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Prostaglandins sensitize nociceptors in cell culture
Neuroscience Letters, 132 (1991) 105-108 © 1991 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/91/$ 03.50 ADONIS 030439409100623Z
105
NSL 08139
Prostaglandins sensitize nociceptors in cell culture Simon Pitchford 1~ and Jon D. Levine 1'2z'~ Departments of 1Medicine, 2Anatomy, 30ral and Maxillofacial Surgery and4Division of Neuroscience, University of California, San Francisco, CA 94143-0724 (U.S.A.) (Received 1 July 1991; Accepted 22 July 1991)
Key words." Prostaglandin; Primary afferent nociceptor; Cell culture; Pain; Hyperalgesia; Capsaicin; Whole-cell patch clamp; Cyclic adenosine monophosphate second messenger Using the whole cell patch clamp technique, a population of nociceptors were identified, by virtue of their small size and capsaicin responsitivity. Response to capsaicin was increased following treatment with the hyperalgesic prostaglandins, PGE2 and PGI2. Treatment of the cells with the cyclic adenosine monophosphate (cAMP) analogues, 8 bromo cAMP and dibutryl cAMP, also resulted in an increase in the capsaicin-induced currents. The effects of the cAMP analogues were greater than that produced by prostaglandin treatment. We conclude that PGE2 and PGI2 act directly on nociceptors, with cAMP as second messenger, to sensitize them to noxious stimulation.
A common symptom of tissue inflammation is hyperalgesia. Hyperalgesia is said to be present when a previously non-noxious stimulus is perceived as painful or if the intensity of pain perceived from a noxious stimulus increases. The exact mechanisms underlying the generation of hyperalgesia are not fully understood but are thought to involve sensitization of nerve fiber terminals of polymodal nociceptors by inflammatory mediators. Prostaglandin E2 and I 2 (PGE2 and PGI2), products of the cyclooxygenase pathway of arachidonic acid metabolism, have been suggested to sensitize nociceptors. In previous studies, which have been almost exclusively in vivo, sensitization has been demonstrated electrophysiologically by recording directly from C-fiber nociceptive afferents [7, 10, 12, 13]. Injection of PGE2 at the site of receptor terminals fails to evoke any response in the fiber, yet the nociceptive threshold to mechanical and chemical stimuli is decreased. The action of PGE2 and PGI2 are thought to be direct since the response has a short latency to onset and the hyperalgesic effects persist after the elimination of the known indirect pathways for the production of hyperalgesia [17, 19]. There is also evidence that these directly-acting agents exert their effects via the cAMP second messenger system since membrane permeable analogues of cAMP also produce hyperalgesia [4, 20] and since phosphodiesterase inhibitors en-
Correspondence: J.D. Levine, Division of Rheumatology, U-426/Box 0724, UCSF, San Francisco, CA 94143, U.S.A.
hance hyperalgesia [20]. Finally, increasing or decreasing the activity of G-proteins, which couple prostaglandin receptors to adenylate cyclase enhances and reduces prostaglandin hyperalgesia, respectively [18]. Baccaglini and Hogan [1], using cultured superior cervical ganglion and dorsal root ganglion cells, demonstrated that after PGE2 the response to a 50 mM K + stimulus was increased. However, these investigators did not identify the cell type affected as a nociceptor. In this study using cultured dorsal root ganglion neurons from adult rat, we used capsaicin to identify nociceptors in vitro. Capsaicin (CAP), the pungent constituent of chili peppers, which selectively activates nociceptors in vivo [5], also selectively activates a population of cultured dorsal root ganglion (DRG) neurons with small cell bodies ( < 30/~m) in vitro; these are presumed to be nociceptors [2, 9, 11]. The activation of nociceptors by capsaicin results in a brief inward current, which reverses at approximately 0 mV [2], suggesting that the current was carried through a non-specific cationic channel. It is not known whether CAP acts at a specific receptor site or at some intracellular site; however, it does not appear to require a second messenger system for its activation [22]. Using patch-clamp techniques we measured the effects of hyperalgesic prostaglandins on the CAPinduced current in these cultured D R G nociceptors and also studied the effect of agents that affect the cAMP second messenger system. Primary cultures of adult rat dorsal root ganglion (DRG) neurons were prepared, as previously described
106
by Winter [21]. Briefly, desheathed L1-L6 ganglia from male 250-350 g Sprague-Dawley rats were placed in Hams F14 media (Imperial Labs., UK) plus 10% heat inactivated fetal calf serum and 1% penicillin/1% streptomycin - F14CS, to which collagenase (Type IV; Sigma, St. Louis) was added to a final concentration of 0.125% for 2 x 90 min at 37°C. The ganglia were then placed in Hanks balanced salt solution (calcium- and magnesium-free) containing 0.25% trypsin and 0.025% versene for 30 min at 37°C. After a wash in F14CS the DRGs were transferred to F 14CS medium containing 80/tg/ml DNase, 100 pg/ml soybean trypsin inhibitor and 2.5 mg/ ml MgSO4 (all Sigma, St. Louis) and mechanically disrupted by trituration using a fire-polished Pasteur pipette. DRG cells were then plated onto glass cover slips, previously coated with 0.1 mg/ml poly-DL-ornithine (Sigma, St. Louis) and 5/~g/ml laminin (Sigma, St. Louis or Collaborative Research, Boston). The cells were incubated in F14CS at 37°C, 3% CO2 and 90% humidity and studied within 3 days of plating. For electrophysiological recordings the cells were bathed in a solution containing (in mM); NaCI 130, KCI 3, CaC12 2.5, MgC12 0.6, NaHCO3 1.2, HEPES 10, glucose 4; pH=7.4, osmolarity = 320 mOsm (corrected with sucrose). Whole cell recordings [6] were made using an Axopatch 1B amplifier controlled by a PDPll/73 computer. Patch pipettes were filled with a solution containing (in mM); KCI 140, MgC1 2, CaC12 0.1, BAPTA 1, HEPES 10; pH=7.4, osmolarity = 310 mOsm. Cells were held at the resting potential, -52.3 + 1.82 mV (mean + S.E.M.) and all tests done from this point. Test agents were pressure ejected towards the cell body from a multi-barreled pipette (tip size of each barrel was 5-10 #m). CAP (500 nM) was ejected for 2 s. This dose of CAP was selected as the test dose for all trials since higher doses of CAP tended to result in rapid desensitization of the inward current. The other test agents used in this study, PGE2 (1 mM), PGI2 (500/tM), 8-bromo cAMP (20 mM), dibutyryl cAMP (10 mM) and IBMX (2 mM), were ejected for 90 s. A stock solution of CAP (1 mg/ml in ethanol) was diluted to the appropriate concentration in recording media. PGE2 and PGI2 were made as a stock of 1 mg/ml in ethanol: recording media (1:9, v:v) and then diluted to final concentrations in recording media. Isobutyl methylxanthine (IBMX) was dissolved in ethanol at an initial concentration of 54 mM and then diluted, in recording media, to its final concentration. The two cAMP analogues were diluted in recording media. Fresh batches of 8-bromo-cAMP were used in each experiment. In whole cell patch-recordings, 60% of adult DRG neurons with diameters < 30/tin responded to CAP
A Vehicle
B
Capsaicin
PRE-
a
_
b
~
POST-
__~ ~ _ V
PGE 2
2
.
8 bromo-cAMP
dibutyryl
e
~
~ . ~
cAMP
IBMX
I Fig. I. A: responses (upper trace) of a dorsal root ganglion (DRG) cell to a 4 s stimulus (lower trace) of either the capsaicin vehicle (left) or 500 nM capsaicin (CAP) (right). Superimposed on the capsaicin trace are brief constant voltage pulses which increase in size during the capsaicin-induced inward current. This indicates an increase in conductance during the CAP treatment. B: CAP-induced inward current in cultured DRG neurons and their modification by hyperalgesic prostaglandins (a, b) and cAMP analogues (c, d) or agents that modify the metabolism of cAMP (e). The traces in the left hand column, labelled PRE-, are control responses to a 2 s CAP stimulus (500 nM) for individual cells. The right hatld column, labelled POST-, contains the responses to the CAP stimulus after a single treatment with the different hyperalgesic agents. The duration of the CAP stimulus is represented by the bars at the bottom of the figure. Hyperalgesic agents used were prostaglandin E2 (PGE2-1 mM) (a), prostaglandin 12 (PGI2-500 nM) (b), 8-bromo cAMP (20 mM) (c), dibutyryl cAMP (10 mM) (d) and, isobutylmethylxanthine (IBMX-2 mM) (e). Bars: vertical = I00 pA; horizontal = 10 s.
(500 nM pipette concentration, 1~5 psi) by generating an inward current, measured as 64 + 7 pA (n= 33, mean + S.E.M.) and an increase in the conductance of the cell (Fig. 1A). Individual cells responded to repeated applications of capsaicin. In some cells the response would decline with time. In these cells, further tests were only carried out once a steady state had been reached. In a small number of cells, the response disappeared after
107 TABLE I EFFECTS OF TEST AGENTS ON CAPSAICIN-INDUCED CURRENT IN CULTURED DRG NEURONS Test agent
Percentage increasein CAP current (mean + S.E.M.)
PGE2 (n = 14) PGI:(n=6) 8-bromo cAMP (n= 13) dibutrylcAMP (n=8) IBMX (n=3)
86 __+ 13 119 + 54 404 + 157 222 + 123 215 + 143
only 1 or 2 injections. These cells were discarded. The reversal potential of this CAP-induced current was approximately 0 mV. In control experiments, of 10 ceils that responded to CAP none responded to the CAP vehicle (Fig. IA). When response to CAP was retested after PGE2 (1 mM) or PGI2 (500 gM) treatment (15 psi, 90 s), 37% (14/ 38) and 66% (6/9) of cells, respectively, showed an increased CAP current (Fig. 1B a, b, Table I) that lasted 1-2 min. Re-exposure of responsive cells to the prostaglandin resulted again in an increased CAP current (unpublished data). PGE2 and PGI2 did not affect the baseline current itself. The prostaglandin vehicle (1.5% alcohol) failed to increase the size of the CAP current in any of 10 cells tested (;(21=4.82, P < 0.05 compared to PGE2 and PGI2). The response to CAP was also enhanced in 62% (13/ 21) of the neurons treated with 8-bromo cAMP (20 mM) and in 50% (8/16) of the neurons treated with dibutyryl cAMP (10 mM) (Fig. 1Bc, d, Table I). After the application of cAMP analogues, the response to CAP increased with time. It was maximal between 20 and 500 s after the treatment and declined more slowly than the increased response after prostaglandins, to approximately 40% of the maximal value by 300-600 s. The CAP current increased similarly on re-exposure to cAMP analogues. Finally, in 3 of 4 neurons tested, IBMX 2 mM also caused an increase in the CAP-induced current (Fig. 1B e, Table I). In the present experiments we have demonstrated, for the first time, that prostaglandins indeed act directly to sensitize nociceptors. Our results also further establish that the in vitro prostaglandin-induced sensitization of nociceptors is mediated by the cAMP second messenger system. Our study used a relatively high concentration of prostaglandin in the applying pipette; these levels, however, would be very much reduced within the recording chamber. In addition, since we are studying responses of the D R G rather than the nerve terminal, the number of receptors for prostaglandins are likely to be
low. Our study is consistent with previous reports of an increased firing frequency of cultured embryonic D R G neurons [1] after bath applied PGE2. We observed that a larger percentage of cells were sensitized by PGI2, than by PGE2. This does not appear to be an artifact of in vitro preparation since in a recent study using experimental neuromas in the rat, we found that PGI2 was significantly more effective than PGE2 at sensitizing C-fiber afferents (unpublished data). In our study a percentage of cells which responded to CAP failed to be affected by prostaglandins. Similar to in vivo studies, in which it has been shown that there is a population of CAP-sensitive fibers which are not sensitized by prostaglandins [10]. Our observation that the PGE2 effects on the CAP current are mimicked by the membrane permeable analogue of cAMP, 8-bromo cAMP, and enhanced by the phosphodiesterase inhibitor, IBMX, suggests that this prostaglandin effect, as in many other of its effects [3, 4, 8, 20] is mediated by the cAMP second messenger system. The mechanism by which CAP currents are increased by prostaglandins has not been delineated. However, cAMP has been shown to modulate the activity of various ion channels, either indirectly, by first activating a cAMP-dependent protein kinase [15, 16], or by phosphorylating an ion channel directly [14]. Importantly, CAP did not affect cAMP levels in dorsal root ganglion neurons [22]. We therefore hypothesize that prostaglandins modify CAP current by producing a cAMP-dependent phosphorylation of ion channels. We would predict that other hyperalgesic agents, which act directly on nociceptors, would, therefore, also be expected to increase CAP currents since they all are assumed to have cAMP as a second messenger. Finally, the experimental model of sensitization we employed, using changes in CAP responses in cultured nociceptors, during patch clamping, provides a powerful tool to investigate the mechanisms of directly-acting hyperalgesic substances. We thank Allan Basbaum, Philip Heller and Yetunde Taiwo for helpful discussions of the data. This work was supported in part by NIH Grants NS21647, AM32634 and DE08973 and the Rita Allen Foundation. 1 Baccaglini,P.L. and Hogan, P.G., Some sensoryneurones in culture express characteristics of differentiated pain sensory cells, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 594-598. 2 Bevan, S. and Forbes, C.A., Membrane effects of capsaicin o n dorsal root ganglionneurons in cell culture, J. Physiol.,398 (1988) 28P. 3 Collier, H.O.J. and Roy, A.C., Morphine-likedrugs inhibit the stimulation by E prostaglandinsof cAMP formation by rat brain homogenates,Nature, 248 (1974) 24-25. 4 Ferreira, S.H. and Nakamura, M., I. Prostaglandinhyperalgesia. A cAMP/Ca2+ dependentprocess, Prostaglandins, 18 (1979) 179190.
108 5 Fitzgerald, M., Capsaicin and sensory neurones - - a review, Pain, 15 (1983) 109-130. 6 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sachs, F.J., Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches, Pflfigers Arch., 391 (1981) 85-100. 7 Handwerker, H.O., Pharmacological modification of the discharge of C-fibers. In Y. Zotterman (Ed.), Sensory Functions of the Skin in Primates, University Press, Oxford, 1976, pp. 427~,39. 8 Hemprecht, D. and Schultz, J., Stimulation by prostaglandin E~ of adenosine 3'5' cyclic monophosphate formation in neuroblastoma cells in the presence of phosphodiesterase inhibitors, Fed. Eur. Biochem. Soc. Lett., 34 (1973) 85-89. 9 Heyman, I. and Rang, H.P., Depolarizing responses to capsaicin in a subpopulation of rat dorsal root ganglion cells, Neurosci. Lett., 56 (1985) 69-75. 10 Martin, H.A., Basbaum, A.I., Kwiat, G.C., Goetzl, E.J. and Levine, J.D., Leukotriene and prostaglandin sensitization of cutaneous high-threshold C- and A-delta mechanonociceptors in the hairy skin of rat hindlimbs, Neuroscience, 22 (1987) 651~59. I1 McLean, M.J., Bennett, P.B. and Thomas, R.M., Subtypes of dorsal root ganglion neurons based on different inward currents as measured by whole-cell voltage clamp, Mol. Cell. Biochem., 80 (1988) 95-107. 12 Mense, S., Sensitization of group IV muscle receptors to bradykinin by 5-hydroxytryptamine and prostaglandin Ez, Brain Res., 225 (1981) 95-105. 13 Mizumura, K., Sato, J. and Kumazawa, T., Effects of prostaglandins and other putative chemical intermediaries on the activity of
the canine testicular polymodal receptors studied in vitro, Pfliigers Arch., 408 (1987) 565-572. 14 Nakamura, T. and Gold, G.H., A cyclic nucleotide-gated conductance in olfactory receptor cilia, Nature, 325 (1987) 442~44. 15 Nestler, E. and Greengard, P., Protein phosphorylation in the brain, Nature, 305 (1983) 583-588. 16 Siegelbaum, S.A. and Tsien, R.W., Modulation of gated ion channels as a mode of transmitter action, Trends Neurosci., 6 (1983) 307 313. 17 Taiwo, Y.O. and Levine, J.D., Prostaglandin effects after elimination of indirect hyperalgesic mechanisms in the skin of the rat, Brain Res., 492 (1989) 397-399. 18 Taiwo, Y.O. and Levine, J.D., Guanine nucleotide regulatory proteins mediate prostaglandin hyperalgesia, Brain Res., 492 (1989) 400403. 19 Taiwo, Y.O., Goetzl, E.J. and Levine, J.D., Hyperalgesia onset latency suggests a hierarchy of action, Brain Res., 23 (1987) 333337. 20 Taiwo, Y.O., Bjerkness, L.K., Goetzl, E.J. and Levine, J.D., Mediation of primary afferent peripheral hyperalgesia by the cAMP second messenger system, Neuroscience, 32 (1989) 577-580. 21 Winter, J., Characterization of capsaicin-sensitive neurones in adult rat dorsal root ganglion cultures, Neurosci. Lett., 80 (1987) 134-140. 22 Wood, J.N., Coote, P.R., Minhas, A., Mullaney, I., McNeill, M. and Burgess, G.M., Capsaicin-induced ion fluxes increase cyclic GMP but not cyclic-AMP levels in rat sensory neurones in culture, J. Neurochem., 53 (1989) 1203-1211.
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NSL 08139
Prostaglandins sensitize nociceptors in cell culture Simon Pitchford 1~ and Jon D. Levine 1'2z'~ Departments of 1Medicine, 2Anatomy, 30ral and Maxillofacial Surgery and4Division of Neuroscience, University of California, San Francisco, CA 94143-0724 (U.S.A.) (Received 1 July 1991; Accepted 22 July 1991)
Key words." Prostaglandin; Primary afferent nociceptor; Cell culture; Pain; Hyperalgesia; Capsaicin; Whole-cell patch clamp; Cyclic adenosine monophosphate second messenger Using the whole cell patch clamp technique, a population of nociceptors were identified, by virtue of their small size and capsaicin responsitivity. Response to capsaicin was increased following treatment with the hyperalgesic prostaglandins, PGE2 and PGI2. Treatment of the cells with the cyclic adenosine monophosphate (cAMP) analogues, 8 bromo cAMP and dibutryl cAMP, also resulted in an increase in the capsaicin-induced currents. The effects of the cAMP analogues were greater than that produced by prostaglandin treatment. We conclude that PGE2 and PGI2 act directly on nociceptors, with cAMP as second messenger, to sensitize them to noxious stimulation.
A common symptom of tissue inflammation is hyperalgesia. Hyperalgesia is said to be present when a previously non-noxious stimulus is perceived as painful or if the intensity of pain perceived from a noxious stimulus increases. The exact mechanisms underlying the generation of hyperalgesia are not fully understood but are thought to involve sensitization of nerve fiber terminals of polymodal nociceptors by inflammatory mediators. Prostaglandin E2 and I 2 (PGE2 and PGI2), products of the cyclooxygenase pathway of arachidonic acid metabolism, have been suggested to sensitize nociceptors. In previous studies, which have been almost exclusively in vivo, sensitization has been demonstrated electrophysiologically by recording directly from C-fiber nociceptive afferents [7, 10, 12, 13]. Injection of PGE2 at the site of receptor terminals fails to evoke any response in the fiber, yet the nociceptive threshold to mechanical and chemical stimuli is decreased. The action of PGE2 and PGI2 are thought to be direct since the response has a short latency to onset and the hyperalgesic effects persist after the elimination of the known indirect pathways for the production of hyperalgesia [17, 19]. There is also evidence that these directly-acting agents exert their effects via the cAMP second messenger system since membrane permeable analogues of cAMP also produce hyperalgesia [4, 20] and since phosphodiesterase inhibitors en-
Correspondence: J.D. Levine, Division of Rheumatology, U-426/Box 0724, UCSF, San Francisco, CA 94143, U.S.A.
hance hyperalgesia [20]. Finally, increasing or decreasing the activity of G-proteins, which couple prostaglandin receptors to adenylate cyclase enhances and reduces prostaglandin hyperalgesia, respectively [18]. Baccaglini and Hogan [1], using cultured superior cervical ganglion and dorsal root ganglion cells, demonstrated that after PGE2 the response to a 50 mM K + stimulus was increased. However, these investigators did not identify the cell type affected as a nociceptor. In this study using cultured dorsal root ganglion neurons from adult rat, we used capsaicin to identify nociceptors in vitro. Capsaicin (CAP), the pungent constituent of chili peppers, which selectively activates nociceptors in vivo [5], also selectively activates a population of cultured dorsal root ganglion (DRG) neurons with small cell bodies ( < 30/~m) in vitro; these are presumed to be nociceptors [2, 9, 11]. The activation of nociceptors by capsaicin results in a brief inward current, which reverses at approximately 0 mV [2], suggesting that the current was carried through a non-specific cationic channel. It is not known whether CAP acts at a specific receptor site or at some intracellular site; however, it does not appear to require a second messenger system for its activation [22]. Using patch-clamp techniques we measured the effects of hyperalgesic prostaglandins on the CAPinduced current in these cultured D R G nociceptors and also studied the effect of agents that affect the cAMP second messenger system. Primary cultures of adult rat dorsal root ganglion (DRG) neurons were prepared, as previously described
106
by Winter [21]. Briefly, desheathed L1-L6 ganglia from male 250-350 g Sprague-Dawley rats were placed in Hams F14 media (Imperial Labs., UK) plus 10% heat inactivated fetal calf serum and 1% penicillin/1% streptomycin - F14CS, to which collagenase (Type IV; Sigma, St. Louis) was added to a final concentration of 0.125% for 2 x 90 min at 37°C. The ganglia were then placed in Hanks balanced salt solution (calcium- and magnesium-free) containing 0.25% trypsin and 0.025% versene for 30 min at 37°C. After a wash in F14CS the DRGs were transferred to F 14CS medium containing 80/tg/ml DNase, 100 pg/ml soybean trypsin inhibitor and 2.5 mg/ ml MgSO4 (all Sigma, St. Louis) and mechanically disrupted by trituration using a fire-polished Pasteur pipette. DRG cells were then plated onto glass cover slips, previously coated with 0.1 mg/ml poly-DL-ornithine (Sigma, St. Louis) and 5/~g/ml laminin (Sigma, St. Louis or Collaborative Research, Boston). The cells were incubated in F14CS at 37°C, 3% CO2 and 90% humidity and studied within 3 days of plating. For electrophysiological recordings the cells were bathed in a solution containing (in mM); NaCI 130, KCI 3, CaC12 2.5, MgC12 0.6, NaHCO3 1.2, HEPES 10, glucose 4; pH=7.4, osmolarity = 320 mOsm (corrected with sucrose). Whole cell recordings [6] were made using an Axopatch 1B amplifier controlled by a PDPll/73 computer. Patch pipettes were filled with a solution containing (in mM); KCI 140, MgC1 2, CaC12 0.1, BAPTA 1, HEPES 10; pH=7.4, osmolarity = 310 mOsm. Cells were held at the resting potential, -52.3 + 1.82 mV (mean + S.E.M.) and all tests done from this point. Test agents were pressure ejected towards the cell body from a multi-barreled pipette (tip size of each barrel was 5-10 #m). CAP (500 nM) was ejected for 2 s. This dose of CAP was selected as the test dose for all trials since higher doses of CAP tended to result in rapid desensitization of the inward current. The other test agents used in this study, PGE2 (1 mM), PGI2 (500/tM), 8-bromo cAMP (20 mM), dibutyryl cAMP (10 mM) and IBMX (2 mM), were ejected for 90 s. A stock solution of CAP (1 mg/ml in ethanol) was diluted to the appropriate concentration in recording media. PGE2 and PGI2 were made as a stock of 1 mg/ml in ethanol: recording media (1:9, v:v) and then diluted to final concentrations in recording media. Isobutyl methylxanthine (IBMX) was dissolved in ethanol at an initial concentration of 54 mM and then diluted, in recording media, to its final concentration. The two cAMP analogues were diluted in recording media. Fresh batches of 8-bromo-cAMP were used in each experiment. In whole cell patch-recordings, 60% of adult DRG neurons with diameters < 30/tin responded to CAP
A Vehicle
B
Capsaicin
PRE-
a
_
b
~
POST-
__~ ~ _ V
PGE 2
2
.
8 bromo-cAMP
dibutyryl
e
~
~ . ~
cAMP
IBMX
I Fig. I. A: responses (upper trace) of a dorsal root ganglion (DRG) cell to a 4 s stimulus (lower trace) of either the capsaicin vehicle (left) or 500 nM capsaicin (CAP) (right). Superimposed on the capsaicin trace are brief constant voltage pulses which increase in size during the capsaicin-induced inward current. This indicates an increase in conductance during the CAP treatment. B: CAP-induced inward current in cultured DRG neurons and their modification by hyperalgesic prostaglandins (a, b) and cAMP analogues (c, d) or agents that modify the metabolism of cAMP (e). The traces in the left hand column, labelled PRE-, are control responses to a 2 s CAP stimulus (500 nM) for individual cells. The right hatld column, labelled POST-, contains the responses to the CAP stimulus after a single treatment with the different hyperalgesic agents. The duration of the CAP stimulus is represented by the bars at the bottom of the figure. Hyperalgesic agents used were prostaglandin E2 (PGE2-1 mM) (a), prostaglandin 12 (PGI2-500 nM) (b), 8-bromo cAMP (20 mM) (c), dibutyryl cAMP (10 mM) (d) and, isobutylmethylxanthine (IBMX-2 mM) (e). Bars: vertical = I00 pA; horizontal = 10 s.
(500 nM pipette concentration, 1~5 psi) by generating an inward current, measured as 64 + 7 pA (n= 33, mean + S.E.M.) and an increase in the conductance of the cell (Fig. 1A). Individual cells responded to repeated applications of capsaicin. In some cells the response would decline with time. In these cells, further tests were only carried out once a steady state had been reached. In a small number of cells, the response disappeared after
107 TABLE I EFFECTS OF TEST AGENTS ON CAPSAICIN-INDUCED CURRENT IN CULTURED DRG NEURONS Test agent
Percentage increasein CAP current (mean + S.E.M.)
PGE2 (n = 14) PGI:(n=6) 8-bromo cAMP (n= 13) dibutrylcAMP (n=8) IBMX (n=3)
86 __+ 13 119 + 54 404 + 157 222 + 123 215 + 143
only 1 or 2 injections. These cells were discarded. The reversal potential of this CAP-induced current was approximately 0 mV. In control experiments, of 10 ceils that responded to CAP none responded to the CAP vehicle (Fig. IA). When response to CAP was retested after PGE2 (1 mM) or PGI2 (500 gM) treatment (15 psi, 90 s), 37% (14/ 38) and 66% (6/9) of cells, respectively, showed an increased CAP current (Fig. 1B a, b, Table I) that lasted 1-2 min. Re-exposure of responsive cells to the prostaglandin resulted again in an increased CAP current (unpublished data). PGE2 and PGI2 did not affect the baseline current itself. The prostaglandin vehicle (1.5% alcohol) failed to increase the size of the CAP current in any of 10 cells tested (;(21=4.82, P < 0.05 compared to PGE2 and PGI2). The response to CAP was also enhanced in 62% (13/ 21) of the neurons treated with 8-bromo cAMP (20 mM) and in 50% (8/16) of the neurons treated with dibutyryl cAMP (10 mM) (Fig. 1Bc, d, Table I). After the application of cAMP analogues, the response to CAP increased with time. It was maximal between 20 and 500 s after the treatment and declined more slowly than the increased response after prostaglandins, to approximately 40% of the maximal value by 300-600 s. The CAP current increased similarly on re-exposure to cAMP analogues. Finally, in 3 of 4 neurons tested, IBMX 2 mM also caused an increase in the CAP-induced current (Fig. 1B e, Table I). In the present experiments we have demonstrated, for the first time, that prostaglandins indeed act directly to sensitize nociceptors. Our results also further establish that the in vitro prostaglandin-induced sensitization of nociceptors is mediated by the cAMP second messenger system. Our study used a relatively high concentration of prostaglandin in the applying pipette; these levels, however, would be very much reduced within the recording chamber. In addition, since we are studying responses of the D R G rather than the nerve terminal, the number of receptors for prostaglandins are likely to be
low. Our study is consistent with previous reports of an increased firing frequency of cultured embryonic D R G neurons [1] after bath applied PGE2. We observed that a larger percentage of cells were sensitized by PGI2, than by PGE2. This does not appear to be an artifact of in vitro preparation since in a recent study using experimental neuromas in the rat, we found that PGI2 was significantly more effective than PGE2 at sensitizing C-fiber afferents (unpublished data). In our study a percentage of cells which responded to CAP failed to be affected by prostaglandins. Similar to in vivo studies, in which it has been shown that there is a population of CAP-sensitive fibers which are not sensitized by prostaglandins [10]. Our observation that the PGE2 effects on the CAP current are mimicked by the membrane permeable analogue of cAMP, 8-bromo cAMP, and enhanced by the phosphodiesterase inhibitor, IBMX, suggests that this prostaglandin effect, as in many other of its effects [3, 4, 8, 20] is mediated by the cAMP second messenger system. The mechanism by which CAP currents are increased by prostaglandins has not been delineated. However, cAMP has been shown to modulate the activity of various ion channels, either indirectly, by first activating a cAMP-dependent protein kinase [15, 16], or by phosphorylating an ion channel directly [14]. Importantly, CAP did not affect cAMP levels in dorsal root ganglion neurons [22]. We therefore hypothesize that prostaglandins modify CAP current by producing a cAMP-dependent phosphorylation of ion channels. We would predict that other hyperalgesic agents, which act directly on nociceptors, would, therefore, also be expected to increase CAP currents since they all are assumed to have cAMP as a second messenger. Finally, the experimental model of sensitization we employed, using changes in CAP responses in cultured nociceptors, during patch clamping, provides a powerful tool to investigate the mechanisms of directly-acting hyperalgesic substances. We thank Allan Basbaum, Philip Heller and Yetunde Taiwo for helpful discussions of the data. This work was supported in part by NIH Grants NS21647, AM32634 and DE08973 and the Rita Allen Foundation. 1 Baccaglini,P.L. and Hogan, P.G., Some sensoryneurones in culture express characteristics of differentiated pain sensory cells, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 594-598. 2 Bevan, S. and Forbes, C.A., Membrane effects of capsaicin o n dorsal root ganglionneurons in cell culture, J. Physiol.,398 (1988) 28P. 3 Collier, H.O.J. and Roy, A.C., Morphine-likedrugs inhibit the stimulation by E prostaglandinsof cAMP formation by rat brain homogenates,Nature, 248 (1974) 24-25. 4 Ferreira, S.H. and Nakamura, M., I. Prostaglandinhyperalgesia. A cAMP/Ca2+ dependentprocess, Prostaglandins, 18 (1979) 179190.
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