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Inositol trisphosphate and cyclic adenosine diphosphate-ribose increase quantal transmitter release at frog motor nerve terminals: possible involvement of smooth endoplasmic reticulum
Neuroscience Vol. 95, No. 4, pp. 927–931, 2000 927 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
IP3 and cADP-ribose increase quantal transmitter release
Pergamon PII: S0306-4522(99)00509-6 www.elsevier.com/locate/neuroscience
Letter to Neuroscience INOSITOL TRISPHOSPHATE AND CYCLIC ADENOSINE DIPHOSPHATE-RIBOSE INCREASE QUANTAL TRANSMITTER RELEASE AT FROG MOTOR NERVE TERMINALS: POSSIBLE INVOLVEMENT OF SMOOTH ENDOPLASMIC RETICULUM E. BRAILOIU* and M. D. MIYAMOTO† Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Box 70577, Johnson City, TN 37614-0577, U.S.A. Key words: inositol trisphosphate, cyclic adenosine diphosphate-ribose, quantal transmitter release, smooth endoplasmic reticulum, frog neuromuscular junction, neurosecretion.
The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca 21. 6,25 The increase in Ca 21 may be due to influx of Ca 21 from the extracellular fluid 15 or release of Ca 21 from intracellular stores such as mitochondria. 1,8,18 Whether Ca 21 utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca 21, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphateribose, 2 so the possibility exists that Ca 21 from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10 24 M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10 24 M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca 21 released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles
at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca 21 and transmitter release. q 1999 IBRO. Published by Elsevier Science Ltd.
Experiments involved intracellular recordings of miniature endplate potentials (MEPPs) from isolated frog neuromuscular junctions. Unbiased estimates of quantal release parameters were used to assess alterations in the components of transmitter release (see Experimental Procedures). Bath application of inositol 1,4,5-trisphosphate (IP3) incorporated into liposomes (10 24 M) produced a rapid increase in the number of quanta released (m) during the first 2 min of administration (Fig. 1A). There appeared to be no significant lag between the time of liposome administration and the initial effect. The increase in m peaked after 2 min and remained at a level of 120–125% of control during the continued administration of IP3. Washing with control Ringer for 40 min returned m to control levels. Examination of the quantal components underlying this effect revealed that the increase in m was due largely to an increase in the number of functional transmitter release sites (n), i.e. there was a similar (magnitude and pattern) increase in n (Fig. 1C), but no changes in the probability of release (p; Fig. 1D) or the spatial variance in p (vars p; Fig. 1B). Application of control liposomes containing only KCl produced no effect. As with IP3, bath application of cyclic adenosine diphosphate-ribose (cADP-r) encapsulated in liposomes (10 24 M) caused a rapid increase in m that was sustained at about 120% of control for the 10-min duration of exposure (Fig. 2A). Again, the increase in m appeared to be due solely to an effect on n: there was a concomitant increase in n (Fig. 2C), but no significant increase in p (Fig. 2D) or vars p (Fig. 2B). Inspection of the recordings obtained with IP3 appeared to indicate an increase in size as well as frequency of MEPPs. Measurements were therefore made of MEPP amplitudes for each time-point in both IP3 and cADP-r experiments. The
†To whom correspondence should be addressed. Tel.: 1 1-423-439-6326; fax: 1 1-423-439-8773. E-mail address: [email protected] (M. D. Miyamoto) *Permanent address: Department of Physiology and Biophysics, School of Medicine, University of Medicine and Pharmacy “Gr. T. Popa”, Iasi, Romania. Abbreviations: cADP-r, cyclic adenosine diphosphate-ribose; IP3, inositol 1,4,5-trisphosphate; m, number of quanta released; MEPP, miniature endplate potential; n, number of functional transmitter release sites; p, probability of release; SER, smooth endoplasmic reticulum; vars p, spatial variance in p. 927
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Fig. 1. Effect of IP3 on the quantal release parameters m (no. of quanta released per unit time), n (no. of functional release sites), p (mean probability of release) and vars p (spatial variance in p). IP3 (10 24 M) was incorporated into multilamellar liposomes and continuously perfused into the tissue chamber between time 0 and 10 min. IP3 caused a significant increase in m that was reversed by 40 min of wash (A). The increase in m was due primarily to an increase in n (C), as there was no significant increase in vars p (B) or p (D). Data from each experiment (except those for vars p) were expressed as percentages of the value at time zero, and the results from six identical experiments averaged (points indicate means ^ S.E.M.). Solid lines in plots of p and vars p represent least squares linear regression, while those for m and n show point-to-point fits. Note the break in the time line during the wash period.
Fig. 2. Effect of cADP-r on quantal parameters of transmitter release. Experimental conditions and plots of results are identical to those shown in Fig. 1, except for the agent employed. Like IP3, cADP-r (10 24 M) caused an increase in m (A) that was due primarily to an increase in n (C), as vars p (B) and p (D) were essentially unaffected.
IP3 and cADP-ribose increase quantal transmitter release
Fig. 3. Effect of IP3 and cADP-r on MEPP amplitude. Results are expressed as percentages of the values at time zero (points show means ^ S.E.M. from six experiments). (A) Exposure to liposomes containing IP3 (10 24 M) caused a 10% increase in MEPP amplitude that was reversed with prolonged wash. (B) Exposure to liposomes containing cADP-r (10 24 M) produced no significant change in MEPP amplitude. Solid line represents linear regression through the points in control, exposure to cADP-r and wash periods.
results confirmed that IP3 produced an increase in MEPP size (10%) and showed that this increase could be reversed with wash (Fig. 3A). By contrast, the results disclosed no change in MEPP size with exposure to cADP-r (Fig. 3B). A role for IP3 in the release of transmitter was suggested previously, based on indirect evidence obtained with a-latrotoxin. 23 In these studies, a-latrotoxin caused an increase in vars p that lagged behind increases in m, n and p. Since the toxin was known to produce phosphoinositide breakdown, 27 the results could be explained by formation of IP3 followed by Ca 21 release from smooth endoplasmic reticulum (SER). 19 The subsequent demonstration of extensive, Ca 21-loaded SER in the frog motor nerve terminal 12 provided additional support for this proposal. However, direct demonstration of the ability of IP3 to enhance transmitter release required a method of delivering the lipid-insoluble compound into the cytoplasmic compartment. In the present study, we used multilamellar liposomes to administer IP3 and cADP-r, as the use of uni- 13,26 and multilamellar liposomes 4,9 is a generally accepted method of delivering drugs into the motor nerve terminal. The final concentration of IP3 and cADP-r in the cytoplasm was estimated to be in the range of 10 26 M, since there was about a 100-fold concentration reduction with this procedure. 4
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Our results show an enhancement of quantal transmitter release (m) with IP3 (Fig. 1) and cADP-r (Fig. 2). Because IP3 and cADP-r activate Ca 21 release from SER (via IP3 and ryanodine receptors, respectively 11,17), this suggests that transmitter release may be modified by Ca 21 release from SER. The increase in m appears to be due primarily to an increase in n, the number of “functional” transmitter release sites. This might occur if the Ca 21 released from SER were to increase the number of primed vesicles that are fusion competent. Alternatively, it may be that Ca 21 from the SER leads to activation of synapsin I and migration of vesicles to the active zone. 14 The results with IP3 (Fig. 1) differ from those of Llina´s et al., 16 who saw no change with injection of the compound into the nerve terminal of the squid giant synapse. This disparity may be due to differences in the model or procedures. However, the results with cADP-r (Fig. 2) are consistent with those of Mothet et al., 21 who found a dose-dependent increase in intracellular Ca 21 concentration and transmitter release with presynaptic injection of cADP-r into the buccal ganglion of Aplysia. We suggested previously that release of Ca 21 from sites located at varying distances from the active zone might produce a spatial variance in p, 22 and our results showed transient but significant increases in vars p with mitochondrial inhibitors. 22,24 By contrast, we did not find an increase in vars p in the present studies. One conjecture is that liposomal delivery of the activators may lead to a low level, even release of Ca 21 from SER, such that there is little variation in the waves of Ca 21 concentration reaching the active zone. The reason for the 10% increase in MEPP size with IP3, but not cADP-r (Fig. 3), is also not clear. Inositol phosphates are involved in regulating clathrin assembly and thus in synaptic vesicle biogenesis. 10,28,29 Accordingly, one explanation is that IP3 or its metabolite(s) (inositol high-polyphosphate series 16) produces a small increase in the size of recycled synaptic vesicles. The alternative explanation of a postsynaptic enhancement with IP3 is less likely, given the small surfaceto-volume ratio of muscle fiber compared with nerve ending. Under these circumstances, it is unlikely that a large enough concentration of IP3 can be delivered to the muscle cytoplasm to alter endplate sensitivity to the neurotransmitter. In conclusion, the present results, showing an increase in quantal transmitter release to direct administration of two SER activators, support the notion that SER may affect transmitter release and that IP3 may be an endogenous second messenger. Further studies are needed to determine whether SER is involved in the components of enhanced transmitter release following tetanic stimulation 7 and whether other organelles such as Golgi are involved. EXPERIMENTAL PROCEDURES
Isolated sciatic–sartorius nerve–muscle preparations from Rana pipiens (J. M. Hazen & Co., Alburg, VT) were used for these experiments. All efforts were made to minimize animal suffering, to reduce the number of animals used and to use alternatives to in vivo techniques if available. Experimental procedures were reviewed and approved by the University Committee for Animal Care: animals were killed by decapitation followed by rapid double pithing, and muscles were mounted in a 5-ml Sylgard-lined Petri dish bath. The bath was continuously perfused with Ringer solution using a dual-chambered roller pump. K 1 concentration in the control Ringer solution was elevated (equimolar substitution of KCl for NaCl) to raise the basal frequency of MEPPs and increase the likelihood of binomial release of transmitter.
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The final control solution contained (mM): NaCl 100.0, KCl 12.5, CaCl2 1.8, tris(hydroxymethyl)aminomethane 2.0 (Tris, to pH 7.2) and glucose 5.6. Preparations were equilibrated for 30 min before use. Each experiment consisted of continuous single junction recording of MEPPs during control, exposure to agent and wash periods. Data from each experiment (except those for vars p) are expressed as percentages of the value at time zero, and the results from six identical experiments averaged (points indicate means ^ S.E.M.). MEPPs were recorded using standard intracellular microelectrode (3 M KCl, 10– 15 MV resistance) techniques. Selection was made from impalements showing focal recording, large size, moderately high frequency, good signal-to-noise ratio, and high and stable muscle resting potential. Experiments were conducted at the ambient room temperature (range 22–258C), which varied less than 28C during any single trial. Only one trial was carried out on each preparation. Bioelectric signals were fed into a high-impedance preamplifier and viewed on a Tektronix oscilloscope. Signal-to-noise ratio was increased using a band-pass filter (0– 1 kHz) and results were recorded on magnetic tape with a modified video cassette recorder. Signals were boosted 20-fold by a rear-output amplifier of the oscilloscope to allow interfacing with an A-D data acquisition unit. Results were then stored on magnetic tape for offline analysis. Digitized data were used to obtain estimates of MEPP size before, during and after exposure to IP3 or cADP-r. MEPP amplitudes were measured using a grid template on a flat-screen monitor, and 100 measurements employed for each time-point. A detailed description of the quantal analysis is described elsewhere. 22 In brief, the number of quanta released by one nerve impulse (m) was replaced by the number of MEPPs in a 50-ms time interval (bin) and 500 sequential bins were used for each quantal estimate. Data
were divided into subgroups of 100 before analysis to minimize nonstationarity, and results that were non-stationary according to statistical tests were discarded. Unbiased estimates of m, n, p and vars p were then computed using analysis of moments. 20 As noted previously, 22 the negative estimate for vars p was due to the presence of some temporal variance, 5 as decreasing bin size (and thus temporal variance) eliminated this problem. The small, systematic underestimate of vars p was considered acceptable for this study. Results were tested using oneway ANOVA, with P , 0.05 indicating a significant difference. Multilamellar liposomes were prepared with 60 mg/ml egg phosphatidylcholine (Sigma Chem. Co.), as described previously. 4 Chemicals to be incorporated into liposomes were dissolved in 140 mM KCl solution at pH 6.9. Vesicles were then treated with diethyl ether (1:6 v/v). To remove non-incorporated solute, liposome batches were dialysed (Sigma dialysis sacs, molecular weight cut-off of 12,400) against Ringer solution (1:600 v/v, 150 min) and the Ringer solution was changed every 30 min. Control liposomes contained 140 mM KCl solution (pH 6.9) only. Liposomes were administered by continuous perfusion after 1:20 v/v dilution in Ringer solution. d-Myo-IP3, hexapotassium salt and cADP-r were obtained from Calbiochem. Liposomes were not altered by the addition of any of the inositol phosphates, including IP3. 3
Acknowledgements—We are extremely grateful to Dr W. L. Stone for the use of his equipment, and to Dr C. Brailoiu and Ms Doris M. Davis for laboratory assistance. This work was supported in part by the Research Development Committee of ETSU.
REFERENCES
1. Alnaes E. and Rahamimoff R. (1975) On the role of mitochondria in transmitter release from motor nerve terminals. J. Physiol., Lond. 248, 285–306. 2. Berridge M. J. (1998) Neuronal calcium signaling. Neuron 21, 13–26. 3. Brailoiu E., Huhurez G., Slatineanu S., Filipeanu C. M., Costuleanu M. and Branisteanu D. D. (1995) TLC characterization of liposomes containing dmyo-inositol derivatives. Biomed. Chromat. 9, 175–178. 4. Brailoiu E. and van der Kloot W. (1996) Bromoacetylcholine and acetylcholinesterase introduced via liposomes into motor nerve endings block increases in quantal size. Pflu¨gers Arch. 432, 413–418. 5. Brown T. H., Perkel D. H. and Feldman M. W. (1976) Evoked neurotransmitter release: statistical effects of nonuniformity and nonstationarity. Proc. natn. Acad. Sci. U.S.A. 73, 2913–2917. 6. Burgoyne R. D. and Morgan A. (1998) Calcium sensors in regulated exocytosis. Cell Calcium 24, 367–376. 7. Cheng H. and Miyamoto M. D. (1999) Effect of hypertonicity on augmentation and potentiation and on corresponding quantal parameters of transmitter release. J. Neurophysiol. 81, 1428–1431. 8. David G., Barrett J. N. and Barrett E. F. (1998) Evidence that mitochondria buffer physiological Ca 21 loads in lizard motor nerve terminals. J. Physiol., Lond. 509, 59–65. 9. de Paiva A. and Dolly J. O. (1990) Light chain of botulinum neurotoxin is active in mammalian motor nerve terminals when delivered via liposomes. Fedn Eur. biochem. Socs Lett. 277, 171–174. 10. Fukuda M. and Mikoshiba K. (1997) The function of inositol high polyphosphate binding proteins. BioEssays 19, 593–603. 11. Golovina V. A. and Blaustein M. P. (1997) Spatially and functionally distinct Ca 21 stores in sarcoplasmic and endoplasmic reticulum. Science 275, 1643–1648. 12. Grohovaz F., Bossi M., Pezzati R., Meldolesi J. and Tarelli F. T. (1996) High resolution ultrastructural mapping of total calcium: electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve–muscle preparation. Proc. natn. Acad. Sci. U.S.A. 93, 4799–4803. 13. Hirsh J. K., Silinsky E. M. and Solsona C. S. (1990) The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings. Br. J. Pharmac. 101, 311–318. 14. Llina´s R., Gruner J. A., Sugimori M., McGuinness T. L. and Greengard P. (1991) Regulation by synapsin I and Ca 21-calmodulin-dependent protein kinase II of transmitter release in squid giant synapse. J. Physiol., Lond. 436, 257–282. 15. Llina´s R. and Moreno H. (1998) Local Ca 21 signaling in neurons. Cell Calcium 24, 359–366. 16. Llina´s R., Sugimori M., Lang E. J., Morita M., Fukuda M., Niinobe M. and Mikoshiba K. (1994) The inositol high-polyphosphate series blocks synaptic transmission by preventing vesicular fusion: a squid giant synapse study. Proc. natn. Acad. Sci. U.S.A. 91, 12,990–12,993. 17. Mackrill J. J. (1999) Protein–protein interactions in intracellular Ca 21-release channel function. Biochem. J. 337, 345–361. 18. Melamed-Book N. and Rahamimoff R. (1998) The revival of the role of the mitochondrion in regulation of transmitter release. J. Physiol., Lond. 509, 2. 19. Meldolesi J. and Pozzan T. (1998) The endoplasmic reticulum Ca 21 store: a view from the lumen. Trends biochem. Sci. 23, 10–14. 20. Miyamoto M. D. (1986) Probability of quantal transmitter release from nerve terminals: theoretical considerations in the determination of spatial variation. J. theor. Biol. 123, 289–304. 21. Mothet J.-P., Fossier P., Meunier F.-M., Stinnakre J., Tauc L. and Baux G. (1998) Cyclic ADP-ribose and calcium-induced calcium release regulate neurotransmitter release at a cholinergic synapse of Aplysia. J. Physiol., Lond. 507, 405–414. 22. Provan S. D. and Miyamoto M. D. (1993) Unbiased estimates of quantal release parameters and spatial variation in the probability of neurosecretion. Am. J. Physiol. 264, C1051–C1060. 23. Provan S. D. and Miyamoto M. D. (1994) Effect of alpha-latrotoxin (a-LTX) on quantal release parameters at motor nerves: support for inositol-1,4,5trisphosphate (IP3)-mediated release of Ca 21 from endoplasmic reticulum (ER). Soc. Neurosci. Abstr. 20, 59. 24. Provan S. D. and Miyamoto M. D. (1995) Real-time detection of mitochondrial inhibition at frog motor nerve terminals using increases in the spatial variance in probability of transmitter release. Neurosci. Lett. 185, 187–190. 25. Silinsky E. M. (1985) The biophysical pharmacology of calcium-dependent acetylcholine secretion. Pharmac. Rev. 37, 81–132. 26. Silinsky E. M., Watanabe M., Redman R. S., Qiu R., Hirsh J. K., Hunt J. M., Solsona C. S., Alford S. and MacDonald R. C. (1995) Neurotransmitter release evoked by nerve impulses without Ca 21 entry through Ca 21 channels in frog motor nerve endings. J. Physiol., Lond. 482, 511–520.
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27. Vicentini L. M. and Meldolesi J. (1984) a-Latrotoxin of black widow spider venom binds to a specific receptor coupled to phosphoinositide breakdown in PC12 cells. Biochem. biophys. Res. Commun. 121, 538–544. 28. Voglmaier S. M., Keen J. H., Murphy J.-E., Ferris C. D., Prestwich G. D., Snyder S. H. and Theibert A. B. (1992) Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2. Biochem. biophys. Res. Commun. 187, 158–163. 29. Zhang B., Koh Y.-H., Beckstead R. B., Budnik V., Ganetsky B. and Bellen H. J. (1998) Synaptic vesicle size and number are regulated by a clathrin adaptor protein required for endocytosis. Neuron 21, 1465–1475. (Accepted 11 October 1999)
IP3 and cADP-ribose increase quantal transmitter release
Pergamon PII: S0306-4522(99)00509-6 www.elsevier.com/locate/neuroscience
Letter to Neuroscience INOSITOL TRISPHOSPHATE AND CYCLIC ADENOSINE DIPHOSPHATE-RIBOSE INCREASE QUANTAL TRANSMITTER RELEASE AT FROG MOTOR NERVE TERMINALS: POSSIBLE INVOLVEMENT OF SMOOTH ENDOPLASMIC RETICULUM E. BRAILOIU* and M. D. MIYAMOTO† Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Box 70577, Johnson City, TN 37614-0577, U.S.A. Key words: inositol trisphosphate, cyclic adenosine diphosphate-ribose, quantal transmitter release, smooth endoplasmic reticulum, frog neuromuscular junction, neurosecretion.
The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca 21. 6,25 The increase in Ca 21 may be due to influx of Ca 21 from the extracellular fluid 15 or release of Ca 21 from intracellular stores such as mitochondria. 1,8,18 Whether Ca 21 utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca 21, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphateribose, 2 so the possibility exists that Ca 21 from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10 24 M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10 24 M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca 21 released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles
at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca 21 and transmitter release. q 1999 IBRO. Published by Elsevier Science Ltd.
Experiments involved intracellular recordings of miniature endplate potentials (MEPPs) from isolated frog neuromuscular junctions. Unbiased estimates of quantal release parameters were used to assess alterations in the components of transmitter release (see Experimental Procedures). Bath application of inositol 1,4,5-trisphosphate (IP3) incorporated into liposomes (10 24 M) produced a rapid increase in the number of quanta released (m) during the first 2 min of administration (Fig. 1A). There appeared to be no significant lag between the time of liposome administration and the initial effect. The increase in m peaked after 2 min and remained at a level of 120–125% of control during the continued administration of IP3. Washing with control Ringer for 40 min returned m to control levels. Examination of the quantal components underlying this effect revealed that the increase in m was due largely to an increase in the number of functional transmitter release sites (n), i.e. there was a similar (magnitude and pattern) increase in n (Fig. 1C), but no changes in the probability of release (p; Fig. 1D) or the spatial variance in p (vars p; Fig. 1B). Application of control liposomes containing only KCl produced no effect. As with IP3, bath application of cyclic adenosine diphosphate-ribose (cADP-r) encapsulated in liposomes (10 24 M) caused a rapid increase in m that was sustained at about 120% of control for the 10-min duration of exposure (Fig. 2A). Again, the increase in m appeared to be due solely to an effect on n: there was a concomitant increase in n (Fig. 2C), but no significant increase in p (Fig. 2D) or vars p (Fig. 2B). Inspection of the recordings obtained with IP3 appeared to indicate an increase in size as well as frequency of MEPPs. Measurements were therefore made of MEPP amplitudes for each time-point in both IP3 and cADP-r experiments. The
†To whom correspondence should be addressed. Tel.: 1 1-423-439-6326; fax: 1 1-423-439-8773. E-mail address: [email protected] (M. D. Miyamoto) *Permanent address: Department of Physiology and Biophysics, School of Medicine, University of Medicine and Pharmacy “Gr. T. Popa”, Iasi, Romania. Abbreviations: cADP-r, cyclic adenosine diphosphate-ribose; IP3, inositol 1,4,5-trisphosphate; m, number of quanta released; MEPP, miniature endplate potential; n, number of functional transmitter release sites; p, probability of release; SER, smooth endoplasmic reticulum; vars p, spatial variance in p. 927
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Fig. 1. Effect of IP3 on the quantal release parameters m (no. of quanta released per unit time), n (no. of functional release sites), p (mean probability of release) and vars p (spatial variance in p). IP3 (10 24 M) was incorporated into multilamellar liposomes and continuously perfused into the tissue chamber between time 0 and 10 min. IP3 caused a significant increase in m that was reversed by 40 min of wash (A). The increase in m was due primarily to an increase in n (C), as there was no significant increase in vars p (B) or p (D). Data from each experiment (except those for vars p) were expressed as percentages of the value at time zero, and the results from six identical experiments averaged (points indicate means ^ S.E.M.). Solid lines in plots of p and vars p represent least squares linear regression, while those for m and n show point-to-point fits. Note the break in the time line during the wash period.
Fig. 2. Effect of cADP-r on quantal parameters of transmitter release. Experimental conditions and plots of results are identical to those shown in Fig. 1, except for the agent employed. Like IP3, cADP-r (10 24 M) caused an increase in m (A) that was due primarily to an increase in n (C), as vars p (B) and p (D) were essentially unaffected.
IP3 and cADP-ribose increase quantal transmitter release
Fig. 3. Effect of IP3 and cADP-r on MEPP amplitude. Results are expressed as percentages of the values at time zero (points show means ^ S.E.M. from six experiments). (A) Exposure to liposomes containing IP3 (10 24 M) caused a 10% increase in MEPP amplitude that was reversed with prolonged wash. (B) Exposure to liposomes containing cADP-r (10 24 M) produced no significant change in MEPP amplitude. Solid line represents linear regression through the points in control, exposure to cADP-r and wash periods.
results confirmed that IP3 produced an increase in MEPP size (10%) and showed that this increase could be reversed with wash (Fig. 3A). By contrast, the results disclosed no change in MEPP size with exposure to cADP-r (Fig. 3B). A role for IP3 in the release of transmitter was suggested previously, based on indirect evidence obtained with a-latrotoxin. 23 In these studies, a-latrotoxin caused an increase in vars p that lagged behind increases in m, n and p. Since the toxin was known to produce phosphoinositide breakdown, 27 the results could be explained by formation of IP3 followed by Ca 21 release from smooth endoplasmic reticulum (SER). 19 The subsequent demonstration of extensive, Ca 21-loaded SER in the frog motor nerve terminal 12 provided additional support for this proposal. However, direct demonstration of the ability of IP3 to enhance transmitter release required a method of delivering the lipid-insoluble compound into the cytoplasmic compartment. In the present study, we used multilamellar liposomes to administer IP3 and cADP-r, as the use of uni- 13,26 and multilamellar liposomes 4,9 is a generally accepted method of delivering drugs into the motor nerve terminal. The final concentration of IP3 and cADP-r in the cytoplasm was estimated to be in the range of 10 26 M, since there was about a 100-fold concentration reduction with this procedure. 4
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Our results show an enhancement of quantal transmitter release (m) with IP3 (Fig. 1) and cADP-r (Fig. 2). Because IP3 and cADP-r activate Ca 21 release from SER (via IP3 and ryanodine receptors, respectively 11,17), this suggests that transmitter release may be modified by Ca 21 release from SER. The increase in m appears to be due primarily to an increase in n, the number of “functional” transmitter release sites. This might occur if the Ca 21 released from SER were to increase the number of primed vesicles that are fusion competent. Alternatively, it may be that Ca 21 from the SER leads to activation of synapsin I and migration of vesicles to the active zone. 14 The results with IP3 (Fig. 1) differ from those of Llina´s et al., 16 who saw no change with injection of the compound into the nerve terminal of the squid giant synapse. This disparity may be due to differences in the model or procedures. However, the results with cADP-r (Fig. 2) are consistent with those of Mothet et al., 21 who found a dose-dependent increase in intracellular Ca 21 concentration and transmitter release with presynaptic injection of cADP-r into the buccal ganglion of Aplysia. We suggested previously that release of Ca 21 from sites located at varying distances from the active zone might produce a spatial variance in p, 22 and our results showed transient but significant increases in vars p with mitochondrial inhibitors. 22,24 By contrast, we did not find an increase in vars p in the present studies. One conjecture is that liposomal delivery of the activators may lead to a low level, even release of Ca 21 from SER, such that there is little variation in the waves of Ca 21 concentration reaching the active zone. The reason for the 10% increase in MEPP size with IP3, but not cADP-r (Fig. 3), is also not clear. Inositol phosphates are involved in regulating clathrin assembly and thus in synaptic vesicle biogenesis. 10,28,29 Accordingly, one explanation is that IP3 or its metabolite(s) (inositol high-polyphosphate series 16) produces a small increase in the size of recycled synaptic vesicles. The alternative explanation of a postsynaptic enhancement with IP3 is less likely, given the small surfaceto-volume ratio of muscle fiber compared with nerve ending. Under these circumstances, it is unlikely that a large enough concentration of IP3 can be delivered to the muscle cytoplasm to alter endplate sensitivity to the neurotransmitter. In conclusion, the present results, showing an increase in quantal transmitter release to direct administration of two SER activators, support the notion that SER may affect transmitter release and that IP3 may be an endogenous second messenger. Further studies are needed to determine whether SER is involved in the components of enhanced transmitter release following tetanic stimulation 7 and whether other organelles such as Golgi are involved. EXPERIMENTAL PROCEDURES
Isolated sciatic–sartorius nerve–muscle preparations from Rana pipiens (J. M. Hazen & Co., Alburg, VT) were used for these experiments. All efforts were made to minimize animal suffering, to reduce the number of animals used and to use alternatives to in vivo techniques if available. Experimental procedures were reviewed and approved by the University Committee for Animal Care: animals were killed by decapitation followed by rapid double pithing, and muscles were mounted in a 5-ml Sylgard-lined Petri dish bath. The bath was continuously perfused with Ringer solution using a dual-chambered roller pump. K 1 concentration in the control Ringer solution was elevated (equimolar substitution of KCl for NaCl) to raise the basal frequency of MEPPs and increase the likelihood of binomial release of transmitter.
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The final control solution contained (mM): NaCl 100.0, KCl 12.5, CaCl2 1.8, tris(hydroxymethyl)aminomethane 2.0 (Tris, to pH 7.2) and glucose 5.6. Preparations were equilibrated for 30 min before use. Each experiment consisted of continuous single junction recording of MEPPs during control, exposure to agent and wash periods. Data from each experiment (except those for vars p) are expressed as percentages of the value at time zero, and the results from six identical experiments averaged (points indicate means ^ S.E.M.). MEPPs were recorded using standard intracellular microelectrode (3 M KCl, 10– 15 MV resistance) techniques. Selection was made from impalements showing focal recording, large size, moderately high frequency, good signal-to-noise ratio, and high and stable muscle resting potential. Experiments were conducted at the ambient room temperature (range 22–258C), which varied less than 28C during any single trial. Only one trial was carried out on each preparation. Bioelectric signals were fed into a high-impedance preamplifier and viewed on a Tektronix oscilloscope. Signal-to-noise ratio was increased using a band-pass filter (0– 1 kHz) and results were recorded on magnetic tape with a modified video cassette recorder. Signals were boosted 20-fold by a rear-output amplifier of the oscilloscope to allow interfacing with an A-D data acquisition unit. Results were then stored on magnetic tape for offline analysis. Digitized data were used to obtain estimates of MEPP size before, during and after exposure to IP3 or cADP-r. MEPP amplitudes were measured using a grid template on a flat-screen monitor, and 100 measurements employed for each time-point. A detailed description of the quantal analysis is described elsewhere. 22 In brief, the number of quanta released by one nerve impulse (m) was replaced by the number of MEPPs in a 50-ms time interval (bin) and 500 sequential bins were used for each quantal estimate. Data
were divided into subgroups of 100 before analysis to minimize nonstationarity, and results that were non-stationary according to statistical tests were discarded. Unbiased estimates of m, n, p and vars p were then computed using analysis of moments. 20 As noted previously, 22 the negative estimate for vars p was due to the presence of some temporal variance, 5 as decreasing bin size (and thus temporal variance) eliminated this problem. The small, systematic underestimate of vars p was considered acceptable for this study. Results were tested using oneway ANOVA, with P , 0.05 indicating a significant difference. Multilamellar liposomes were prepared with 60 mg/ml egg phosphatidylcholine (Sigma Chem. Co.), as described previously. 4 Chemicals to be incorporated into liposomes were dissolved in 140 mM KCl solution at pH 6.9. Vesicles were then treated with diethyl ether (1:6 v/v). To remove non-incorporated solute, liposome batches were dialysed (Sigma dialysis sacs, molecular weight cut-off of 12,400) against Ringer solution (1:600 v/v, 150 min) and the Ringer solution was changed every 30 min. Control liposomes contained 140 mM KCl solution (pH 6.9) only. Liposomes were administered by continuous perfusion after 1:20 v/v dilution in Ringer solution. d-Myo-IP3, hexapotassium salt and cADP-r were obtained from Calbiochem. Liposomes were not altered by the addition of any of the inositol phosphates, including IP3. 3
Acknowledgements—We are extremely grateful to Dr W. L. Stone for the use of his equipment, and to Dr C. Brailoiu and Ms Doris M. Davis for laboratory assistance. This work was supported in part by the Research Development Committee of ETSU.
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