stimulated [3H]inositol monophosphate and [ 3H]inositol bisphosphate accumulations at every age tested but the enhancement of both [JH]inositol phosphates was greater at 7 days than at 40 days. A marked, time-dependent inhibition of [3H]inositol trisphosphate and [JH]inositol tetrakisphosphates accumulations, i.e. 29-33 and 76-79%, respectively, was produced by lithium at every age tested. Lithium also inhibited both [3H]inositol-l,3,4-trisphosphate and [3H]inositol1,4,5-trisphosphate by 29-38%. There were no developmental differences in the ECs0 values for lithium-induced potentiations of [JH]inositol mono- and bisphosphate accumulations (i.e. 0.4-0.6 and 4-6 mM, respectively). Similarly, negligible changes in the ECs0 values for carbachol-induced [ 3H]inositol mono- and bisphosphate accumulations were observed in the presence or absence of lithium at every age tested. Models of receptor coupling and the sensitivity of inositol polyphosphate dephosphorylation to lithium block during development are considered. Muscarinic receptors; Inositol polyphosphates; (Development)
1. Introduction It is now clear that a large variety of neurotransmitter receptors, including muscarinic receptors, are linked, via a guanine nucleotide-sensitive mechanism, to a phospholipase C-mediated hydrolysis of phosphoinositides in various regions of the central nervous system (Abdel-Latif, 1986; Downes, 1986; Nahorski et al., 1986; Rooney and Nahorski, 1986). Such a phosphodiesteratic cleavage produces the two putative second messengers inositol-l,4,5-trisphosphate (InsP3) and diacylglycerol (Berridge and Irvine, 1984). The former
Correspondence to: Dr. T.A. Rooney, Department of Pathology and Cell Biology,Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19102, U.S.A.
metabolite can mobilise intracellular bound calcium in many cells, including those of neuronal origin (Ueda et al., 1986), probably by a stereoselective interaction with a receptor recently reported in cerebellum (Willcocks et al., 1987). Recent evidence indicates that InsP3 can undergo either dephosphorylation to inositol-l,4-bisphosphate (InsP2) (Storey et al., 1984), or phosphorylation to inositol-l,3,4,5-tetrakisphosphate (InsP4) (Hawkins et ai., 1986; Irvine et al., 1986) and that this inositol phosphate may regulate Ca 2÷ homeostasis at the level of the plasma membrane (Irvine and Moor, 1986). Thus, the proportion of InsP 3 undergoing phosphorylation or dephosphorylation and its regulation would seem to be crucial to the functional significance of several cell surface receptors in a variety of different situations. The immature brain with its developing
0922-4106/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)
426
innervation and receptor density provides an opportunity to investigate the relationship between receptor activation and phosphoinositide responsiveness. In a previous report we have shown that by assaying total [3H]inositol phosphates in the presence of lithium, carbachol produces supramaximal phosphoinositide responses despite very low density of muscarinic receptors within the first week of development which decline to adult values at > 21 days when receptor density matures (Rooney and Nahorski, 1987). In the present study we have extended these striking observations by investigating inositol polyphosphate responses in the developing rat cortex.
2. Materials and methods
The methods used were essentially those previously described by Brown et al. (1984), with modifications (Batty et al., 1985; Batty and Nahorski, 1985). Male Sprague-Dawley rats of various ages (7, 14, 21 and 40 days) were killed by cervical dislocation, decapitated, the brains removed and the cerebral cortex dissected (Glowinski and Iversen, 1966) on ice. The cerebral cortex was crosschopped into 350 × 350 /~m slices and incubated for 60 rain in a modified Krebs-bicarbonate buffer, equilibrated previously with 95% 02-5% CO2, pH 7.4, at 37 °C in a shaking water bath. Samples (50 /~1) of packed slices were then incubated for 60 min with 1.0 /~M [3H]inositol either in the presence or absence of appropriate lithium concentration before labelled slices were exposed to agonist stimulation. Incubations were terminated by addition of 300 #1 of 7% (v/v) perchloric acid, tissue sedimented by centrifugation and the supernatants neutralised by addition of a freshly prepared 1 : 1 (v/v) mixture of Freon/tri-n-octylamine, as previously described (Downes et al., 1986). Inositol phosphates were separated on columns of Dowex-1 anion-exchange resin (200-400 mesh; formate form) or by gradient-elution high performance liquid chromatography (HPLC) as previously described (Batty and Nahorski, 1985).
2.1. Drugs [2-3H]Myo-inositol (15 Ci/mmol) was obtained from New England Nuclear (Boston, MA, U.S.A.). Tri-n-octylamine was obtained from Aldrich Chemical Co. Ltd. (Dorset, England). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). or Fisons p.l.c. (Loughborough, England).
2.2. Analysis of data The concentration of agonist producing halfmaximal stimulation (ECs0) was obtained by subjecting the concentration-effect curve to a four parameter logistic model using computer-assisted iterative curve-fitting (ALLFIT) (De Lean et al., 1978).
3. Results
3.1. Developmental profiles for carbachol-induced inositol polyphosphate accumulation The relative proportions of each individual inositol phosphate accumulating under stimulation by carbachol was investigated. The measurements of individual [3H]inositol phosphates were performed in the absence and presence of lithium over a 30 min stimulation period. It is clear from fig. 1 that lithium enhances carbachol-stimulated [ 3H]inositol monophosphate ([ 3H]InsP1) and [ 3H]inositol bisphosphate ([3HllnsP2) accumulations at every age tested. Moreover, lithium has a marked inhibitory effect on [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4) accumulations (i.e. by 36-46% and 6779% respectively) at every age. Lithium also produces a greater enhancement of [3H]InsP1 and [3H]InsP2 at the earlier ages (7 and 14 days) than in the more mature brain (21 and 40 days). Lithium enhances [3H]InsP1 accumulation by 9.1-, 6.6-, 4.5- and 4.3-fold at 7, 14, 21 and 40 days respectively. Lithium also enhances [3H]InsP2 accumulations by 2.6-, 2.5-, 2.3- and 2.1-fold at 7, 14, 21 and 40 days respectively. There are also significant accumulations of each of the [3H]inositol phos-
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Fig. 1. Measurements of individual [3H]inositol phosphates at various stages (age in days) of postnatal ontogeny in rat cerebral cortex. The effects of carbachol (1 mM) at 30 rain in the presence of 5 mM LiC1 are shown. The striped bars show the responses obtained in the presence of LiCI and the open bars show the responses in the absence of LiC1. Samples were incubated with [3H]inositol (5 t~Ci/tube) in the presence or absence of 5 mM lithium for 60 min and then stimulated with carbachol. The results are expressed as cpm/2 ml fraction of each batch sample. Each bar in the histogram represents the mean _+S.E.M, of three to six experiments, each performed in triplicate.
phates in the absence of lithium following carbachol stimulation. These measurements indicate that the stimulated levels of [3H]InsP3 and particularly [3H]InsP4 in the absence of lithium are significantly greater at the earlier ages (7 and 14 days) than those obtained at 21 and 40 days. There also seems to be a slightly greater accumulation of [3H]InsP2 in the absence of lithium at the earlier ages, but any differences in the levels of [3H]InsP1 are less obvious. Under these conditions, lithium enhanced basal [3H]InsPl, [3H]InsP2 and [3H]InsP3 accumulations at each age but had a negligible effect on basal [3H]InsP4 accumulation.
3.2. Dose-response curves for carbachol-stimulated inositol polyphosphate accumulations during developrnent In a previous report we have shown that doseresponse curves for carbachol obtained by measuring total [3H]inositol phosphates (>90% [3H] InsP1) show no variability in ECs0 during development (Rooney and Nahorski, 1987). These observations were further investigated in the present studies by performing dose-response curves to carbachol for each [3H]inositol phosphate. Thus, it was possible to observe whether agonist-stimu-
428
lated accumulations of total [3H]inositol phosphates faithfully reflect receptor activation and whether any variability in the coupling of the muscarinic-induced phosphoinositide responses could be detected by measuring inositol phosphate products which accumulate upstream from total [3H]inositol phosphate accumulations. To do this, dose-response curves for each [3H]inositol phosphate were obtained under carbachol stimulation both in the absence and presence of lithium. These dose-response curves were performed at two time points. Consequently, dose-response curves to 120"
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Fig. 2. Dose-response curves to carbachol for inositol polyphosphate accumulation in the immature (7 days) cerebral cortex. Responses obtained in the presence (shaded circles) and absence (shaded triangles) of 5 mM LiC1 are shown. Slices were incubated with [3H]inositol (5 vCi/tube) in the presence or absence of 5 mM lithium and then incubated for 5 min (A) or 30 rain (B) in the presence of carbachol. The 100% values in terms of cpm/2 ml fraction are shown as maximal values. Each dose-response curve represents the mean+S.E.M, of three experiments, each performed in triplicate. Agonist concentrations above 1 mM did not significantly further increase the production of inositol phosphates.
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- Log Carb Log Carb - Log Carb Fig. 3. Dose-response curves to carbachol for inositol polyphosphate accumulation in cerebral cortex (40 days). Responses were obtained in the presence (shaded circles) and absence (shaded triangles) of 5 mM LiCI. Incubations were performed at 5 min (A) and 30 rain (B) in the presence of carbachol. Slices were incubated with [3H]inositol (5/~Ci/tube) for 60 rain either in the presence or absence of 5 mM lithium before stimulation by agonist. The 100% values in terms of cpm/2 ml fraction are shown as maximal values. Each dose-response curve represents the mean 5-S.E.M. of three experiments, each performed in triplicate. Agonist concentrations above 1 mM did not significantly further increase the production of inositol phosphates. - Log Carb
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absence and presence of lithium (figs. 2B and 3B). Perhaps the most important results to note are that the ECs0 values for carbachol in p r o m o t i n g the accumulation of each of the [3H]inositol phosphates in the presence and absence of lithium in b o t h 7- and 40-day-old rats are almost identical (table 1). The enhancements p r o d u c e d by lithium on [3H]InsP1 and [3H]InsP2, as well as the inhibitory effects on [3H]InsP3 and [3H]InsP4, shown in the dose-response curves at b o t h ages, are also compatible with the effects observed in fig. 1 using
a single maximal dose of carbachol. The dose-response curves to carbachol performed in the absence and presence of lithium in b o t h 7- and 40-day-old rats do show some differences at a 5 min stimulation period. Firstly, although lithium still enhances [3H]InsP] and [3H]InsP2 accumulation more in the 7- than in the 40-day-old animal, it does so less m a r k e d l y than at the 30 min stimulation period (figs. 2A and 3A). Secondly, lithium does not seem to be able to inhibit [3H]InsP3 and [3H]InsP4 accumulations, in either the 7 day or
430 TABLE 1 Postnatal ontogeny of ECs0 values for carbachol-induced polyphosphate accumulation. The values represent the means + S.E.M. of two to three experiments. Cerebral cortical slices were labelled with [3H]inositol (5 # O / t u b e ) for 60 min either in the presence or absence of 5 m M LiC1. Samples were further incubated for 5 or 30 rain after addition of carbachol or Krebs medium. Samples were extracted and triplicates combined as described in Materials and methods. The ECs0 values for carbachol-induced polyphosphate accumulation were calculated as described in Materials and methods. Carbachol incubation
InsP 1
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Stim. period
Rat age
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ECso (/~M)
(min)
(days)
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the 40 day animals, at the 5 rain stimulation period. However, the ECs0 values for carbachol-induced accumulation of each of the [3H]inositol phosphates are very similar to those obtained at the 30 rain stimulation period (table 1).
3.3. Measurement of [3H]lnsP3 isomers during development In order to investigate the effects of lithium on [3H]InsP3 in more detail, measurements of [3H]Ins-l,3,4-P3 and [3H]Ins-l,4,5-P3 were made. Table 2 shows the effect of carbachol on these isomers in both 7- and 40-day-old rats. It is clear from this table that lithium inhibits the stimulated accumulation of both [3H]Ins-l,3,4-P3 and [3H]Ins-l,4,5-P3 by 29-38% at both ages with a 30 min stimulation period. No effect on the levels of the [3H]InsP3 isomers was observed at the 5 rain stimulation period. Carbachol produced a 5- and 3-fold stimulation of [3H]Ins-l,4,5-P3 accumulation at both time points in the absence of lithium in 7- and 40-day-old rats, respectively. In the presence of lithium, carbachol produced a 6- and 3-fold stimulation of [3H]Ins-l,4,5-P3 at both ages with a 5 min stimulation period. Carbachol also produced a 17- and ll-fold enhancement of [3H]Ins-l,3,4-P3 levels at both ages with a 5 min stimulation. It is difficult to determine the percentage stimulation produced by carbachol on [3H]Ins-l,3,4-P3 in the absence of lithium due to the undetectable control levels under such conditions. Lithium also pro-
InsP4 ECs0 ( # M )
duced a slight enhancement of both [3H]Ins-l,3,4P3 and [3H]Ins-l,4,5-P3 basal levels under all conditions examined.
TABLE 2 Effects of 5 m M LiC1 on control and carbachol-stimulated (1 mM) accumulations of [3HllnsP3 isomers. Cerebral cortical slices were labelled with [3H]inositol ( 5 / ~ C i / t u b e ) for 60 min either in the presence or absence of 5 m M LiC1. Samples were further incubated for 5 or 30 min after addition of carbachol or Krebs medium. Samples were extracted and triplicates combined as described in Materials and methods and subsequently analysed by H P L C for [3HllnsP3. In order to perform this study in triplicate, tissue was obtained by pooling cerebral tissue from 7-8 animals of both ages. The data represent combined triplicates so as to facilitate the detection of both inositol phosphate isomers at both ages. Similar results have been obtained on at least one other occasion. Incubation
7-day-old rat Control (5 min) Carbachol (5 min) Control (30 rain) Carbachol (30 rain)
Inositol phosphates (cpm) Ins-l,3,4-P 3
Ins-l,4,5-P 3
+Li +
-Li +
+Li +
-Li +
34 573 72 357
547 562
573 3 380 694 2150
554 3001 653 3 263
47 497 50 348
476 507
507 1448 666 1024
493 1528 583 1750
40-day-old rat Control (5 min) Carbachol (5 min) Control (30 min) Carbachol (30 min)
431
3.4. Effects of lithium on carbachol-stimulated inositol polyphosphate accumulation during development Having firmly established in the previous sections that the supramaximal responses produced by carbachol at early ages cannot be explained by a more efficiently coupled muscarinic receptor in these animals, the possibility of an altered sensitivity to lithium during development was examined by performing dose-response curves to lithium for each [3H]inositol phosphate. These dose-response curves were performed in both 7- and 40-day-old rats at a 30 rain Stimulation period. Lithium produced a dose-related potentiation of [3H]InsP1 in both 7- and 40-day-old rats (fig. 4). In the 7-dayold rat lithium produced a 10-11-fold enhancement with a maximal and half-maximal effect at 3-5 and 0.6 mM, respectively. In the 40-day-old rat, lithium produced a 5-fold enhancement with maximal and half-maximal effects at 3-5 and 0.4 mM, respectively. Similarly, lithium produced a significant, though less potent, enhancement of [3H]InsP2 by 5- and 2-fold at a concentration of 15-30 mM in both 7- and 40-day-old rats respectively. The half-maximal InsP2 response to lithium was produced at a concentration of 4-6 mM at both ages. In contrast to the potentiating effects on [3H]-
InsPa and [3H]InsP2, lithium produced a dose-related inhibition of both [3H]InsP3 and [3H]InsP4 in 7- and 40-day-old rats. The effect on [3H]InsP3 was evident between 0.1-0.3 mM and was halfmaximal by 0.4-0.5 mM at both ages. The inhibition was maximal by 3-5 mM and represented a 29-33% decrease of both ages. The effect on [3H]InsP4 was evident between 0.1-0.3 mM and was half-maximal by 0.3-0.4 mM at both ages. This represented a 76-79% decrease at both ages and was maximal by 3-5 mM.
4. Discussion
The results presented in this study indicate, that upon activation, the muscarinic receptor in young rats produces supramaximal polyphosphoinositide responses compared to adult. However, the muscarinic receptor does not appear to be more efficiently coupled to phosphoinositide hydrolysis as the ECs0 values for carbachol-induced inositol polyphosphate accumulation are similar to those obtained in adults. These results are consistent with previous measurements of total [3H]InsPs which also show supramaximal responses to carbachol with no shift in its dose-response curve in rat cerebral cortex of young rats (Balduini et al., 1987; Rooney and Nahorski, 1987).
,.-.. 700O0..~ > 600000 .,O ,~ 50000C •~ 40000~
14000InsP1
12000-
2100InaP2
28oo-
InsP3
24002000-
10000/
1200-
1600"
6000-
l ~
900-
1200-
4000-
t
600
800-
6000-
30000-
/
~
20000"
E 10000Q.
40day
-
Log LiCL
lnsP4
7aay
2000-
400-
-Log
LiCL
-Log
LiCL
_ Log LiCL
Fig. 4. Dose-related effects of lithium on carbachol-stimulated inositol polyphosphate accumulations in cerebral cortex from 7- and 40-day-old rats. Slices were incubated with [3H]inositol (5/~Ci/tube) in the presence of appropriate lithium concentration for 60 min. Samples were then incubated with carbachol (1 mM) for a further 30 rain. The results are expressed as cpm/2 ml fraction of each batch sample. Data obtained in the absence of lithium (not shown) was not significantly different to that observed at 10-4 M lithium. Results represent the means_+S.E.M. of three experiments, each performed in triplicate.
432 Furthermore, the relative intrinsic activity of the muscarinic partial agonist, arecoline, has been shown to remain constant throughout development (Rooney and Nahorski, 1987). It should be noted, however, that the present data do not agree with a recent report by Heacock et al. (1987) who suggest an enhanced coupling (exhibited as a lower ECs0 value for agonists) of the muscarinic receptor to cortical phosphoinositide metabolism in the young rat. The reasons for these differences are not known, though it is noteworthy that the low ECs0 value for carbachol reported in young rats by these authors is identical to that obtained at all ages in the present study. The present studies attempt to elucidate the basis of this markedly elevated muscarinic receptor-stimulated inositol phosphate production. The developmental changes in muscarinic receptor-induced inositol polyphosphate accumulation could relate to a greater ability of lithium to enhance [ 3H]InsPl and [ 3H]insP2 accumulation in young animals. However, this greater enhancing property of lithium in younger animals does not seem to be due to an increased sensitivity to lithium at these ages. Lithium has the same ECs0 value in 7- and 40-day-old rats for both [3H]InsPl and [3H]InsP2 accumulations (i.e. 0.4-0.6 and 4-6 raM, respectively). These results are consistent with previous reports in brain (Berridge et al., 1982; Brown et al., 1984; Batty and Nahorski, 1985) as well as in hepatocytes and pituitary GH 3 cells (Drummond et al., 1984; Thomas et al., 1984). It is therefore clear that the respective phosphatases regulating InsP 1 and InsP2 dephosphorylation, although sensitive to lithium during development, do not change with regard to their sensitivity for lithium block. The greater enhancement produced by lithium, predominantly on [3H]InsP1 accumulation in younger animals is also accompanied by a dose-dependent inhibition of [ 3H]insP 3 and [ 3H]insP4 accumulation at the more prolonged stimulation times. This inhibition of [3H]InsP3 and [3H]InsP4 accumulation is also evident in 40-day-old rats where the lithium enhancement of [3H]InsP1 accumulation is less than in 7-day-old rats. Lithium produces a 30-40% inhibition of [3H]InsP3 accumulation and a 67-79% inhibition of [3H]InsP4 accumulation in both 7- and
40-day-old rats. The differential enhancing properties of lithium on [3H]InsP1 could be explained if the various isomers of this inositol phosphate which could potentially be formed from both [3H]Ins-l,4,5-P3 and Ins-l,3,4-P 3 dephosphorylation were produced, in part, via lithium-insensitive dephosphorylation pathways (see Majerus et al., 1988). Thus, it is possible there is a change in the relative activity of these phosphatases as the animals mature. Such possibilities would result in the lower [3H]InsP1 accumulation in the presence of lithium observed with no shift in the dose-response curve to carbachol. As an examination of the isomeric forms of [3H]InsP1 produced at the various stages of development was not performed in the present studies it is impossible to tell which isomers are potentiated by lithium or indeed formed at the various stages of development. These questions are the focus of current investigations in this laboratory. Although lithium has been shown to potentiate carbachol-induced [3H]InsP1 and [3H]InsP2 accumulations, it seems to have an inhibitory effect on both stimulated [3H]InsP3 and [3H]InsP4 levels. A similar inhibitory effect on [3H]InsP3 accumulation was originally reported by Batty and Nahorski, 1985, although it was subsequently shown that a large part of the inhibition was due to a reduction in the levels of [3H]InsP4 which represented a contaminant of the original [3H]InsP3 fraction (Batty and Nahorski, 1987). It is also important to note that the effect of lithium on both [3H]InsP3 and [3H]InsP4 accumulations seems to be time-dependent. Thus, lithium inhibits both InsP3 and InsP4 at the 30 min but not the 5 min stimulation period. The effect of lithium is also dependent on the length of preincubation period with lithium. For example, we previously found no effect on [3 H]insP 3 accumulation if agonist and lithium were added together (Batty and Nahorski, 1987) rather than having lithium present during the labelling period with [3H]inositol before agonist stimulation. As yet, the reasons for lithium's inhibitory effects on both [3H]InsP3 and [3H]InsP4 accumulations are unknown although some possibilities have been discussed (Batty and Nahorski, 1987). For example, the effects could be explained by a depletion of a receptor-associated phospholipid
433
pool, although this seems unlikely as labelled inositol is present in the incubation buffer throughout the period of agonist and lithium treatments. Moreover, lithium produces its inhibitory effect on [3H]InsP3 accumulations at a concentration where its potentiation of [3H]InsP1 accumulations is still linear. If it is assumed that all of the InsP~ measured is derived from InsP 3, as is suggested by the similar ECs0 values for both inositol phosphates upon agonist activation, then it is unlikely that the inhibitory effects of lithium on InsP3 and InsP4 can be explained by a decrease in the pool of receptor-associated PtdlnsP 2. Another possibility, consistent with the observed lag period associated with the onset of lithium's inhibitory action on both InsP 3 and InsP4, is that the effects could be mediated via protein kinase C activation by an elevation of diacylglycerol following observed lithium treatment (Drummond and Raeburn, 1984; Downes and Stone, 1986). Such an activation could have negative feedback effects on the various phosphoinositide kinases and phosphatases or directly at the level of the receptor. For example, phorbol esters have been shown to inhibit muscarinic receptorinduced inositol polyphosphate accumulation (Orellana et al., 1985) and to increase the hydrolysis of Ins-l,4,5-P 3 by an activation of a 5-phosphatase (Molina y Vedia and Lapetina, 1986). Since it is likely that the same 5-phosphatase is involved in dephosphorylating both Ins-1,4,5-~ and Ins-l,3,4,5-P4 then this may, in part, explain the inhibitory effect produced by lithium on both of these inositol phosphates. Such an indirect effect of lithium is further supported by the fact that the Ins-l,4,5-P 3 5-phosphatase has been shown to be insensitive to any direct effects of lithium (Downes et al., 1982; Seyfred et al., 1984; Connolly et al., 1985). There are some anomalies in the effects of lithium on both InsP 3 and InsP4. Lithium inhibits InsP 4 accumulation by 67-79% but only inhibits InsP 3 levels by 36-46%. A more detailed analysis of InsP3 accumulations shows that Ins-l,3,4-P 3 and Ins-l,4,5-P3 are inhibited to the same degree by lithium. Thus, a 67-79% inhibition of InsP4 only results in a 30-38% inhibition of Ins-l,3,4-P 3. As Ins-l,3,4-P 3 has been shown to be formed
solely from a 5-phosphatase attack on Ins-l,3,4,5P4 (Batty et al., 1985; Hansen et al., 1986; Hawkins et al., 1986) the disproportionate decreases in InsP4 and Ins-l,3,4-p~ produced by lithium could be explained if lithium also enhanced Ins-l,3,4-P 3 accumulation. This possibility has been reported by Burgess et al. (1985), and indeed lithium has been shown to elevate total InsP 3 levels in hepatocytes and G H 3 cells (Drummond et al., 1984; Thomas et al., 1984). In conclusion, it is clear from these results that the supramaximal responses produced in young rats cannot be explained by an enhanced coupling of the muscarinic receptor or by a differential sensitivity to lithium. However, the extent to which the various pathways of inositol polyphosphate dephosphorylation are used a n d / o r blocked by lithium may vary during development. Moreover, the time-dependent inhibitory effects of lithium on both InsP 3 and lnsP 4 require further investigation in order to obtain a greater understanding of how lithium perturbs the phosphoinositide signalling system in brain. Future studies should be focussed on a more detailed analysis of the effects of lithium on the separated isomeric forms of inositol phosphates produced during development.
Acknowledgements The authors would like to thank Dr. I.H. Batty for running of HPLC samples and Jenny Bell for help in the preparation of the manuscript.
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