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Stimulation of phosphoinositide hydrolysis in myelin by muscarinic agonist and potassium
Brahi Research, 436 (1987) 357-362 Elsevier
357
BRE 22590
Short Communications
Stimulation of phosphoinositide hydrolysis in myelin by muscarinic agonist and potassium Jorge N. Larocca, Agostino Cervone and Robert W. Ledeen Departments of Neurology and Biochemistry, Albert Einstein College of Medicine, Bron.~, NY 10461 (U.S.A.)
(Accepted 11 August 1987) Key words: Myelin; Phosphoinositide; Muscarinic receptor; Myelin phosphoinositide
Slices of rat brainstem that had been prelabeled by in vivo injection of [3H]inositol were stimulated with carbachol in the presence of lithium and changes measured in the radioactivity of inositol lipids and water-soluble inositol phosphates. For the latter, significant increases were seen for inositol mono- and bisphosphate but not inositol trisphosphate. Analysis of whole tissue phosphoinositides revealed significantly reduced radioactivity in phosphatidylinositorl and phosphatidylinositol 4-phosphate, whereas myelin showed decreases in those as well as phosphatidylinositoi 4,5-bisphosphate. These effects were blocked by atropine. Stimulation of the tissue slices with elevated K + resulted in increased formation of inositol phosphate and decreased radioactivity in phosphatidylinositol. The effect was not blocked by atropine and in the presence of thins agent, which reduced background reaction, all 3 phosphoinositides showed significant K+-induced loss of label. Elevated K+ and carbachol thus function through different mechanisms in this system. Carbaehol is believed to affect myelin phosphoinositides through direct interaction with muscarinic receptors which were recently shown to be present in this membrane. The role of myelin as facilitator of saltatory conduction has made it a membrane of considerable interest to neuroscientists. Although previously thought to be metabolically inert, it is now viewed in a somewhat different light owing to the discovery of numerous intrinsic enzymes involved in lipid metabolism, ion transport, phosphorylation/dephosphorylation, protein degradation, etc (for review see refs. 25, 34, 41). The recent discovery of high affinity muscarinic cholinergic receptors in myelin purified from rat brainstem 24 provides further indication of an active membrane. Those experiments pointed to the presence of both M l and M 2 rece t ubtypes in myelin and thus raised the possibility of linkage to phosphoinesitide metabolism. Brain as a whole contains large amounts of the substrates and enzymes of inositol lipid turnover ~2a4 and the same appears to be true of myelin ~tself. Thus the latter has been reported to be highly enriched in phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol
(Pl) 10"18"34, and to contain phosphoesterase and kinase activities involved in their metabolism 7'9. We have sought to test the possibility that these myelin-localized receptors control inositol lipid metabolism within the membrane by stimulating prelabeled white matter slices with a cholinergic agonist and looking for evidence of enhanced breakdown of myelin phosphoinositides. This was done by measuring decreases in inositol lipid label as well as increases in inositol phosphate products. We have also studied the effects of high potassium on the metabolism of these lipids. Preliminary reports of this work have been pre~,nted 22"23. In a typical experiment, four 21-day-old S p r a g u e Dawley rats were injected intracerebrally with [23H]myoinositol (15 Ci/mmol, American Radiolabeled Chemicals, St. Louis, MO); 25/~Ci in 5 #! saline was injected into each hemisphere (50~Ci total). After approximately 16-18 h the animals were decapitated and the brains rapidly removed and dissected in the cold. Brainstems were cross-chopped into 350 # m
Correspondence: R.W. Ledeen, Departments of Neurology and Biochemistry. Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A.
0006-8993/87/$03.50 @') 1987 Elsevier Science Publishers B.V. (Biomedical Division)
358 slices with a tissue chopper and the slices transferred to 10 ml Erlenmeyer flasks. Each flask contained slices from one-half brainstem. These were incubated in 4 ml of modified Krebs-Ringer bicarbonate (KRB) with the following composition (mM); NaCI 106, KCI 4.7, KH2PO4 1.2, Na,SO4 1.2, MgCI2 1.2, LiCI 10, CaCI 2 1.3, N a H C O 3 25, glucose 11, inositol 5; this was equilibrated with O~_/CO2 (19:1 v/v) to a final pH of 7.4. The slices were gently agitated in a shaking water bath at 37 °C for 1 h, in the presence or absence of 2 mM carbachol. Some samples were preincubated 5 min in the presence of 10/~M atropine before addition of carbachol. In studies on the effect of K ÷, 60 mM of the NaCI was replaced by an equivalent amount of KCI in the modified KRB medium (4 ml per flask). In other experiments designed to test the effect of atropine during K ÷ depolarization, the brainstem slices were first incubated for 5 min in 3 ml of regular KRB buffer containing 10 ~M atropine, followed by addition of 3.1 ml of modified KRB with 101~M atropine in which all the NaCI was isotonically replaced with KCI (final KCI = ca. 60 mM). This was reincubated at 37 °C for 1 h. The tissue suspensions were centrifuged 5 min at 1000 rpm in a HG-4L Sorvall rotor and the resulting supernatant discarded. The pellet was homogenized in 4 ml of 0.30 M sucrose buffered with 20 mM Tris-HCl (pH 9.5) and aliPIP ~S
40-
30PIP2
till
PI
~, 20 u i11
r.,
10
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-10 ~J Fig. 1. Effects of carbachol and carbachol plus atropine on whole tissue phosphoinositides. Brainstem slices previously labeled with [3H]inositol were incubated 1 h at 37 °C with either added buffer, carbachol (2 mM) or carbachol (2 mM) plus atropine (10 ltM). The atropine was added 5 min before stimulation with carbachol. Radioactivity levels in whole tissue phosphoinositides were determined as described in the text. Results are expressed as % of decrease with respect to control; the amounts counted corresponded to approximately 200 l~g of tissue protein. Each bar in the histogram represents the mean + S.E.M. of 4-8 experiments. Open bars represent atropine experiments. *P < 0.05 (Student's two-tailed t-test).
40
-
PIP2
PIP
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Fig. 2. Effects of carbachol and carbachol ph:s atropine on myelin phosphoinositides. Brainstem slices previously labeled with [3H]inositol were incubated under co;lditions described in Fig. 1. The brainstem slices were then homogenized and myelin isolated. Radioactivity levels in myelin phosphoinositides were determined as described in the text. Results are expressed as % decrease with respect to control: the amounts counted corresponded to approximately 120!tg of myelin protein. Each bar in the histogram represents the mean + S.E.M. of 4-6 experiments. Open bars represent atropine experiments. *P < 0.05 (Student's two-tailed t-test). quots removed for protein determination by a modified Lowry procedure 26. One-fourth of the sample was treated with 10% (w/v) trichloroacetic acid (TCA) and the remainder set aside for myelin isolation (see below). The precipitate was centrifuged and the resulting pellet re-extracted with 1 ml of 10% TCA. The combined supernatants were extracted 4 times with an equal volume of ethyl ether to remove TCA and then applied to a Dowex 1 x 8-400 column for separation of inositol phosphates 3. The pellet was extracted with acidified chloroform-methanol according to the entire procedure of Bell et al. 1, except that 0.2 mg/ml of butylated hydroxytoluene was present as antioxidant. The lipid extract was evaporated to dryness with N, and redissolved in 0.3 ml of the solvent employed for TLC: c h l o r o f o r m - m e t h a n o l - w a t e r - c o n c . NH 3 (90:90:22:7, v/v). Aliquots were applied to Merck silica gel 60 HPTLC plates, 10 cm x 20 cm ( V W R Scientific, South Plainfield, N J), and the plates developed with the above solvent in paper-lined tank. Standards of PI, PIP, and PIP 2 (Sigma, St. Louis, MO) were co-chromatographed for identification. The iipids were detected with I2 vapor and bands parallel to the standards were scraped and counted. The remaining three-fourths of the sample (see above) was employed for myelin isolation according to Haley et al. 17. This is based on the procedure of
359 IP
40-
showing the greatest decrease (ca. 30-35%) with PI and PIP 2 radiolabel decreasing about half as much (Fig. 1). The change recorded for the latter lipid was not significant. Absolute levels of radioactivity for PIP.,, PIP, and PI in control samples were 476, 1140, and 12,600 dpm/mg protein, respectively. A somewhat different pattern was observed in the myelin subsequently isolated from brainstem after carbachoi stimulation (Fig. 2) in that all 3 phosphoinositides showed significant loss of radioactivity (ca. 2028%). Absolute counts for myelin PIP,, PIP and PI in control samples were 896,482 and 7220 dpm/mg protein, respectively. As seen in Figs. 1 and 2, these hydrolytic reactions were effectively blocked by the presence of 10 IbM atropine in the incubation medium. The apparent "over-compensation" seen with atropine was possibly a reflection of the phosphoinositide breakdown occurring in control sampies which would be diminished by this agent. Further evidence for stimulated hydrolysis came from analysis of inositol phosphates isolated from whole brainstem (Fig. 3). The increase (32%) in radioactivity of inositol phosphate (IP) was approximately twice that of inositol bisphosphate (IP2), while the change for inositol trisphosphate (IP3) was not significant. Absolute levels of radioactivity for IP3, IP2 and IP in control samples were 173, 1570, and 3820 dpm/mg protein, respectively. As with the lipids, the effect was blocked by atro: ine. A somewhat similar pattern of inositol phosphate release was reported with rat cerebral cot-
30 IP2
u., 2O u Z
10
IPa ,,,
W
-I0I -20
Fig. 3. Effect of carbachol and carbachoi plus atropine on accumulation of inositol phosphates. Brainstem slices previously labeled with [3Hlinositol were incubated under conditions described in text and Fig. 1. lnositol phosphates were separated by anion exchange chromatography. Results are expressed as % of increase with respect to control; the amounts counted corresponded to approximately 500 !~g of tissue protein. Each bar in the histogram represents the mean + S.E.M. of 4-8 experiments. Open bars represent atropine experiments. *P < 0.05 (Student's two-tailed t-test).
Norton and Poduslo 33 with an additiona] floating up gradient for higher purity ~7. To the labeled tissue was added an approximately equivalent amount of unlabeled brains:.em. Lipids were extracted and chromatographed from the resulting myelin as described above. The results indicated that carbachol enhanced phosphoinositide hydrolysis in whole brainstem. PIP A
B PIP2
40 LU Or)
< 30 LU n" rO
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IP2
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~--] WITHATROPINE
~ NOATROPINE
Fig. 4. Effects of elevated K + on phosphoinositide hydrolysis. Brainstem slices previously labeled with [~H]inositol were incubated 1 h at 37 °C vdth either buffer or buffer containing 60 m'M KCI. Atropine when present (It) !4M) was added 5 rain before elevation of K-. A: decrease of label in mx'elin phosphoinositides. B: increase of radiolabeled inositol phosphates. The results are expressed as the q change with respect to control: the amounts counted corresponded to approximately 120.ug of myelin protein for phosphoinositides and to 500 !~g of tissue protein for inositol phosphates. Each bar in the histogram represents the mean + S.E.M. of 4-7 experiments. *P < 0.05 (Student's two-tailed t-test).
360 tex slices following carbachol stimulation-. Inositol phosphate patterns reflect not only the products of phosphoinositide hydrolysis but also the conversion via phosphatase to forms with less phosphate; the presence of Li ÷ blocks further hydrolysis beyond inositol monophosphate 3. Treatment of the pre-labeled brainstem slices with elevated K ÷ also caused enhanced breakdown of phosphoinosltidc~, as seen in the significant elevation of released radiolabeled iP ~,~t:~,,.,~4). IP-_ and IP3 remained unchanged and atropine was without effect on IP formation. Among the phosphoinositides only PI showed significant loss of radiolabel in the absence of atropine but all 3 were significantly decreased in the presence of this blocking agent. Absolute counts for inositol phosphates and myelin PIP: in control samples were approximately 65-100% higher than those mentioned above while those for PIP and PI were similar. As mentioned, atropine is seen as reducing the level of background activity and since it does not block K + stimulation the magnitud~ of this effect is considered more meaningful in its presence. The inositol phosphates liberated through carbachol, as well as the phosphoinositide changes observed in whole tissue slices, very likely reflected the results of cholinergic stimulation of diverse elements in the brainstem. However, in considering myelin as a specifically targeted membrane, the fact that inositol phospholipids of this structure showed significant loss of radioactivity following carbachol treatment is strongly suggestive of effector-mediated phosphoinositide breakdown in that membrane• Considering that phosphoinositide hydrolysis would not likely occur in one membrane as a result of receptor activation in another (although that may be theoretically possible through some kind of exchange process), the most plausible mechanism to explain our data is direct stimulation of the above-mentioned muscarinic receptor in myelin itself. We believe the same interpretation can be applied to the recent abstract report 21 that cholinergic stimulation of rat cortical brain slices caused enhanced incorporation of 32p into phosphatidic acid and phosphoinositides of myelin. Work in pr6gress in our laboratory (Golly, Larocca and Ledeen, in preparation) has indicated breakdown of prelabeled phosphoinositides of myelin through direct carbachol stimulation of isolated myelin• Earlier work 8,39 had demonstrated the •
"
"~0
rapidity with which 32pi is incorporated into phosphoinositides of myelin, either in vivo or in vitro. Studies by Deshmukh and coworkers 7 pointed to the presence in myelin of phosphoinositide phosphodiesterase, the key enzyme activated by agonist-receptor interaction, along with phosphomonoesterases and phosphoinositide kinases 7. One of the latter enzymes, PI kinase, was recently isolated from bovine CNS myelin 36. Such enzymes present in other brainstem components could account for the different patterns of phosphoinositide depletion noted in the present study. For example, failure of PIP2 in brainstem slices to show significant loss of radiolabel (Fig. 1) might conceivably result from compensating resynthesis from PIP via PIP kinase. Alternatively, the data might reflect the presence of a specific phosphodiesterase for PIP; a number of studies have indicated the presence of different phospholipase C activities that catalyze direct breakdown of PI and PIP 27. Either of these mechanisms could also be operative in myelin which showed equivalent loss of all 3 phosphoinositides. Although the pattern of myelin phosphoinositide depletion was sinn|ar for elevated K + as for carbachol, the mechanisms were apparently different as shown by the opposite responses to atropine. Elevated K ÷ and carbachol were previously shown to facilitate phosphoinositide turnover, sometimes in synergistic fashion, in brain slices and isolated nerve endingsll.16.19,37.43. The physiological significance of the receptorlinked phosphoinositide system in myelin is not known. While in brain such systems have been ascribed primarily to neurons 15, recent work has shown their presence in several glial cell preparations as well 5'6'28'3°'35. A key feature of this phenomenon is phosphodiesterase cleavage of PIP 2 to form two second messengers, IP 3 and diacylglycerol 2,32.38. It is not clear at present how messenger-activated release of Ca 2÷ (via IP3) might occur in myelin, although the presence of smooth endoplasmic reticulum has been noted in the adaxonal components of myelin (e.g. lateral loops) 13. A possible role for diacylglycerol is easier to visualize since the kinase which it activates is known to be present in myelin and to utilize the myelin basic proteins as substrates 4,29,31,4°,42. Further study of the way in which phosphoinositides mediate
361 signal transduct:.on in myelin should provide additional insight into the metabolic properties of this membrane.
( R . W . L . ) , and National Multiple Sclerosis Society Grant RG 1941-A-1 (J.N.L.). We are happy to acknowledge receiving helpful suggestions from by Dr. Maynard Makman.
This work was supported by N I H Grant NS-16181
I Bell, M.E., Peterson, R.G. and Eichberg, Metabolism of phospholipids in peripheral nerve from rats with chronic streptozotocin-induced diabetes: increased turnover of phosphatidylinositol-4,5-bisphosphate, J. Neurochem., 39 (1982) 192-200. 2 Berridge, M.J., Inositol trisphosphate and diacylglycerol as second messengers, Bioc~em. J., 220 (1984) 345-360. 3 Berridge, M.J., Downes, C.P. and Hanley, M.R., Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands, Biochem. J., 206 (1982) 587-595. 4 Carnegie, P.R., Dunkley, P.R., Kemp, B.E. and Murray, A.W., Phosphorylation of selected serine and threonine residues in myelin basic protein by endogenous and exogenous protein kinases, Nature (Lond.), 249 (1974) 147-150. 5 DeGeorge, J.J., Moreli, P., McCarthy, K.D. and Lapetina, E.G., Cholinergic stimulation of arachidonic acid and phosphatidic acid metabolism in C62B glioma cells, J. Biol. Chem., 261 (1986) 3428-3433. 6 DeGeorge, J.J., Moreil, P., McCarthy, K.D. and Lapetina, E.G., Adrenergic and cholinergic stimulation of arachidonic and phosphatidate metabolism in cultured astroglial cells, Neurochem. Res., l l (1986) 1061-[071. 7 Deshmukh, D.S., Bear, W.D. and Brockerhoff, H., Polyphosphoinositide biosynthesis in three subfractions of rat brain myelin, J. Neurochem., 30 (1978) 1191-1193. 8 Deshmukh, D.S., Kuizon, S., Bear, W.D. and Brockerhoff, H., Rapid incorporation in vivo of intracerebraUy injected 32pi into polyphosphoinositides of three subfractions of rat brain myelin, J. Neurochem., 36 (1981) 594-601. 9 Deshmukh, D.S., Kuizon, S., Bear, W.D. and Brockerhoff, H., Polyphosphoinositide mone, and diphosphoesterases of three subfractions of rat brain myelin, Neurochem. Res., 7 (1982) 617-626. 10 Eichberg, J. and Dawson, R.M.C., Polyphosphoinositides in myelin, Biochem. J., 96 (1965) 644-650. 11 Eva, C. and Costa, E., Potass;um ion facilitation of phosphoinositide turnover activation by muscarinic receptor agonists in rat brain, J. Neurochem., 46 (1986) 1429-1435. 12 Fisher, S.K. and Agranoff, B.W., Receptor activation and inositol lipid hydrolysis in neural tissues, J. Neurochem., 48 (1987) 999-1017. 13 Friedrich, Jr. V.L., Massa, P., and Mugnaini, E., Fine structure of oligodendrocytes and central myelin sheaths. In A. Boese (Ed.), Search for the Cause of Multiple Sclerosis and other Chronic Diseases of the Central Nervous System, Chemie, Deerfield Beach, FL, 1980, pp. 27-49. 14 Gonzales, R.A. and Crews, F.T., Characterization of the cholinergic stimulation of phosphoinositide hydrolysis in rat brain slices, J. Neurosci., 4 (1984) 3120-3127. 15 Gonzales, R.A., Feldstein, J.B., Crews, F.T. and Raizada, M.K., Receptor-mediated inositide hydrolysis is a neuronal response: comparison of primary neuronal and glial cultures, Brain Research, 345 (1985) 350-355. 16 Gurwitz, D. and Sokolovsky, M., Dual pathways in musca-
rinic receptor stimulation of phosphoinositide hydrolysis. Biochemistry, 26 (1987) 633-638. 17 Haley, J.E., Samuels, F.G. and Ledeen, R.W., Study of myelin purity in relation to axonal contaminants, Cell Mol. Neurobiol., 1 (1981) 175-187. 18 Hauser, G., Eichberg, J. and Gonzales-Sastre, F., Regional distribution of polyphosphoinositides in rat brain, Biochim. Biophys. Acta, 248 (1971) 87-95. 19 Hawthorne, J.N. and Bleasdale, J.E., Phosphatidic acid metabolism, calcium ions, and transmitter release from electrically stimulated synaptosomes, Mol. Cell. Biochem., 8 (1975) 83-87. 20 Jacobson, M.D., Wusteman, M. and Downes, C.P., Muscarinic receptors and hydrolysis of inositol phospholipids in rat cerebral cortex and parotid gland, J. Neurochem., 44 (1985) 465-472. 21 Kahn, D. and MoreU, P., Cholinergic stimulation of phosphoinositide metabolism in rat brain myelin, Trans. Am. Soc. Neurochem., 17 (1986) 315 (Abstr.). 22 Larocca, J.N., Makman, M.H., Golly, F. and Ledeen, R.W., The presence of muscarinic receptors linked to phospholipase C and adenylate cyclase in purified myelin. In Phospholipids in the Nervous System: Biochemical and Molecular Pathology, Satellite Symposium of the ISN-ASN Joint Meeting (Abstr.), Caracas, 1987. 23 Larocca, J.N. and Ledeen, R.W.. Enhanced breakdown of myelin phosphoinositides by carbachol and potassium: further evidence for a muscarinic receptor in myelin. J. Neurochem., 48 (1987) S119 (Abstr.). 24 Larocca, J.N., Ledeen, R.W., Dvorkin, B. and Makman, M.H., Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat t~rain myelin, J. Neurosci., in press. 25 Ledeen, R.W., Lipid-metabol;.zmg enzymes of myelin and their relation to the axon, J. Lipid Res., 25 (1984) 1548-1554. 26 Lees, M.B. and Paxman, S., Modification of the Lowry procedure for the analysis of proteolipid protein, Anal. Biochem., 47 (1972) 184-192. 27 Majerus, P.W., Connolly, T.M., Deckmyn, H., Ross, T.S., Bross, T.E., Ishii, H., Bansai, V.S. and Wilson, D.B., The metabolism of phosphoinositide-derived messenger molec, des, Science, 234 (1986) 1519-1526. 28 Masters, S.B., Harden, T.K. and Brown, J.H., Relationships between phosphoinositide and calcium responses to muscarinic agonists in astrocytoma cells, Mol. Pharmacol.. 26 (1984) 149-155. 29 Miyamoto, E., and Kakiuchi, S., In vitro and in vivo phosphorylation of myelin basic protein by exogenous and endogenous adenosine 3",5'-monophosphate-protein kinases in brain, J. Biol. Chem., 249 (1974) 2769-2777. 30 Murphy, S., Pearce, B. and Morrow, C., Astrocytes have both M r and M2 muscarinic receptor subtypes, Brain Research, aM i le~;~ !77-180. 31 Murray, N. and Steck, A.J., Activation of myelin protein
362
32 33 34 35
36
37
kinase by diacylglycerol and 4-phorbol 12-myristate 13-acetate,/. Neurochem., 46 (1986) 1655-1657. Nishizuka, Y., Turnover of inositol phospholipids and signal transductior:,;S~.ience, 225 (1984) 1365-1370. Norton, W.T. ~nd Poduslo, S., Myelination in rat brain: method of myelin isolation, J. Neurochem., 21 (1973) 749-751. Norton, W.T., and Cammer, W., Isolation and characterization of myelin. In P. Morell (Ed.), Myelin, Plenum, New York, 1984, pp. 147-195. Pearce, B., Cambray-Deakin, M., Morrow, C., Grimble, J. and Murphy, S., Activation of muscarinic and of alpha-1 adrenergic receptors on astrocytes results in the accumulation of inositol phosphates, J. Neurochem., 45 (1985) 1534-1540. Saltiel, A.R., Fox, J.A., Sherline, P., Sahyoun, N. and Cuatrecasas, P., Purification of phosphatidylinositol kinase from bovine brain myelin, Biochem. J., 241 (1987) 759-763. Schacht, J. and Agranoff, B.W., Stimulation of hydrolysis of phosphatidic acid by cholinergic agents in guinea pig syn-
aptosomes, J. Biol. Chem., 249 (1974) 1551-1557. 38 Sekar, M.C. and Hokin, L.E., The role of phosphoinositides i~ signal transduction, J. Membr. Biol., 89 (1986) 193-210. 39 Sheltawy, A. and Dawson, R.M.C., The metabolism of polyphosphoinositides in hen brain and sciaticnerve, Biochem. J., 111 (1969) 157-165. 40 Steck, AoJ. and Appel, S.H., Phosphorylation of myelin basic protein, I. Biol. Chem., 249 (1974) 5416-5420. 41 Suzuki, K., Myelin-associated enzymes. In N. Baumann (Ed.), Neurological Mutations Affecting Myelination, Elsevier, Amsterdam, 1980, pp. 333-347. 42 Turner, R.S., Chou, C.H.J., Kibler, R.F. and Kuo, J.F., Basic protein in brain myelin is phosphorylated by endogenous phospholipid-sensitive Ca2+-dependent protein kinase, J. Neuroche~z., 39 (1982) 1397-1404. 43 Yandrasitz, J.R., Berry, G., Cipriano, V.M., Hwang, S.M. and Segal, S., Effect of elevated potassium on phospholipid and inositol metabolism of isolated nerve endings, Neurochem. Int.,9 (1986) 295-304.
357
BRE 22590
Short Communications
Stimulation of phosphoinositide hydrolysis in myelin by muscarinic agonist and potassium Jorge N. Larocca, Agostino Cervone and Robert W. Ledeen Departments of Neurology and Biochemistry, Albert Einstein College of Medicine, Bron.~, NY 10461 (U.S.A.)
(Accepted 11 August 1987) Key words: Myelin; Phosphoinositide; Muscarinic receptor; Myelin phosphoinositide
Slices of rat brainstem that had been prelabeled by in vivo injection of [3H]inositol were stimulated with carbachol in the presence of lithium and changes measured in the radioactivity of inositol lipids and water-soluble inositol phosphates. For the latter, significant increases were seen for inositol mono- and bisphosphate but not inositol trisphosphate. Analysis of whole tissue phosphoinositides revealed significantly reduced radioactivity in phosphatidylinositorl and phosphatidylinositol 4-phosphate, whereas myelin showed decreases in those as well as phosphatidylinositoi 4,5-bisphosphate. These effects were blocked by atropine. Stimulation of the tissue slices with elevated K + resulted in increased formation of inositol phosphate and decreased radioactivity in phosphatidylinositol. The effect was not blocked by atropine and in the presence of thins agent, which reduced background reaction, all 3 phosphoinositides showed significant K+-induced loss of label. Elevated K+ and carbachol thus function through different mechanisms in this system. Carbaehol is believed to affect myelin phosphoinositides through direct interaction with muscarinic receptors which were recently shown to be present in this membrane. The role of myelin as facilitator of saltatory conduction has made it a membrane of considerable interest to neuroscientists. Although previously thought to be metabolically inert, it is now viewed in a somewhat different light owing to the discovery of numerous intrinsic enzymes involved in lipid metabolism, ion transport, phosphorylation/dephosphorylation, protein degradation, etc (for review see refs. 25, 34, 41). The recent discovery of high affinity muscarinic cholinergic receptors in myelin purified from rat brainstem 24 provides further indication of an active membrane. Those experiments pointed to the presence of both M l and M 2 rece t ubtypes in myelin and thus raised the possibility of linkage to phosphoinesitide metabolism. Brain as a whole contains large amounts of the substrates and enzymes of inositol lipid turnover ~2a4 and the same appears to be true of myelin ~tself. Thus the latter has been reported to be highly enriched in phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol
(Pl) 10"18"34, and to contain phosphoesterase and kinase activities involved in their metabolism 7'9. We have sought to test the possibility that these myelin-localized receptors control inositol lipid metabolism within the membrane by stimulating prelabeled white matter slices with a cholinergic agonist and looking for evidence of enhanced breakdown of myelin phosphoinositides. This was done by measuring decreases in inositol lipid label as well as increases in inositol phosphate products. We have also studied the effects of high potassium on the metabolism of these lipids. Preliminary reports of this work have been pre~,nted 22"23. In a typical experiment, four 21-day-old S p r a g u e Dawley rats were injected intracerebrally with [23H]myoinositol (15 Ci/mmol, American Radiolabeled Chemicals, St. Louis, MO); 25/~Ci in 5 #! saline was injected into each hemisphere (50~Ci total). After approximately 16-18 h the animals were decapitated and the brains rapidly removed and dissected in the cold. Brainstems were cross-chopped into 350 # m
Correspondence: R.W. Ledeen, Departments of Neurology and Biochemistry. Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A.
0006-8993/87/$03.50 @') 1987 Elsevier Science Publishers B.V. (Biomedical Division)
358 slices with a tissue chopper and the slices transferred to 10 ml Erlenmeyer flasks. Each flask contained slices from one-half brainstem. These were incubated in 4 ml of modified Krebs-Ringer bicarbonate (KRB) with the following composition (mM); NaCI 106, KCI 4.7, KH2PO4 1.2, Na,SO4 1.2, MgCI2 1.2, LiCI 10, CaCI 2 1.3, N a H C O 3 25, glucose 11, inositol 5; this was equilibrated with O~_/CO2 (19:1 v/v) to a final pH of 7.4. The slices were gently agitated in a shaking water bath at 37 °C for 1 h, in the presence or absence of 2 mM carbachol. Some samples were preincubated 5 min in the presence of 10/~M atropine before addition of carbachol. In studies on the effect of K ÷, 60 mM of the NaCI was replaced by an equivalent amount of KCI in the modified KRB medium (4 ml per flask). In other experiments designed to test the effect of atropine during K ÷ depolarization, the brainstem slices were first incubated for 5 min in 3 ml of regular KRB buffer containing 10 ~M atropine, followed by addition of 3.1 ml of modified KRB with 101~M atropine in which all the NaCI was isotonically replaced with KCI (final KCI = ca. 60 mM). This was reincubated at 37 °C for 1 h. The tissue suspensions were centrifuged 5 min at 1000 rpm in a HG-4L Sorvall rotor and the resulting supernatant discarded. The pellet was homogenized in 4 ml of 0.30 M sucrose buffered with 20 mM Tris-HCl (pH 9.5) and aliPIP ~S
40-
30PIP2
till
PI
~, 20 u i11
r.,
10
0
kT.J
-10 ~J Fig. 1. Effects of carbachol and carbachol plus atropine on whole tissue phosphoinositides. Brainstem slices previously labeled with [3H]inositol were incubated 1 h at 37 °C with either added buffer, carbachol (2 mM) or carbachol (2 mM) plus atropine (10 ltM). The atropine was added 5 min before stimulation with carbachol. Radioactivity levels in whole tissue phosphoinositides were determined as described in the text. Results are expressed as % of decrease with respect to control; the amounts counted corresponded to approximately 200 l~g of tissue protein. Each bar in the histogram represents the mean + S.E.M. of 4-8 experiments. Open bars represent atropine experiments. *P < 0.05 (Student's two-tailed t-test).
40
-
PIP2
PIP
PI
30--
20
,N ~o 0
-lO -
J
W
Fig. 2. Effects of carbachol and carbachol ph:s atropine on myelin phosphoinositides. Brainstem slices previously labeled with [3H]inositol were incubated under co;lditions described in Fig. 1. The brainstem slices were then homogenized and myelin isolated. Radioactivity levels in myelin phosphoinositides were determined as described in the text. Results are expressed as % decrease with respect to control: the amounts counted corresponded to approximately 120!tg of myelin protein. Each bar in the histogram represents the mean + S.E.M. of 4-6 experiments. Open bars represent atropine experiments. *P < 0.05 (Student's two-tailed t-test). quots removed for protein determination by a modified Lowry procedure 26. One-fourth of the sample was treated with 10% (w/v) trichloroacetic acid (TCA) and the remainder set aside for myelin isolation (see below). The precipitate was centrifuged and the resulting pellet re-extracted with 1 ml of 10% TCA. The combined supernatants were extracted 4 times with an equal volume of ethyl ether to remove TCA and then applied to a Dowex 1 x 8-400 column for separation of inositol phosphates 3. The pellet was extracted with acidified chloroform-methanol according to the entire procedure of Bell et al. 1, except that 0.2 mg/ml of butylated hydroxytoluene was present as antioxidant. The lipid extract was evaporated to dryness with N, and redissolved in 0.3 ml of the solvent employed for TLC: c h l o r o f o r m - m e t h a n o l - w a t e r - c o n c . NH 3 (90:90:22:7, v/v). Aliquots were applied to Merck silica gel 60 HPTLC plates, 10 cm x 20 cm ( V W R Scientific, South Plainfield, N J), and the plates developed with the above solvent in paper-lined tank. Standards of PI, PIP, and PIP 2 (Sigma, St. Louis, MO) were co-chromatographed for identification. The iipids were detected with I2 vapor and bands parallel to the standards were scraped and counted. The remaining three-fourths of the sample (see above) was employed for myelin isolation according to Haley et al. 17. This is based on the procedure of
359 IP
40-
showing the greatest decrease (ca. 30-35%) with PI and PIP 2 radiolabel decreasing about half as much (Fig. 1). The change recorded for the latter lipid was not significant. Absolute levels of radioactivity for PIP.,, PIP, and PI in control samples were 476, 1140, and 12,600 dpm/mg protein, respectively. A somewhat different pattern was observed in the myelin subsequently isolated from brainstem after carbachoi stimulation (Fig. 2) in that all 3 phosphoinositides showed significant loss of radioactivity (ca. 2028%). Absolute counts for myelin PIP,, PIP and PI in control samples were 896,482 and 7220 dpm/mg protein, respectively. As seen in Figs. 1 and 2, these hydrolytic reactions were effectively blocked by the presence of 10 IbM atropine in the incubation medium. The apparent "over-compensation" seen with atropine was possibly a reflection of the phosphoinositide breakdown occurring in control sampies which would be diminished by this agent. Further evidence for stimulated hydrolysis came from analysis of inositol phosphates isolated from whole brainstem (Fig. 3). The increase (32%) in radioactivity of inositol phosphate (IP) was approximately twice that of inositol bisphosphate (IP2), while the change for inositol trisphosphate (IP3) was not significant. Absolute levels of radioactivity for IP3, IP2 and IP in control samples were 173, 1570, and 3820 dpm/mg protein, respectively. As with the lipids, the effect was blocked by atro: ine. A somewhat similar pattern of inositol phosphate release was reported with rat cerebral cot-
30 IP2
u., 2O u Z
10
IPa ,,,
W
-I0I -20
Fig. 3. Effect of carbachol and carbachoi plus atropine on accumulation of inositol phosphates. Brainstem slices previously labeled with [3Hlinositol were incubated under conditions described in text and Fig. 1. lnositol phosphates were separated by anion exchange chromatography. Results are expressed as % of increase with respect to control; the amounts counted corresponded to approximately 500 !~g of tissue protein. Each bar in the histogram represents the mean + S.E.M. of 4-8 experiments. Open bars represent atropine experiments. *P < 0.05 (Student's two-tailed t-test).
Norton and Poduslo 33 with an additiona] floating up gradient for higher purity ~7. To the labeled tissue was added an approximately equivalent amount of unlabeled brains:.em. Lipids were extracted and chromatographed from the resulting myelin as described above. The results indicated that carbachol enhanced phosphoinositide hydrolysis in whole brainstem. PIP A
B PIP2
40 LU Or)
< 30 LU n" rO
,,, 20 Q
;P
il"
PIP
PI
IP3
IP2
IP
-
40
LU
T'k
-30
< ILl re tO
-.~20 Z
lO
~--] WITHATROPINE
~ NOATROPINE
Fig. 4. Effects of elevated K + on phosphoinositide hydrolysis. Brainstem slices previously labeled with [~H]inositol were incubated 1 h at 37 °C vdth either buffer or buffer containing 60 m'M KCI. Atropine when present (It) !4M) was added 5 rain before elevation of K-. A: decrease of label in mx'elin phosphoinositides. B: increase of radiolabeled inositol phosphates. The results are expressed as the q change with respect to control: the amounts counted corresponded to approximately 120.ug of myelin protein for phosphoinositides and to 500 !~g of tissue protein for inositol phosphates. Each bar in the histogram represents the mean + S.E.M. of 4-7 experiments. *P < 0.05 (Student's two-tailed t-test).
360 tex slices following carbachol stimulation-. Inositol phosphate patterns reflect not only the products of phosphoinositide hydrolysis but also the conversion via phosphatase to forms with less phosphate; the presence of Li ÷ blocks further hydrolysis beyond inositol monophosphate 3. Treatment of the pre-labeled brainstem slices with elevated K ÷ also caused enhanced breakdown of phosphoinosltidc~, as seen in the significant elevation of released radiolabeled iP ~,~t:~,,.,~4). IP-_ and IP3 remained unchanged and atropine was without effect on IP formation. Among the phosphoinositides only PI showed significant loss of radiolabel in the absence of atropine but all 3 were significantly decreased in the presence of this blocking agent. Absolute counts for inositol phosphates and myelin PIP: in control samples were approximately 65-100% higher than those mentioned above while those for PIP and PI were similar. As mentioned, atropine is seen as reducing the level of background activity and since it does not block K + stimulation the magnitud~ of this effect is considered more meaningful in its presence. The inositol phosphates liberated through carbachol, as well as the phosphoinositide changes observed in whole tissue slices, very likely reflected the results of cholinergic stimulation of diverse elements in the brainstem. However, in considering myelin as a specifically targeted membrane, the fact that inositol phospholipids of this structure showed significant loss of radioactivity following carbachol treatment is strongly suggestive of effector-mediated phosphoinositide breakdown in that membrane• Considering that phosphoinositide hydrolysis would not likely occur in one membrane as a result of receptor activation in another (although that may be theoretically possible through some kind of exchange process), the most plausible mechanism to explain our data is direct stimulation of the above-mentioned muscarinic receptor in myelin itself. We believe the same interpretation can be applied to the recent abstract report 21 that cholinergic stimulation of rat cortical brain slices caused enhanced incorporation of 32p into phosphatidic acid and phosphoinositides of myelin. Work in pr6gress in our laboratory (Golly, Larocca and Ledeen, in preparation) has indicated breakdown of prelabeled phosphoinositides of myelin through direct carbachol stimulation of isolated myelin• Earlier work 8,39 had demonstrated the •
"
"~0
rapidity with which 32pi is incorporated into phosphoinositides of myelin, either in vivo or in vitro. Studies by Deshmukh and coworkers 7 pointed to the presence in myelin of phosphoinositide phosphodiesterase, the key enzyme activated by agonist-receptor interaction, along with phosphomonoesterases and phosphoinositide kinases 7. One of the latter enzymes, PI kinase, was recently isolated from bovine CNS myelin 36. Such enzymes present in other brainstem components could account for the different patterns of phosphoinositide depletion noted in the present study. For example, failure of PIP2 in brainstem slices to show significant loss of radiolabel (Fig. 1) might conceivably result from compensating resynthesis from PIP via PIP kinase. Alternatively, the data might reflect the presence of a specific phosphodiesterase for PIP; a number of studies have indicated the presence of different phospholipase C activities that catalyze direct breakdown of PI and PIP 27. Either of these mechanisms could also be operative in myelin which showed equivalent loss of all 3 phosphoinositides. Although the pattern of myelin phosphoinositide depletion was sinn|ar for elevated K + as for carbachol, the mechanisms were apparently different as shown by the opposite responses to atropine. Elevated K ÷ and carbachol were previously shown to facilitate phosphoinositide turnover, sometimes in synergistic fashion, in brain slices and isolated nerve endingsll.16.19,37.43. The physiological significance of the receptorlinked phosphoinositide system in myelin is not known. While in brain such systems have been ascribed primarily to neurons 15, recent work has shown their presence in several glial cell preparations as well 5'6'28'3°'35. A key feature of this phenomenon is phosphodiesterase cleavage of PIP 2 to form two second messengers, IP 3 and diacylglycerol 2,32.38. It is not clear at present how messenger-activated release of Ca 2÷ (via IP3) might occur in myelin, although the presence of smooth endoplasmic reticulum has been noted in the adaxonal components of myelin (e.g. lateral loops) 13. A possible role for diacylglycerol is easier to visualize since the kinase which it activates is known to be present in myelin and to utilize the myelin basic proteins as substrates 4,29,31,4°,42. Further study of the way in which phosphoinositides mediate
361 signal transduct:.on in myelin should provide additional insight into the metabolic properties of this membrane.
( R . W . L . ) , and National Multiple Sclerosis Society Grant RG 1941-A-1 (J.N.L.). We are happy to acknowledge receiving helpful suggestions from by Dr. Maynard Makman.
This work was supported by N I H Grant NS-16181
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32 33 34 35
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37
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