0022-3042~79/0501-159YY02OO/O
Journal of Neumchamialr) Val. 32. pp. IS99 to 1601 Pergamon Press Ltd 1979. Printed in Great Britain Q
International Society lor Neurochemistry Ltd
SHORT COMMUNICATION Post-synaptic PI-effect of nerve growth factor in rat superior cervical ganglia (Received 11 September 1978. Revised 7 December 1978. Accepted 19 December 1978)
NEUROTRANSMITTERS, hormones and drugs are known to GANG, N.I.H. Acetylcholine chloride was purchased from elicit an increased turnover or phosphorylinositol group Sigma. Rats of Sprague-Dawley strain were used. SCG removed from 10 day old rats were desheathed of phosphatidylinositol (PI) in various tissues (HAWTHORNE, 1973: MICHELL, 1975). This effect is often rcferred and maintained in organ culture at 37°C for 10 h as described earlier (LAKSHMANAN, 1978). Briefly, three pairs of to as ‘PI-effect’ or ‘inositide-effect’. Recently, nerve growth SCG (per well) were placed in 250p1 of BGJb medium. factor (NGF) has been shown to elicit a PI-effect in rat supcrior cervical ganglia (SCG) (LAKSHMANAN, 1978). NGF Fitton-Jackson modification without phcnol red. The is a protein which is known to play a key role in the medium was supplemented with 0.1% bovine serum d b u growth, development and maintenance of sympathetic min, Fraction V, and an antibiotic mixture which included penicillin (100 units/ml) and streptomycin (50 pg/ml). neurones through all ages of animal life (LEVI-MONTALCINI & ANGELETTI,1968; VARON,1975). The PI-effect elicited Freshly prepared ascorbic acid (0.1 mg/ml) and glutamine by NGF in rat SCG appears to be a receptor specific event (2 mM) were added just before use. All drugs except QNB since (a) no other hormone could substitute for NGF, (b) were added just before the addition of buffer or NGF in M while NGF-induced PI-effect is sensitive to NGF-antiserum, (c) 5 pI volume to a final concentration of 5 x the phenomenon is observed in the physiological range QNB was 5 x lo-’ M. At the end of incubation the ganglia of NGF concentration, and (d) a high concentration of were removed and washed with 2 m l of ice-cold 0.9% growth hormone which is known to inhibit the binding saline. Pairs of ganglia were then homogenized in 4 ml of NGF to membrane preparation of rabbit sympathetic chloroform- methanol mixture (2: I v/v). The lipid extract ganglia decreased the NGF-induced PI-turnover. The was washed 4 times with 0.2 vol of 0.9% containing NGF-receptor associated PI-effect is observed as early as M-unlabelled myoinositol. Finally, the organic layer 4 h using [”P]orthophosphate as a precursor to study the was transferred to scintillation vials and then dried under turnover of phosphatides and the effect appears to be more a stream of nitrogen. Fifteen millilitres of scintillation cocktail (5 g PPO and 0.5 g POPOP in 1000 ml toluene) specific in that NGF did not stimulate the incorporation of labelled orthophosphate into phosphatides other than was added before counting. A duplicate sample of the phosphatidylinositol (LAKSHMANAN, 1978). One of the ganglion-lipid extract was mixed with rat brain synaptospecific effects of NGF-antiserum in young animals soma1 lipid fraction (0.54.75pmol lipid P) as a carrier appears to be a blockade of synaptic transmission (LARRA- and then subjected to two dimensional thin layer chromaBEE, 1972). Furthermore, the ability of exogenous NGF to tography as described earlier (LAKSHMANAN, 1978). The prevent the synaptic depression after axotomy suggests a majority of the radioactivity (95S98%) was found to be normal physiological role of NGF in synaptic transmission present in the phosphatidylinositol fraction. The radio(PURVES activity in PI was expressed as d.p.m./pair of ganglia, com& NJA. 1976). These studics prompted the present investigation on the site (pre- or post-synaptic) at which puted using an internal standard. For studying the site of NGF-induced stimulation of NGF elicits the PI-effect, a phenomenon related to permeability changes accompanying action-potential (DURELL PI-turnover, bilateral decentralization of SCG was perel ul., 1969; HAWTHORNE & KEMP.1964). formed at 28 days before the rats were taken for the experiment. Male rats (50-60g) were anaesthetized with pentobarbital (20 mg/kg., i.p). A short length of preganglionic MATERIALS AND METHODS nerve (0.5 mm) was removed approx 5 mm below the gangNGF, 2-5s form, was prepared by the method of Boc- lion. The success of the operation was determined by the appearance of ptosis on both sides when the rats awakCHINI & ANGELETTI(1969). Culture medium and antibiotics ened. Further proof of denervation was assessed by were obtained from Grand Island Biological Co., New York. P 3 H IMyoinositol (2 Ci/mmol) was purchased from measuring the acetylcholine esterase activity on the day the Radiochemical Centre, Amersham. Atropine, quinucli- of the experiment. Usually, 4 pairs of sham-operated and dinylbenzilate (QNB), d-tubocurarine, chlorisandamine denervated ganglia were pooled separately, desheathed, and hexomethonium were kindly provided by Dr. WOLF- and freed of attached lengths of nerve. Pairs of ganglia were homogenized in 400 pl of ‘homogenizing buffer’ [lo0 mm-Tris (hydroxymethyljaminomethane (Tris) buffer, Abbreciations used: PI, phosphatidylinositol; PI-effect, pH 7.4 containing 1% Triton X-100 (v/v)], at 4°C using increased turnover of phosphorylinositol group of phos- a glass-glass tissue homogenizer. The homogenates of phatidylinositol: NGF, nerve growth factor; SCG, superior ganglia were assayed for acetylcholine esterase activity by determining the amount of acetylcholine hydrolyzed in the cervical ganglia; QNB, quinuclidinylbenzilate. 1599
1600
Short communication
reaction mixture. The reaction mixture contained 4 mMacetylcholine chloride. 6 mM-MgCI, and 40 mM-Tris-HCI buffer, pH 7.4. In general the detergent-solubilized protein, equivalent to 200pg protein, was added and the final volume was 1 ml. After 1Omin of incubation at 3 7 T , the acetylcholine was determined according to the calorimetric hydroxamic acid method of HESTRIN(1949). The protein was measured according to LOWRYet a/. (1951), against a standard solution of bovine serum albumin. The specific activity of acetylcholine esterase is defined as pmol of acetylcholine hydrolyzed/h/mg of detergent-solubilized proteins. RESULTS AND DISCUSSION The addition of N G F ( 5 x lo-' M) increased the incorporation of labelled myoinositol into PI by 155% of control in rat SCG (Fig. I). However, this could not be blocked by the muscarinic antagonists atropine (5 x M) or QNR ( 5 x to-' M) or by the nicotinic antagonists such as d-tubocurarine (5 x M) or chlorisandamine (5 x M) used. Hexomothonium ( 5 x lo-' M), a ganglionic blocking agent, also did not prevent the PIeffect of NGF. It appears from the results that both muscarinic- and nicotinic-cholinergic receptors of rat SCG are not involved in mediating the PI-effect of NGF. This is in distinct contrast to the reported property of stimulated PI-breakdown in rat SCG by electrical stimulation of preganglionic nerve, which is known to liberate acetylcholine (LARRABEE & LEICHT,1965; PICKARD et al., 1977). However, none of the ganglion-blocking drugs at the concentrations used in the present study had any effect on PIturnover in the absence of NGF. During the course of the investigation an increase of NGF-mediated inositideeffect was noticed in the presence of atropine. This problem is currently under study. In order to ascertain the site at which NGF elicits the stimulation of PI-turnover, SCG from decentralized rats were used. Since preganglionic denervation of SCG in rats was reported to decrease the activity of acetylcholine esteref al., 1976), the activity of this enzyme ase (FUMAGALLI was measured in SCG of decentralized and sham-operated rats. The acetylcholine esterase activity in decentralized SCG was 17.8 f 1.8pmol/h/mg protein while that of sham-operated SCG was 35.1 2.2 pmol/h/mg protein. These data on the decrease in acetylcholine esterase activity observed after decentralization are in accordance
A
B
C
D
E
F
+
+
et al. (1976). These with the experiments of FUMAGALLI results together with the persistence of ptosis in the operated animals cstablish that the decentralization procedure was successful. The results presented in Table 1 provide evidence that N G F elicits an inositide-effect similar to that in shamoperated animals. Thus it appears that the post-synaptic structures are the primary locus at which NGF elicits the PI-effect in rat SCG. Electrical stimulation of preganglionic cholinergic nerve & LEICHT,1965; PICKARD et al., 1977) fibers (LARRABEE
TABLE1. NGF-INDUCEDPHOSPHATIDYLINOSITOL TURNOVER;
EFFECT OF SURGICAL
DENERVATION
Addition to cu 1t ure None NGF
G
FIG. 1. Effect of ganglion blocking agents on nerve growth Factor-induced phosphatidylinosilol turnover in rat superior cervical ganglia. Three pairs of superior cervical ganglia (per well) of 10 day old rats were organ cultured in 250pl of BGJb medium, 2.5s NGF (5 x 1 0 - ' ~ ) and [2-3H]myoinositol (1 5 pCi/well) were added simultaneously. AIL drugs except QNB were added before the addition of buffer or N G F in 5 pl vol to a final concentration of 5 x M, while QNB was 5 x ~ O - ' M . For other details see text. Results were expressed as d.p.m./pair of ganglia. Each bar S.E. of at least 16 pairs of ganglia. represents the mean * P < 0.001, @ P < 0.01 compared to control as determined by Student's t test. A = control? B = NGF, C = N G F + atropine, D = NGF + QNB, E = N G F + tubocurarine, F = N G F chlorisandamine, G = N G F hexamethonium. None of the ganglion-blocking agents have any effect on PI-turnover in the absence of NGF.
Radioactivity in phosphatidylinositol (d.p.m./pair SCG) ShamBilaterallyoperated decentralized 264 & 22 (100%) 401 k 30 (151%)*
230 361
28 (100%)
k 18 (156%)*
Thirty-two male rats (5&60 g) either sham-operated or bilaterally decentralized, werc used 28 days after the operation. Pairs of ganglia (1 pair per well) were cultured in 250pl of BGJb medium in the absence or presence of N G F ( 5 x 1 0 - ' ~ ) . The incubation was continued in the presence of 12.5 pCi of [2-3H]myoinositol for 10 h at 37°C. A t the end of incubation the ganglia were removed, washed, and processed as described in text. Each value represents the mean & S.E. from 6 pairs of superior cervical ganglia. * P < 0.01 compared to their respective controls.
Short communication
1601
HAWTHORNE J. N. & KEMPP. (1964) The brain phosphoinositides. Adu. Lipid. Res. 2, 127-166. HESTRIN S. (1949) The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application. J . hiol. Chem. 180, 249-260. HOKINM. R., HOKINL. E. & SHELPW. D. (1960) The effect of acetylcholine on the turnover of phosphatidic acid and phosphoinositide in sympathetic ganglia and in various parts of the central nervous system in tjirro. J . Gen. Physiol. 44, 217-226. LAKSHMANAN J. (1978) Nerve growth factor induced turnover of phosphatidylinositol in rat superior cervical Acknowledgements-I would like to thank Dr. WOLFGANG ganglia. Biochem. biophys. Res. Commun. 82. 767-775. M. G. (1972) Early effects of antiserum to the for the gift of drugs, Dr. SRINATH for the supply of surgi- LARRABEE cally denervated rats, Drs. PADMANABAN and JAMALUDDIN nerve growth factor on ganglion metabolism and synapfor critical reading of the manuscript, Dr. BALAKAM for tic transmission, in Nerue Growth Factor and its Antihis interest and CSIR for the financial support. serum (ZAMAB E. & KNIGHT .I. eds.) , pp. 177-184. The Athelone Press of University of London. LARRABEE M. G. & LEICHTW. S. (1965) Metabolism of Molecular Biophysics Unit, J. LAKSHMANAN phosphotidylinositol and other lipids in active neurones Indian Institute of Science, of sympathetic ganglia and other peripheral nervous tisBangalore-560 012, sues. The site of inositidc effect. J . Neurochem. 12, 1-13. India LEVI-MONTALCINI R. & ANGELETTI P. u. (1968) Nerve growth factor. Physiol. Rev. 48, 534-569. REFERENCES LOWRY0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol BOCCHINI v. & ANGELETTI P. u. (1969) The nerve growth reagent, J. biol. Chem. 193, 265-275. factor purification as a 30,000 molecular weight protein. Proc. natn. Acud. Sci., U.S.A. 64, 787-794. MICHELLR. H. (1975) Inositol phospholipids and cell surDURELL J., GARLAND T. J. & FRIEDEL R. 0.(1969) Acetylface receptor function. Biochim. biophys. Acta. 415, choline action: biochemical aspects. Science 165, 81-147. M. R., HAWTHORNE J. N., HAYASHI E. & YAMADA 862-866. PICKARD FUMAGALLI L., DERENZIS G. & NIANIN. (1976) AcetylchoS. (1977) Effects of surugatoxin and other nicotinic and line receptors: number and distribution in intact and muscarinic antagonists on phosphatidylinositol metahdeafferentiated superior cervical ganglion of the rat. J . o h m in active sympathetic ganglia. Biuchem. Pharmuc. Neurochem. 27, 47-52. 26, 448450. PURVES D. & NJA D. (1976) Effects of nerve growth factor HAWTHORNE J. N. (1973) Phospholipid metabolism and transport of materials across cell membrane, in Form on synaptic transmission after axotomy. Nature, Lond. and Function of Phospholipids (ANSELLG. B., DAWSON 260, 535-536. R. M. C. & HAWTHORNE J. N., eds.) pp. 423-440. Else- VARON S. (1975) Nerve growth factor. Expl Neurol. 48, 75-92. vier, Amsterdam.
and addition of acetylcholine (HOKINe f al., 1960) have been shown to elicit a PI-effect in rat SCG through specific receptors on the post-synaptic structures. Although N G F appears to bring about a similar increase in PI turnover, independently of neurotransmitter (acetylcholine), the phenomenon does occur on the post-synaptic structures. This property of N G F is in common with the reported property of acetylcholine in this tissue. Further work is in progress to elucidate whether N G F acts on the cell body or dendrites in SCG and the functional significance of NGF-induced PI-effect in this target tissue.
Journal of Neumchamialr) Val. 32. pp. IS99 to 1601 Pergamon Press Ltd 1979. Printed in Great Britain Q
International Society lor Neurochemistry Ltd
SHORT COMMUNICATION Post-synaptic PI-effect of nerve growth factor in rat superior cervical ganglia (Received 11 September 1978. Revised 7 December 1978. Accepted 19 December 1978)
NEUROTRANSMITTERS, hormones and drugs are known to GANG, N.I.H. Acetylcholine chloride was purchased from elicit an increased turnover or phosphorylinositol group Sigma. Rats of Sprague-Dawley strain were used. SCG removed from 10 day old rats were desheathed of phosphatidylinositol (PI) in various tissues (HAWTHORNE, 1973: MICHELL, 1975). This effect is often rcferred and maintained in organ culture at 37°C for 10 h as described earlier (LAKSHMANAN, 1978). Briefly, three pairs of to as ‘PI-effect’ or ‘inositide-effect’. Recently, nerve growth SCG (per well) were placed in 250p1 of BGJb medium. factor (NGF) has been shown to elicit a PI-effect in rat supcrior cervical ganglia (SCG) (LAKSHMANAN, 1978). NGF Fitton-Jackson modification without phcnol red. The is a protein which is known to play a key role in the medium was supplemented with 0.1% bovine serum d b u growth, development and maintenance of sympathetic min, Fraction V, and an antibiotic mixture which included penicillin (100 units/ml) and streptomycin (50 pg/ml). neurones through all ages of animal life (LEVI-MONTALCINI & ANGELETTI,1968; VARON,1975). The PI-effect elicited Freshly prepared ascorbic acid (0.1 mg/ml) and glutamine by NGF in rat SCG appears to be a receptor specific event (2 mM) were added just before use. All drugs except QNB since (a) no other hormone could substitute for NGF, (b) were added just before the addition of buffer or NGF in M while NGF-induced PI-effect is sensitive to NGF-antiserum, (c) 5 pI volume to a final concentration of 5 x the phenomenon is observed in the physiological range QNB was 5 x lo-’ M. At the end of incubation the ganglia of NGF concentration, and (d) a high concentration of were removed and washed with 2 m l of ice-cold 0.9% growth hormone which is known to inhibit the binding saline. Pairs of ganglia were then homogenized in 4 ml of NGF to membrane preparation of rabbit sympathetic chloroform- methanol mixture (2: I v/v). The lipid extract ganglia decreased the NGF-induced PI-turnover. The was washed 4 times with 0.2 vol of 0.9% containing NGF-receptor associated PI-effect is observed as early as M-unlabelled myoinositol. Finally, the organic layer 4 h using [”P]orthophosphate as a precursor to study the was transferred to scintillation vials and then dried under turnover of phosphatides and the effect appears to be more a stream of nitrogen. Fifteen millilitres of scintillation cocktail (5 g PPO and 0.5 g POPOP in 1000 ml toluene) specific in that NGF did not stimulate the incorporation of labelled orthophosphate into phosphatides other than was added before counting. A duplicate sample of the phosphatidylinositol (LAKSHMANAN, 1978). One of the ganglion-lipid extract was mixed with rat brain synaptospecific effects of NGF-antiserum in young animals soma1 lipid fraction (0.54.75pmol lipid P) as a carrier appears to be a blockade of synaptic transmission (LARRA- and then subjected to two dimensional thin layer chromaBEE, 1972). Furthermore, the ability of exogenous NGF to tography as described earlier (LAKSHMANAN, 1978). The prevent the synaptic depression after axotomy suggests a majority of the radioactivity (95S98%) was found to be normal physiological role of NGF in synaptic transmission present in the phosphatidylinositol fraction. The radio(PURVES activity in PI was expressed as d.p.m./pair of ganglia, com& NJA. 1976). These studics prompted the present investigation on the site (pre- or post-synaptic) at which puted using an internal standard. For studying the site of NGF-induced stimulation of NGF elicits the PI-effect, a phenomenon related to permeability changes accompanying action-potential (DURELL PI-turnover, bilateral decentralization of SCG was perel ul., 1969; HAWTHORNE & KEMP.1964). formed at 28 days before the rats were taken for the experiment. Male rats (50-60g) were anaesthetized with pentobarbital (20 mg/kg., i.p). A short length of preganglionic MATERIALS AND METHODS nerve (0.5 mm) was removed approx 5 mm below the gangNGF, 2-5s form, was prepared by the method of Boc- lion. The success of the operation was determined by the appearance of ptosis on both sides when the rats awakCHINI & ANGELETTI(1969). Culture medium and antibiotics ened. Further proof of denervation was assessed by were obtained from Grand Island Biological Co., New York. P 3 H IMyoinositol (2 Ci/mmol) was purchased from measuring the acetylcholine esterase activity on the day the Radiochemical Centre, Amersham. Atropine, quinucli- of the experiment. Usually, 4 pairs of sham-operated and dinylbenzilate (QNB), d-tubocurarine, chlorisandamine denervated ganglia were pooled separately, desheathed, and hexomethonium were kindly provided by Dr. WOLF- and freed of attached lengths of nerve. Pairs of ganglia were homogenized in 400 pl of ‘homogenizing buffer’ [lo0 mm-Tris (hydroxymethyljaminomethane (Tris) buffer, Abbreciations used: PI, phosphatidylinositol; PI-effect, pH 7.4 containing 1% Triton X-100 (v/v)], at 4°C using increased turnover of phosphorylinositol group of phos- a glass-glass tissue homogenizer. The homogenates of phatidylinositol: NGF, nerve growth factor; SCG, superior ganglia were assayed for acetylcholine esterase activity by determining the amount of acetylcholine hydrolyzed in the cervical ganglia; QNB, quinuclidinylbenzilate. 1599
1600
Short communication
reaction mixture. The reaction mixture contained 4 mMacetylcholine chloride. 6 mM-MgCI, and 40 mM-Tris-HCI buffer, pH 7.4. In general the detergent-solubilized protein, equivalent to 200pg protein, was added and the final volume was 1 ml. After 1Omin of incubation at 3 7 T , the acetylcholine was determined according to the calorimetric hydroxamic acid method of HESTRIN(1949). The protein was measured according to LOWRYet a/. (1951), against a standard solution of bovine serum albumin. The specific activity of acetylcholine esterase is defined as pmol of acetylcholine hydrolyzed/h/mg of detergent-solubilized proteins. RESULTS AND DISCUSSION The addition of N G F ( 5 x lo-' M) increased the incorporation of labelled myoinositol into PI by 155% of control in rat SCG (Fig. I). However, this could not be blocked by the muscarinic antagonists atropine (5 x M) or QNR ( 5 x to-' M) or by the nicotinic antagonists such as d-tubocurarine (5 x M) or chlorisandamine (5 x M) used. Hexomothonium ( 5 x lo-' M), a ganglionic blocking agent, also did not prevent the PIeffect of NGF. It appears from the results that both muscarinic- and nicotinic-cholinergic receptors of rat SCG are not involved in mediating the PI-effect of NGF. This is in distinct contrast to the reported property of stimulated PI-breakdown in rat SCG by electrical stimulation of preganglionic nerve, which is known to liberate acetylcholine (LARRABEE & LEICHT,1965; PICKARD et al., 1977). However, none of the ganglion-blocking drugs at the concentrations used in the present study had any effect on PIturnover in the absence of NGF. During the course of the investigation an increase of NGF-mediated inositideeffect was noticed in the presence of atropine. This problem is currently under study. In order to ascertain the site at which NGF elicits the stimulation of PI-turnover, SCG from decentralized rats were used. Since preganglionic denervation of SCG in rats was reported to decrease the activity of acetylcholine esteref al., 1976), the activity of this enzyme ase (FUMAGALLI was measured in SCG of decentralized and sham-operated rats. The acetylcholine esterase activity in decentralized SCG was 17.8 f 1.8pmol/h/mg protein while that of sham-operated SCG was 35.1 2.2 pmol/h/mg protein. These data on the decrease in acetylcholine esterase activity observed after decentralization are in accordance
A
B
C
D
E
F
+
+
et al. (1976). These with the experiments of FUMAGALLI results together with the persistence of ptosis in the operated animals cstablish that the decentralization procedure was successful. The results presented in Table 1 provide evidence that N G F elicits an inositide-effect similar to that in shamoperated animals. Thus it appears that the post-synaptic structures are the primary locus at which NGF elicits the PI-effect in rat SCG. Electrical stimulation of preganglionic cholinergic nerve & LEICHT,1965; PICKARD et al., 1977) fibers (LARRABEE
TABLE1. NGF-INDUCEDPHOSPHATIDYLINOSITOL TURNOVER;
EFFECT OF SURGICAL
DENERVATION
Addition to cu 1t ure None NGF
G
FIG. 1. Effect of ganglion blocking agents on nerve growth Factor-induced phosphatidylinosilol turnover in rat superior cervical ganglia. Three pairs of superior cervical ganglia (per well) of 10 day old rats were organ cultured in 250pl of BGJb medium, 2.5s NGF (5 x 1 0 - ' ~ ) and [2-3H]myoinositol (1 5 pCi/well) were added simultaneously. AIL drugs except QNB were added before the addition of buffer or N G F in 5 pl vol to a final concentration of 5 x M, while QNB was 5 x ~ O - ' M . For other details see text. Results were expressed as d.p.m./pair of ganglia. Each bar S.E. of at least 16 pairs of ganglia. represents the mean * P < 0.001, @ P < 0.01 compared to control as determined by Student's t test. A = control? B = NGF, C = N G F + atropine, D = NGF + QNB, E = N G F + tubocurarine, F = N G F chlorisandamine, G = N G F hexamethonium. None of the ganglion-blocking agents have any effect on PI-turnover in the absence of NGF.
Radioactivity in phosphatidylinositol (d.p.m./pair SCG) ShamBilaterallyoperated decentralized 264 & 22 (100%) 401 k 30 (151%)*
230 361
28 (100%)
k 18 (156%)*
Thirty-two male rats (5&60 g) either sham-operated or bilaterally decentralized, werc used 28 days after the operation. Pairs of ganglia (1 pair per well) were cultured in 250pl of BGJb medium in the absence or presence of N G F ( 5 x 1 0 - ' ~ ) . The incubation was continued in the presence of 12.5 pCi of [2-3H]myoinositol for 10 h at 37°C. A t the end of incubation the ganglia were removed, washed, and processed as described in text. Each value represents the mean & S.E. from 6 pairs of superior cervical ganglia. * P < 0.01 compared to their respective controls.
Short communication
1601
HAWTHORNE J. N. & KEMPP. (1964) The brain phosphoinositides. Adu. Lipid. Res. 2, 127-166. HESTRIN S. (1949) The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application. J . hiol. Chem. 180, 249-260. HOKINM. R., HOKINL. E. & SHELPW. D. (1960) The effect of acetylcholine on the turnover of phosphatidic acid and phosphoinositide in sympathetic ganglia and in various parts of the central nervous system in tjirro. J . Gen. Physiol. 44, 217-226. LAKSHMANAN J. (1978) Nerve growth factor induced turnover of phosphatidylinositol in rat superior cervical Acknowledgements-I would like to thank Dr. WOLFGANG ganglia. Biochem. biophys. Res. Commun. 82. 767-775. M. G. (1972) Early effects of antiserum to the for the gift of drugs, Dr. SRINATH for the supply of surgi- LARRABEE cally denervated rats, Drs. PADMANABAN and JAMALUDDIN nerve growth factor on ganglion metabolism and synapfor critical reading of the manuscript, Dr. BALAKAM for tic transmission, in Nerue Growth Factor and its Antihis interest and CSIR for the financial support. serum (ZAMAB E. & KNIGHT .I. eds.) , pp. 177-184. The Athelone Press of University of London. LARRABEE M. G. & LEICHTW. S. (1965) Metabolism of Molecular Biophysics Unit, J. LAKSHMANAN phosphotidylinositol and other lipids in active neurones Indian Institute of Science, of sympathetic ganglia and other peripheral nervous tisBangalore-560 012, sues. The site of inositidc effect. J . Neurochem. 12, 1-13. India LEVI-MONTALCINI R. & ANGELETTI P. u. (1968) Nerve growth factor. Physiol. Rev. 48, 534-569. REFERENCES LOWRY0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol BOCCHINI v. & ANGELETTI P. u. (1969) The nerve growth reagent, J. biol. Chem. 193, 265-275. factor purification as a 30,000 molecular weight protein. Proc. natn. Acud. Sci., U.S.A. 64, 787-794. MICHELLR. H. (1975) Inositol phospholipids and cell surDURELL J., GARLAND T. J. & FRIEDEL R. 0.(1969) Acetylface receptor function. Biochim. biophys. Acta. 415, choline action: biochemical aspects. Science 165, 81-147. M. R., HAWTHORNE J. N., HAYASHI E. & YAMADA 862-866. PICKARD FUMAGALLI L., DERENZIS G. & NIANIN. (1976) AcetylchoS. (1977) Effects of surugatoxin and other nicotinic and line receptors: number and distribution in intact and muscarinic antagonists on phosphatidylinositol metahdeafferentiated superior cervical ganglion of the rat. J . o h m in active sympathetic ganglia. Biuchem. Pharmuc. Neurochem. 27, 47-52. 26, 448450. PURVES D. & NJA D. (1976) Effects of nerve growth factor HAWTHORNE J. N. (1973) Phospholipid metabolism and transport of materials across cell membrane, in Form on synaptic transmission after axotomy. Nature, Lond. and Function of Phospholipids (ANSELLG. B., DAWSON 260, 535-536. R. M. C. & HAWTHORNE J. N., eds.) pp. 423-440. Else- VARON S. (1975) Nerve growth factor. Expl Neurol. 48, 75-92. vier, Amsterdam.
and addition of acetylcholine (HOKINe f al., 1960) have been shown to elicit a PI-effect in rat SCG through specific receptors on the post-synaptic structures. Although N G F appears to bring about a similar increase in PI turnover, independently of neurotransmitter (acetylcholine), the phenomenon does occur on the post-synaptic structures. This property of N G F is in common with the reported property of acetylcholine in this tissue. Further work is in progress to elucidate whether N G F acts on the cell body or dendrites in SCG and the functional significance of NGF-induced PI-effect in this target tissue.
