The Journal of Neuroscience, May 1992, 12(5): 1917-1927

Prostaglandin E, Increases Calcium Conductance and Stimulates Release of Substance P in Avian Sensory Neurons

0. D. Nicol, D. K. Klingberg, and M. R. Vasko Department of Pharmacology and Toxicology, School of Medicine, Indiana University, Indianapolis, Indiana 46202

Prostaglandins are known to lower activation threshold to of dogs produces hyperalgesia as indicated by a lowering of the thermal, mechanical, and chemical stimulation in small-di- pressure necessary to elicit a withdrawal or by the degree ofjoint ameter sensory neurons. Although the mechanism of pros- incapacitation, respectively (Willis and Cornelsen, 1973; Fer- taglandin action is unknown, agents known to elevate intra- reira et al., 1978; Taiwo and Levine, 1989). In humans, PGs in cellular calcium produce a sensitization that is similar to that micromolar concentrations intensify bradykinin- or histamine- produced by prostaglandins. Consistent with the idea of induced pain (Ferreira, 1972). In addition, the accepted mech- prostaglandin-induced elevations in calcium, prostaglandins anism for pain relief produced by aspirin and other nonsteroidal might also stimulate the release of neurotransmitter from antinflammatory drugs is the inhibition of cyclooxygenase, the sensory neurons. We therefore examined whether prosta- enzyme that catalyzes the conversion of arachidonic acid to PGs glandin E, (PGE,) could enhance the release of the putative (Collier, 1971; Smith and Willis, 1971; Vane, 1971). sensory transmitter substance P (SP) from isolated neurons Presumably, PG-induced hyperalgesia involves a direct action of the avian dorsal root ganglion grown in culture. Utilizing of these eicosanoids on sensory neurons. PGE, and PGE, sen- the whole-cell patch-clamp recording technique, we also ex- sitize small-diameter sensory nerve fibers to thermal and me- amined whether POE, could alter calcium currents in these chanical stimulation (Handwerker, 1976; Pateromichelakis and cells. Exposure of sensory neurons to PGE, produced a dose- Rood, 1982; Martin et al., 1987) and facilitate activation of dependent increase in the release of SP. One micromolar these neurons by potassium, capsaicin, and bradykinin (Chahl POE, increased release approximately twofold above basal and Iggo, 1972; Baccaglini and Hogan, 1983; Yanagisawa et al., release, whereas 5 and 10 PM PGE, increased release by 1986; Schaible and Schmidt, 1988). Although the cellular mech- about fourfold. The release evoked by these higher concen- anisms for the action of PGs on sensory neurons remain un- trations of POE, was similar in magnitude to the release known, early studies suggested that calcium may have an im- induced by 50 mM KCI. Neither arachidonic acid (10 PM), portant regulatory role. Agents that are known to alter the prostaglandin F, (10 PM), nor the lipoxygenase product leu- intracellular concentration of calcium produce hyperalgesia that kotriene 6, (1 PM) significantly altered SP release. The ad- is similar to that produced by PGE, (Ferreira and Nakamura, dition of 1 FM PGE, increased the peak calcium currents by 1979). In addition, recent studies utilizing bovine chromaffin 1.8-fold and 1.4-fold for neurons held at potentials of -80 cells and NG108-15 neuroblastoma-glioma hybrid cells dem- and -90 mV, respectively. The action of PGE, was rapid with onstrate that PGE, facilitates the entry of calcium through ac- facilitation occurring within 2 min. As with release studies, tivation of neuronal membrane calcium channels (Koyama et arachidonic acid, prostaglandin Fza, and leukotriene B, had al., 1988; Miwa et al., 1988; Mochizuki-Oda et al., 1991). no significant effect on the amplitude of the calcium current. Consistent with this enhanced influx of calcium, it is possible These results suggest that PGE, can stimulate the release that PGs could initiate the release of neurotransmitter from of SP through the activation or facilitation of an inward cal- sensory neurons. The enhanced release of neurotransmitter could cium current. The capacity of PGE, to facilitate the calcium in turn contribute to PG-induced hyperalgesia and to PG-in- current in these sensory neurons may be one mechanism to duced inflammatory responses. Ueda et al. (1985) and Uda et account for the ability of prostaglandins to sensitize sensory al. (1990) have proposed that enhanced release of one putative neurons to physical or chemical stimuli. sensory neurotransmitter, substance P (SP), by PGs could be the mechanism to explain the PG-induced hyperalgesia and PG- Prostaglandins (PGs) appear to be important mediators in- induced potentiation ofthe stimulated contraction ofiris sphinc- volved in lowering the pain threshold during tissue injury and ter muscle. To date, however, no studies have been performed inflammation, that is, in producing hyperalgesia. Injection of to determine if PGs actually stimulate transmitter release from PGE, or PGI, into the hind paw of the rat or into the knee joints sensory neurons. We sought to examine whether PGE, or other eicosanoids could alter the release of SP from avian sensory neurons grown Received July 26, 1991; revised Dec. 4, 1991; accepted Dec. 23, 1991. in culture. Our studies indicate that PGE, can enhance the re- This work was supported by PHS 2-S07-RR-5371 to G.D.N. and by PHS l-ROl-NS21697 and a grant from the Arthritis Foundation to M.R.V. We thank lease of SP from avian sensory neurons grown in culture. In J. Crider for her technical assistance. contrast, neither PGF,,, arachidonic acid, nor the lipoxygenase Correspondence should be addressed to Dr. G. D. Nicol, Department of Phar- macology and Toxicology, 635 Bamhill Drive, School of Medicine, Indiana Uni- metabolite leukotriene B, (LTB,) has any significant effect on versity, Indianapolis, IN 46202. release. To examine a possible mechanism for the enhanced Copyright 0 1992 Society for Neuroscience 0270-6474/92/121917-l 1$05.00/O release of SP, we utilized the whole-cell patch-clamp technique 1918 Nicol et al. * PGE, Stimulates lc. and Releases SP

(Hamill et al., I98 1) to examine the actions of PGE, and other concentration of acetic acid is 2 N. In each experiment, a number of eicosanoids on calcium currents in these sensory neurons. PGE, wells were used as controls and consisted of the measurement of the basal release and the release induced by high extracellular potassium. enhanced the voltage-dependent calcium current, suggesting that If potassium did not evoke the release of SP, the experiment was dis- the facilitated calcium current might be responsible for increas- carded. Neither vehicle, ethanol, nor I-methyl-2-pyrrolidinone, at the ing release of SP. Again, neither PGF,,, arachidonic acid, nor concentrations used in the eicosanoid experiments, had any effect on LTB, had any significant effect on calcium currents. either the basal or potassium-stimulated release of SP. For experiments Preliminary findings from these studies have appeared in ab- involving indomethacin, cells were incubated in Krebs’ buffer contain- ing IO &ml indomethacin for 30 min prior to initiating the release stract form (Vasko et al., 1989; Nicol and Vasko, 199 1). protocol. After collection of the release solutions, the samples were lyophilticd and reconstituted in an assay buffer of 0. I M phosphate buffer (pH 7.4- Materials and Methods 7.5) containing 0.9% NaCl, 0. I% gelatin, and 2 ppm phenol red. Five Experiments were performed using primary cultures of neurons har- to nine microliters of 2 N NaOH were added to each sample to adjust vested from the dorsal root ganglia (DRG) of 8-10 d old chicken em- the pH to approximately 7. Neutralized solutions were centrifuged and bryos. Eicosanoids were purchased from Cayman Chemical Co. (Ann supematant was assayed for SP as described below. In all experiments, Arbor, Ml), peptides from Peninsula Laboratory (Belmont, CA), nerve release by a test substance was compared to basal release and results growth factor from Collaborative Biochemical Products Inc. (Bedford, were expressed as total picograms of SP released. MA), other supplies for culture medium from GIBCO Laboratories Measurement ofsubstance P content in DRG neurons. Following the (Grand Island, NY), and routine chemicals from Sigma Chemical Com- release protocol, the content of SP in these neurons was determined in pany (St. Louis, MO). each culture dish. One milliliter of 2 N acetic acid was added to each Cell culture. Avian dorsal root ganglia neurons were harvested and dish containing DRG neurons and incubated at room temperature for grown using a modification of the method of Dichter and Fischbach 15 min. The solution was then removed and the procedure repeated a ( 1977). Briefly, fertilized chicken eggs were incubated for 8-l 0 d at 39°C. second time. The extracts were combined and diluted 1:20, 1:50, and Embryos were removed and DRG dissected out. The ganglia were placed 1:lOO with 2 N acetic acid. Samples were frozen, lyophilized, and as- in a calcium-free, magnesium-free, phosphate-buffered saline solution sayed for SP as described below. (Puck’s saline) at 4°C. Routinely, 12-20 ganglia were removed from Radioimmunoassay for substance P. Substance P was assayed using each embryo’ and 12-l 8 embryos were dissected at one time. After a modification of our previously described radioimmunoassay (Pang dissection, the ganglia were transferred to a sterile 15 ml centrifuge tube, and Vasko, 1986). Briefly, the radioimmunoassay was performed by washed with Puck’s saline. and then incubated at 37°C for 30 min in adding 25 PI of the I:8000 dilution of the anti-substance P serum (57P) Puck’s saline containing 0.6 1% crude collagenase. The ganglia were then and 25 ~1 of 12’I-8-Tyr-SP containing 4000-5000 cpm to 300 ~1 of 0. I gently pelleted by centrifugation at 500 x g for 5 min, the supematant M phosphate buffer containing known amounts of SP (ranging from 2.5 discarded, and the ganglia resuspended in growth medium. The growth to 500 pg) or to 300 ~1 of the reconstituted unknown samples. This medium was Dulbecco’s Modified Eagle’s Medium supplemented with mixture was then incubated for 16-18 hr at 4°C. After incubation, 500 2 mM glutamine, 50 U/ml penicillin, 50 j&ml streptomycin, 10% (v/ ~1 of a charcoal solution consisting of 1% Norite I charcoal suspended v) heat-inactivated fetal calf serum, 5% (v/v) chicken embryo extract in 0. I M phosphate buffer (pH 7.5) containing 0.3% NaCI, and 0. I% (Dichter and Fischbach, 1977), and 100 or 200 @ml of 7s nerve growth bovine serum albumin were added to each tube. This solution was factor (Collaborative Research Inc., Lexington, MA). Individual cells centrifuged at 1500 x g for 15 min at 4°C to remove unbound peptides. were dissociated from the ganglia by trituration using a fire-polished The supematant was decanted and the radioactivity determined by Pasteur pipette until a cloudy suspension was observed. The cells were gamma scintillation spectrometry. The amounts ofSP in unknown sam- counted using a hemocytometer and diluted in growth medium, and ples were estimated by comparing unknown samples to the “logit-log” approximately 300,000-400,000 cells were plated on 60 mm collagcn- linearized standard curve (Rodbard and Leward, 1970). Using this coated Falcon brand culture dishes. For electrophysiological experi- method, the minimal detectable limit (95% confidence interval) was 5 ments, small plastic coverslips were placed on the bottom of the culture pg of SP with a 50% displacement of bound peptide at approximately dishes. They were collagen coated, and cells were plated directly onto 75 Pg. these slips. The cells are grown for 3 d at 37°C in a 5% CO, atmosphere, With each chemical manipulation, standard curves for SP were de- using growth medium containing either IO PM cystosine P-D-arabino- termined in the presence of the highest concentration of test compound furanoside, or 50 PM 5-fluoro-2’-deoxyuridine and 150 PM uridine to to confirm that the experimental manipulation did not alter the standard minimize growth of non-neuronal cells. After 3 d ofexposure to mitotic curve. Anti-substance P serum was raised in our laboratory from rabbits inhibitors and every other day thereafter, neurons were fed with 2 ml using thyroglobulin-bound SP (Pang and Vasko, 1986). The antibody of medium without mitotic inhibitors. Using this protocol, neuronal used does not cross-react to any significant degree with a number of cultures make extensive neurite processes by the third day in culture, other peptides including the tachykinins: neurokinin A, neurokinin B, with most non-neuronal cells dying and no longer adhering to the culture kassinin, eledoisin, or physalacmin; nor does it cross-react with ara- dish. Typically by day 6 in culture, approximately 90% of the viable chidonic acid metabolites. cells are neurons. Chromatography. Neurons were exposed to I ml of Krebs’ bicarbon- Substance P release. After 5-7 d of growth, release studies were per- ate buffer containing 50 mM KCI for 5 min to release SP. This solution formed using a modification of the method of Mudge et al. (1979). was then acidified using acetic acid such that the final concentration Medium was gently aspirated from the culture dish, and the cells were was 2 N. frozen, and lyophilized. After lyophilization, the sample was washed with I ml of a modified Krebs’ bicarbonate buffer consisting of resuspended in mobile phase consisting of 0.2 ml of 69Oh H,O, 30% 135 mM NaCI, 3.5 mM KC], I mM MgCI,, 20 mM NaHCO,, 2.5 mM acetonitrile, and I % phosphoric acid (v/v), and the sample was injected CaCI,, 3.3 mM dextrose, 100 FM ascorbic acid, 20 PM bacitracin, and onto an 8 mm i.d. 4 PM Cl 8 radial Pak L.C. cartridge (Waters Associates, 0.5% bovine serum albumin, aerated with 95% 0,. 5% CO,, pH 7.4- Milford, MA) that was preequilibrated with mobile phase. Peptide was 7.5, and maintained at 36-37°C. Cells were then incubated for two eluted with the mobile phase at a flow rate of I ml/min. Fractions were consecutive 5 min periods with I ml of Krebs’ buffer to measure resting collected at 30 XC intervals, the organic phase evaporated under a release (referred to in the text as basal release), followed by one 5 min nitrogen stream, and the aqueous phase frozen. Frozen samples were exposure to Krebs’ buffer containing either 40 mM or 50 mM KCI (sub- lyophilized, resuspended in assay buffer (see above), and assayed for SP stituted for equimolar sodium), PGE,, PGF,, arachidonic acid, or LTB,. as described above. A parallel run was performed with the SP standard. Cells were then reexposed for two 5 min incubations with Krebs’ buffer Measuremenl of calcium currents. Avian DRG neurons were plated containing 3.5 mM KCI to reestablish basal release. For all experiments, and cultured on small plastic coverslips as described above. After a eicosanoids were initially dissolved in 70% ethanol or in I-methyl-2- period of 5-7 d, a coverslip was removed from the culture dish and ovrrolidinone (Aldrich Chemical Co.. Milwaukee, WI) and diluted to transferred to the bottom of a small recording chamber. In order to appropriate concentrations with Krebs’ buffer. During each 5 min in- eliminate sodium and potassium currents, the neurons were bathed in cubation, cells were rctumed to the incubator. After each 5 min incu- a solution ofthe followingcomposition (in mM): 105 CsCI, 20 tetraethyl- bation, the Krebs’ buffer was carefully aspirated and pipetted into 12 ammonium chloride, IO BaCI,, I MgCl,, 10 HEPES, 10 glucose, and 1 x 75 mm culture tubes containing glacial acetic acid such that the final PM tctrodotoxin (pH adjusted to 7.4 with either free base arginine or The Journal of Neuroscience, May 1992, f2(5) 1919

50 mM KCI 50 mM KCI A 2.5 mM Calcium B No Added Calcium/P.0 mM EGTA

* 160 160 N=8 N=9 = g 140 140 z w 120 120 5 40) 100 loo

2 80 80 z d 60 60 P 8 40 mc ;i 20 20 9 v) 0 cl 0 Basal Basal Stlm Basal Basal Basal Basal Stlm Basat Basal

Figure 1. Potassium-stimulated release of SP from avian DRG neurons is dependent on extracellular calcium. The ordinate represents the amount of SP released/dish of neurons/5 min incubation. Each column represents the mean f SEM of the resting or basal release (Basal, open columns) or the release in the presence of 50 mM KC1 (Slim, hafched columns). A is the release in buffer containing 2.5 mM calcium, whereas B represents the release in buffer containing no added calcium and 2 mM EGTA. The number of dishes examined is indicated in each panel. The asterisk indicates statistically significant differences (p < 0.05) from the basal release.

CsOH). The recording chamber was then placed on the stage of an for each test was set at 0.05. Evoked release is defined as the amount inverted microscope for viewing the cells and placement ofthc recording of SP released during the 5 min of stimulation minus the average of the electrode. The recording chamber had an approximate volume of 500 basal releases prior to stimulation. ~1 and was superfused by a gravity flow/aspiration setup. The calcium current (Ic.) was recorded from chick DRG neurons by utilizing the whole-cell patch-clamp technique (Hamill et al., I98 I). Results Membrane currents were recorded with a EPC-7 patch-clamp amplifier Characterization of substance P release (List Electronic, Darmstadt, Germany). The recording pipettes were Experiments were first performed to characterize potassium- pulled from borosilicate disposable pipettes and were filled with the following solution (in mM): 50 CsCI, 50 N-methyl-D-glucamine chloride, stimulated releaseof SP. As can be seenin Figure 1A, exposure IO tetraethylammonium chloride, 2 MgCI,, IO EGTA, IO HEPES, and of DRG neuronsto Krebs’ buffer containing 3.5 mM KC1 results 4 Na,-ATP (pH adjusted to 7.2 with free base arginine or CsOH). The in a resting or basal releaseof 30 + 6 and 24 -t 6 pg/dish of pipette solution had an osmolality of about 275 mOsm. These pipettes cells/5 min for the first two incubation periods, respectively. typically had resistances of 2-4 MQ. When the extracellular concentration of KC1 is increasedto 50 The recording pipette was placed against the surface of the DRG neuron, and gentle suction was applied by mouth until a sealing resis- mM, the releaseofSP is increasedapproximately four- to fivefold tance of 2-10 CR was obtained. Additional suction was then applied to to 138.4 -t 15 pg/dish/5 min (Fig. 1A). The potassium-stimu- the pipette until there was a large increase in the pipette capacitance, lated releasedoes not occur when neurons are incubated with indicating attainment of the whole-cell configuration. The cell capaci- Krebs’ buffer containing no added calcium and 2 mM EGTA tance was compensated nominally by approximately 70% and had typ- ical series resistance values of 3-5 MR. The whole-cell current recordings (Fig. lB), suggestingthat the evoked releaseof SP is calcium were sampled at 0.5 msec per point. The cells were held at resting dependent. Basalrelease of SP is not altered by the extracellular potentials of either -60 or -90 mV. Depolarizing voltage steps of IO calcium concentration. The total content of SP in each dish of mV and 175 msec in duration were computer generated with the soft- cells ranged from 2 to 4 ng, with basal releasebeing approxi- ware package of pcuhfp (Axon Instruments, Inc., Foster City, CA). mately 1% of total content, whcrcas potassium-stimulatedre- Data storage and analysis were accomplished with the ~CLAMP package. In all the current traces shown, the capacitive and linear leakage currents leaseis approximately 4% of total SP content. have been subtracted by averaging and scaling four hyperpolarizing steps To ascertain if the immunoreactive product released from one-fourth the amplitude of the test potential. All experiments were DRG neurons is SP, we determined whether it cochromato- done at room temperature (22-25°C). graphswith authentic SP usingreverse-phase HPLC. As shown Statistics. All release data are presented as the mean f SEM for four to nine dishes of neurons. To compare basal and stimulated SP release, in Figure 2, the majority of the SP-like immunoreactivity that analysis of variance and Bonferroni’s procedure for multiple t tests were is releasedfrom neurons exposed to Krebs’ bicarbonate buffer performed on basal versus stimulated release (Wallenstein et al., 1980). cochromatographswith authentic SP. Because the first period of basal release was not significantly different from the second period of basal release in any of the experiments, Eflect of PGE, on substance P release statistical comparisons were made between the initial basal release and the stimulated release. Student’s f test was used to compare potassium- BecausePGE, produceshyperalgesia and lowers the threshold evoked release with release evoked by test substance. Significance level of activation in sensoryneurons, it seemslikely that the eicosa- 1920 Nicol et al. * PGE, Stimulates lc. and Relbases SP

Substance P Standard the releaseof neurotransmitter is usually dependent on calcium influx. Consequently, we determined whether PGE, could fa- cilitate the inward calcium current in these neurons. The ad- dition of PGE, produced a significant increasein the amplitude of I,, as recorded in these neurons by utilizing the whole-cell patch-clamp technique. These results are illustrated in Figure 4. Under control conditions, an inward current whosemaximum amplitude of -412 pA is obtained at 0 mV (Fig. 4A,C). When the perfusate is changedto one containing 1 PM PGE,, there is a large and rapid increasein Z,, recorded with this voltage pro- tocol (Fig. 4B,C). The maximum value of Zcaunder thesecon- ditions is increasedto -854 pA. As in the control recording, the maximal value is obtained at 0 mV, indicating no significant changein the activation profile for this inward I,,. The PGE,- induced increasein I,, is observed after 2 min of exposure to the eicosanoid. However, the increasein Zc, was not sustained and returned to normal levels within 10 min of exposure to the Cell Perfusate PG (data not shown). The amplitude of I,, and its dependence 1 on the applied voltage are presentedin the current-voltage re- lation shown in Figure 4C. The current-voltage curves for this DRG neuron demonstrate that the addition of PGE, enhances I,, at all voltage stepsexcept where the driving forces are likely to be small (-+60 and k50 mV). The maximum value of I,, is obtained at 0 mV and undergoes a twofold elevation in the presenceof PGE,. The capacity of PGE, to enhance I,, was observed in seven OJA bwwa--? of nine DRG neuronsstudied, and theseresults are summarized in the current-voltage relations illustrated in Figure 5, A and B. 0 2 4 6 8 10 12 14 16 18 20 The data are normalized to the peak values of I,, under the Fraction Number control conditions. For a holding potential of -60 mV (Fig. Figure 2. The immunoreactive product released by potassium stim- 5A), the mean peak value of I,, (n = 4) for each voltage step ulation of avian DRG neurons coelutes with authentic SP. Samples were was normalized to -376 pA at + 10 mV. In neurons exposed chromatographed using HPLCas discussed in the Materialsand Meth- ods.The ordinaterepresents the SP-like immunoreactivity in pg/frac- to PGEI, the mean I,, value is enhanced about 1.8-fold (Fig. tion measured by the radioimmunoassay. The abscissa represents the 5A). In a similar manner, when the holding potential was -90 fractions collected every 0.5 min. mV (Fig. SB), the peak value of I,, (n = 3) is increasedby about 1.4-fold upon the addition of PGE,. For both holding potentials, noid could increasethe releaseof neurotransmitter from these the PGE,-induced elevation in I,, was not observed for voltage neurons. Consequently, we studied whether PGE, could stim- stepsbelow -20 mV. The lack of any significant increasein I,, ulate the releaseof SP from sensoryneurons grown in culture. for voltage stepsbelow -20 mV (especially at the holding po- The releaseprotocol was performed on dishesof neuronswhere tential of -90 mV) suggeststhat PGE, is affecting primarily the cells were exposed during the third incubation period to L-type calcium channels rather than the T-type calcium chan- either Krebs’ buffer containing 40 mM KC1 or buffer containing nels (Fox et al., 1987). For a holding potential of -60 mV, various concentrations of PGE, (3.5 mM KCl). Resultsof these exposure to PGE, appearsto shift the voltage at which the peak studies are illustrated in Figure 3. Exposure of cells to PGE, value of I,, is obtained. For the control current-voltage curve, producesa dose-dependentincrease in the releaseof SP above the peak I,, occurred at + 10 mV, whereasthe peak value of I,, basalrelease. PGE, at a concentration of 1 PM stimulates the in the presenceof PGE, occurred at 0 mV. This 10 mV shift releaseof SP approximately twofold over basalrelease, whereas may not be significant and may reflect the variability of Z,, for both 5 and 10 PM PGE, increasesthe releaseapproximately the small sample size. This shift was not observed for the -90 fourfold. The evoked release,that is, stimulated releaseminus mV holding potential. These resultsdemonstrate that PGE, can basal release,caused by 1 PM PGE, is 23 f 8 pg/dish/5 min, enhancethe activation of I,-, in DRG neuronsgrown in culture. by 5 FM PGE, is 66 * 7 pg/dish/5 min, and by 10 PM PGE, is 63 -t 3 pg/dish/5 min. Evoked releaseproduced by 40 mM KC1 Effects of arachidonic acid is 45 + 3 pg/dish/5 min (Fig. 3) whereasthe releasecaused by To determine if the effects of PGE, are selective and not the 50 mM KC1 is .113 + 10 pg/dish/5 min (seeFig. 1). For neurons result of formation of other metabolic products of arachidonic exposed to either 1 I.LM or 5 I.LM PGE,, the releaseof SP returns acid, we studied whether the metabolic precursor of PGE,, ar- to the basallevel when the buffer is replacedwith Krebs’ buffer achidonic acid, could alter the releaseof SP or calcium currents lacking PGE,. In contrast, releaseof SP after exposure to 10 FM in DRG neurons. Releaseexperiments were performed using PGE, remains elevated even after the eicosanoid is removed. different groups of cells with or without indomethacin pretreat- ment. The results from theseexperiments are shown in Figure Efects of PGE, on calcium currents 6. When neurons are exposed to 10 PM arachidonic acid, there The PGE,-induced increase in SP release observed in DRG is no significant increase in the releaseof SP (Fig. 6B). Basal neuronsmay be due to an increasein the influx of calcium since releasefor the 5 min period prior to exposure to arachidonic The Journal of Neuroscience, May 1992, V(5) 1921

40 mM KCI 1 lM PGE2 100 N=7

60

60

Basal Basal Stim Basal Basal Basal Basal Stim Basal Basal Figure 3. Various concentrations of ii PGE, stimulate the release of SP from z 5 @l*PGE2 10 pM PGE2 avian DRG neurons. The ordinatesrep- LB loo- 100 resent the amount of SP released/dish N=6 * of neurons/5 min incubation. Each col- T umn represents the mean k SEM of the 60 release of SP for each 5 min incubation. In each case, the data are presented showing the basal release during a 5 min 60 * exposure to Krebs’ buffer containing 3.5 mM KC1 (Basal,open cohnns), or re- lease from neurons exposed for 5 min 40 to buffer containing 40 mM KCl, or for 5 min to Krebs’ buffer containing 3.5 20 20 mM KC1 and various concentrations of PGE, as indicated (Mm, hatchedcol- umns). The number of dishes examined 0 0 .lzl, is indicated in each panel. The asterisk indicates statistically significant differ- Basal Basal Stim Basal Basal Basal Basal Stim Basal Basal ences from basal release. acid is 26 + 4 pg/dish/5 min, whereas the release in the presence range, -322 to -2637 PA) whereas2-5 min after the addition ofarachidonic acid is 3 1 f 4 pg/dish/5 min. In neuronal cultures of 10 KM arachidonic acid the peak value of I,-, is - 1360 + 348 prepared from the same DRG cell harvests, a significant increase pA (mean f SEM; n = 7; range, - 32 1 to - 276 1 PA). For both in release of SP from 34 + 3 pg/dish/5 min to 129 + 14 pg/ the control and 10 PM arachidonic acid conditions, the peak dish/5 min is observed after exposure to 50 mM KC1 (Fig. 6A), value of I,, for each voltage step was normalized to the maximal demonstrating that the absence of any arachidonic acid effect is value of I,, under its respective control condition. The nor- not due to a lack of neuronal viability. Basal release of SP from malized values were then averaged, theseresults are illustrated neurons exposed to arachidonic acid for 5 min does not return in Figure 7. The current-voltage curves for both the control and to the levels observed prior to arachidonic acid, but increases arachidonic acid are very similar. The peak values of I,, for to 44 + 4 and 48 f. 2 pg/dish/5 mitt, which is significantly both the control and arachidonic acid conditions occur at 0 mV. higher than the initial basal release. However, this late increase Arachidonic acid also doesnot have any significant effect on I, in SP release is not observed when cells are pretreated with 10 over the long term. In two of the seven neurons where I,, was &ml indomethacin for 30 min (Fig. 6C). Basal release for the examined at 10 and 20 min after the addition of 10 PM ara- 5 min period prior to exposure to arachidonic acid is 14 -+ 1 chidonic acid, the peak value of I,, under the control condition pg/dish/5 min. Exposure to 10 PM arachidonic acid does not is - 1020 pA (mean of two neurons) whereas 10 and 20 min alter the release and has a value of 15 f 2 pg/dish/5 min. After after the addition of arachidonic acid, I, is -972 and -936 arachidonic acid is removed, the release remains similar to that pA, respectively. observed for the control condition, having values of 11 f 1 and Similar results occur when neurons are exposed to 1 PM ar- 10 + 2 pg/dish/5 min. Potassium-stimulated release is not al- achidonic acid. The peak value for the amplitude of I,, under tered by indomethacin pretreatment (data not shown). These the control condition is - 674 + 113 pA (mean + SEM; n = 9; results suggest that this late rise in SP release observed after range, - 196 to - 1297 PA), whereas 10 min after the addition exposure to 10 .+.LM arachidonic acid may result from the for- of 1 PM arachidonic acid the peak value of I,-., is -693 + 133 mation of a product of the metabolism of arachidonic acid by pA (mean f SEM; n F 9; range, - 196 to - 1308 PA). These cyclooxygenase. results indicate that arachidonic acid has no direct action on We also examined the effects of arachidonic acid on calcium the amplitude or voltage dependence of I,, in these sensory currents in DRG neurons. The addition of either 1 PM or 10 neurons. PM arachidonic acid does not cause any significant increase in I,. In experiments utilizing the larger concentration of arachi- Efects of PGF,, and LTB, donic acid, the peak value for the amplitude of I, under the There is evidence that both PGF,, and LTB, also produce hy- control condition is - 1313 + 293 pA (mean + SEM; n = 7; peralgesia,although this action doesnot appear to be the result 1922 Nicol et al. - PGE, Stimulates lc. and Releases SP

A C MEMBRANE POTENTIAL (mV)

-800 -lwk,k I,,,,,, =,a,,,,,, !/f CURRENT 0 so loo 150 200 250 TIME (ms) B

0 50 loo 150 200 250 TIME (ms) Figure 4. PGE, enhances the calcium current in a single avian DRG neuron. For A and B, the ordinate represents the membrane current in pA, whereas the abscissa is the time in milliseconds. The cell was held at -60 mV, incremental voltage steps of 10 mV were then applied until the maximal value of +60 mV was reached. The timing and duration of the voltage step are shown in the bottom truce. A represents recording of Ic. from a DRG neuron in whole-cell mode under control conditions. B shows Ic, in the same cell 2 min after the addition of 1 PM PGE, to an identical series of voltage steps. This allows sufficient time for the bath volume to be turned over four times. C represents the dependence of 1, on the applied voltage step for both the control condition (open circles) and after addition of PGE, (solid triangles).

of a direct effect on sensory neurons (Levine et al., 1984; Taiwo of 3.5 mM) has no significant effect on the releaseof SP. Basal and Levine, 1986). Based on this finding, and to determine releasein these neurons is 23 t 3 and 19 k 3 pg/dish/5 min further the specificity of the effects of PGE,, we also examined compared to a releaseof 24 f 1 pg/dish/5 min in the presence the effectsof PGF,, and LTB, on DRG neuronsgrown in culture. of PGF,, (Fig. 8B). In a similar manner, exposure of neurons Releasestudies were performed as describedabove. Dishes of to 1 PM LTB, doesnot significantly alter releaseabove the basal neuronswere exposedto either Krebs’ buffer containing 3.5 mM levels. Basalrelease in these neurons is 23 f 4 pg/dish/5 min, KCl, buffer containing 50 mM KCl, or buffer containing 3.5 mM whereasthe releasein the presenceof LTB, is 28 +- 4 p&dish/5 KC1 and 10 PM PGF,, or 1 PM LTB,. The results of these ex- min (Fig. SC’). periments are illustrated in Figure 8. As previously observed, The addition of PGF,, to the medium bathing the DRG neu- exposure of neurons to 50 mM KC1 results in an approximate rons did not alter the amplitude of I,,. In the control condition, fivefold increasein the releaseof SP from a basalrelease of 20 I,, has a maximum value of - 1179 k 126 pA (mean f SEM; L 5 pg/dish/5 min to 102 + 6 pg/dish/5 min (Fig. 8A). The n = 12; range, -436 to -2063 PA), whereas after a 2 min addition of 10 pM PGF,, to the Krebs’ buffer (KC1 concentration exposure to 10 PM PGF,,, Z, has a maximum value of - 1259 The Journal of Neuroscience, May 1992, 12(5) 1923

A MEMBRANE POTENTIAL (mv) MEMBRANE POTENTIAL (mv)

NORMALIZED NORMALIZED -2.0 CURRENT -15 CURRENT Figure 5. Normalized current-voltage relationships for control and PGE,-treated DRG neurons. The values of Zc, obtained for each voltage step were normalized to their respective maximal value of Ze. under control conditions. The values of Zc, obtained after the addition of 1 ELMPGE, (range, 1S-6 min) were then normalized to their respective peak values for the control Z&. The ordinate represents the normalized current values, and the abscissa, the applied voltage step. A and B represent neurons with holding potentials of -60 mV (n = 4) and -90 mV (n = 3), respectively. Open circles represent Ze. from control cells, and solid triangles represent neurons treated with 1 PM PGE,. f 143 pA (mean f SEM; n = 12; range, -480 to -2019 PA). spectively, for the two neurons. No effect is observed after 1 For both the control and PGF,, conditions, the peak value of min. This slight stimulatory action of LTB, may reflect a phys- I,, for each voltage step was normalized to the maximum value iological role for the lipoxygenase product of arachidonic acid, of Z,, under its respective control condition. The normalized although there is little effect compared to the action of PGE,. values were then averaged; theseresults are shown in Figure 9. More likely, it is a nonspecific effect. The lack of a significant The presenceof PGF,, hasno significant effect on the amplitude action of LTB, within minutes of its administration and the lack of I,, recorded at all voltage steps. of effect of either PGF,, or arachidonic acid are consistent with LTB, also does not significantly alter I,,. The addition of 1 the notion that the in situ metabolic products of either the cy- PM LTB, has only a slight effect on I,,. In recordings from two clooxygenaseor lipoxygenase pathways are not responsiblefor DRG neurons,the peak values of I,, are - 13 13 and - 1896 pA the enhanced Zcaobserved upon the application of PGE,. for a holding potential of -90 mV under control conditions (data not shown). In recordings from nine other neurons held Discussion at -90 mV, the peak values of I,, ranged from -800 to -2500 In initial experiments, we characterized the releaseof SP from PA. After 15 min of exposure to 1 I.LMLTB,, the peak value of avian DRG neuronsgrown in culture. In a majority of the dishes I,-= had increasedabout 1.1-fold to - 1530 and - 2067 pA, re- of cells used, the neurons contained approximately 2-4 ng of

50 mM KCI 10pM Arachidonic Acid A 10 MM Arachidonic Acid B C lndomethacin Pretreatment n=6 140 ( n=6 n=6 120. I 100. I 60.

60. 40 20 0Kl : QIxzizmn~ -iEr Basal Basal Stim Basal Basal Basal Basal Stim Basal Basal

Figure 6. Arachidonic acid does not alter the release of SP from DRG neurons. The ordinates represent the amount of SP released/dish of neurons/5 min incubation. Each column is the mean * SEM for cells exposed to Krebs’ buffer containing either the control or the experimental solutions. A represents the control experiments with neurons exposed to buffers containing either 3.5 mM KC1 (Basal, open columns) or 50 mM KC1 (Stim, hatched column). B illustrates the release from neurons exposed to Krebs’ buffer containing either 3.5 mM KC1 (Basal, open columns) or 3.5 rnM KC1 and 10 PM arachidonic acid (Stim, hatched column). C represents the release from cells pretreated with indomethacin (10 &ml) for 30 min and then exposed to 10 PM arachidonic acid; open and hatched columns are the same as in B. The number of dishes examined is indicated in each panel. The asterisk indicates statistically significant differences from the basal release prior to arachidonic acid exposure. 1924 Nicol et al. - PGE, Stimulates IC. and Releases SP

MEMBRANE POTENTIAL (mV) MEMBRANE POTENTIAL (mV)

NORMALIZED NORMALIZED CURRENT CURRENT Figure 7. Arachidonic acid has no effect on calcium currents in DRG Figure 9. PGF,, does not significantly alter the calcium current in DRG neurons. The points in the current-voltage relation represent the nor- neurons. The points in this current-voltage relation represent the nor- malized mean values of I,, for both the control condition (open circles) malized mean values of Zc, for both the control condition (open circles) and after the addition of 10 PM arachidonic acid (solid triangles) for and after the addition of 10 PM PGF,, (solid triangles) for 12 DRG seven DRG neurons. The peak values of Zc, for each voltage step under neurons. The peak values of Zc, for each voltage step under control and control and arachidonic acid conditions were normalized to the max- PGF,, conditions were normalized to the maximum value of Zc. ob- imum value of Zc, obtained under the control condition for each re- tained under the control condition for each respective neuron. These spective neuron. These normalized values were then averaged and are normalized values were then averaged and are shown in the current- shown in the current-voltage relation above. The experimental record- voltage relation above. The experimental recordings were obtained 2 ings were obtained between 2 and 5 min after the addition of arachidonic min after the addition of PGF,,. The holding potential was -60 mV. acid. The holding potential was -60 mV. tracellular calcium. These results are comparable to releaseof SP per dish of cells. This is similar to the total tissue content SP from other neuronal preparations (Jesse11and Iversen, 1977; reported by Mudge et al. (1979) for avian DRG neuronsgrown Gamse et al., 1981; Pang and Vasko, 1986) and suggestthat in cell culture. In addition, both the basal(resting) and potas- sensoryneurons in culture are a valid model for studying trans- sium-stimulated release of SP observed in these experiments mitter release.The immunoreactive substancereleased by the are similar in magnitude to values reported by Mudge et al. cells and measuredusing our radioimmunoassayappears to be (1979) and Holz et al. (1988). BasalSP releaseis approximately authentic SP becauseits elution profile on HPLC is similar to 1% of the total tissuecontent of the peptide, whereasthe release that of the SP standard and becausethe antisera we use in the evoked by 50 mM KC1 representsapproximately 4% of the total assay does not cross-react to any significant degree with any content. This potassium-stimulatedrelease is dependent on ex- known mammalian tachykinin.

60 mM KCI 10 pM PGF 2a 1 pM LTB4 A B C .; 140 1 n=7 Tn=6 n=4

Jzl~rl~E!igYl.n4 Basal Basal Stim Basal Basal Basal Basal Stim Basal Basal Basal Basal Stim Basal Basal

Figure 8. The eicosanoids, PGF,, and LTB, do not alter SP release from DRG neurons. The ordinates represent the amount of SP released/dish of neurons/5 min incubation. Each column is the mean * SEM for cells exposed to Krebs’ buffer containing either the control or the experimental solutions. A represents control experiments for neurons exposed to Krebs’ buffer containing either 3.5 mM KC1 (Basal, open columns) or 50 mM KC1 (Stim, hatched column) to evoke release. B illustrates the release obtained for neurons exposed to either the control conditions of 3.5 mM KC1 (Basal, open columns) or 3.5 mM KC1 and 10 PM PGF,, (Stim, hatched column). C illustrates the release from neurons exposed to either 3.5 rnM KC1 (Basal, open columns) or 3.5 mM KC1 and 1 PM LTB, (Stim, hatched column). The number of dishes examined is indicated in each panel. The asterisk indicates statistically significant differences from the basal release. The Journal of Neuroscience. May 1992. f2(5) 1925

Extensive experimental evidence suggests that PGE, is a direct calcium channels have inactivated. One obvious limitation of chemical mediator involved in sensitizing sensory neurons; con- the current results is that only one concentration of PGE, was sequently, we sought to determine if this cicosanoid could en- examined. We chose to use 1 PM PGE, basedon the rclcase hance neurotransmittcr release from sensory neurons. We chose studies showing that this was the lowest concentration tested to study the release of SP from DRG cells because abundant that evoked release.Because this low concentration waseffective evidence suggests that this peptide is an important neuroactive in enhancing I,,, higher concentrations were not examined. compound in nociceptive sensory neurons (see reviews by Salt In both chick and rat DRG neurons, the release of SP is and Hill, 1983; Cuello, 1987). In addition, SP appears to be an believed to be mediated by L-type calcium channels. The po- important mediator of components of the inflammatory re- tassium-stimulated release of SP from these neurons can be sponse, especially neurogenic inflammation (Payan, 1989). Our blocked with the addition of dihydropyridine calcium channel results demonstrate that PGE, stimulates the release of SP from antagonistssuch as nitrendipine and nifedipine (Pemey et al., avian DRG neurons in a dose-dependent manner. Exposure of 1986; Rane et al., 1987). Thus, our findings that PGE, can neurons to 1 PM PGE, results in a small but significant increase increase both the releaseof SP and I,-, in chick DRG neurons in SP, whereas at higher concentrations the evoked release is is consistent with the notion that PGE, may be modulating the similar to that observed with elevated concentrations of extra- activity of L-type calcium channels. This is basedon the fact cellular potassium. The concentrations of PGE, utilized in the that the capacity of PGE, to enhanceI,, was similar for holding current studies are similar to those required to sensitize sensory potentials of either -60 or -90 mV (seeFig. 54,B) and this neurons to noxious stimuli as observed in previous reports. For enhancement occurred only for voltage stepsthat were greater example, Yanagisawa et al. (1986) demonstrated that 0.84 PM than -20 mV (seeFigs. 4C, 5A). Also, the inactivation of I,., PGE, enhanced the nociceptive reflex in an isolated spinal cord- was relatively slow and lacked the rapid transient relaxation tail preparation from newborn rat. A lesser concentration of (data not shown) indicative of N-type channel activity (Fox et PGE, (0.1 PM) potentiated the potassium-induced firing of rat al., 1987). sensory neurons grown in culture (Baccaglini and Hogan, 1983). It seemslikely that the enhancement of I,, by PGE, may In addition, micromolar concentrations of PGE, are required contribute to the increasein SP releaseproduced by this eicosa- to increase the influx of calcium in neuroblastoma-glioma hy- noid. However, !his was not specifically determined in our ex- brid cells (Miwa et al., 1988) and to stimulate catecholamine periments, becausenot all the neurons grown in cell culture release from bovine adrenal chromaffin cells (Koyama et al., synthesize and releaseSP. Therefore, it is possiblethat the ob- 1988). servedchanges in I,, are recorded from neuronsother than those The PGE,-induced increase in SP release is relatively selective containing SP. An analogousmechanism for the enhancement for this prostanoid because neither PGF,,, arachidonic acid, nor of transmitter releaseby activation of I,, has been observed in the lipoxygenase metabolic product LTB, significantly enhances bovine chromaffin cells and may be particularly relevant to these SP release upon acute exposure. Neurons exposed to 10 PM nociceptive sensoryneurons. Addition ofdopamine to the chro- arachidonic acid for 5 min show a slight but significant increase maffin cell stimulates a specific set of calcium channels whose in SP release during the two 5 min collections following removal activity appearsto be controlled by CAMP and CAMP-depen- of arachidonic acid, an effect that is attenuated by the indo- dent protein kinase (Artalejo et al., 1990). This facilitation of methacin pretreatment. This latter effect suggests that arachi- I,-, may contribute to the releaseof more neurotransmitter, that donic acid is being metabolized to a PG by the cells in culture. is, a positive feedback mechanismto boost epinephrine levels In fact, Vesin and Droz (199 1) have demonstrated that after a beyond thosenormally released.In sensoryneurons, PGE, may 10 min incubation period with labeled arachidonic acid, avian act through a similar type of pathway where PGE, acts to en- DRG neurons grown in culture can metabolize arachidonic acid hance Ica and thus facilitate the releaseof SP. to various PGs, especially PGE,. Of primary importance, how- The mechanismof action for the increase in transmitter re- ever, is our result demonstrating that arachidonic acid does not leaseand the activation of calcium channels remainsunknown. have an immediate action (at least within 10 min), supporting It is possible that PGE, activates calcium channels directly, the idea that the enhanced release of SP is specific for PGE,. although to our knowlcdgc, there is no evidence available to The results presented in this study demonstrate that PGE, support this possibility. However, PGs are known to modulate can increase the amplitude of I,, elicited by voltage steps in the activity of many different types of secondmessenger systems these sensory neurons. As with the release studies, the effect of (seereviews by Halushka et al., 1989; Shimizu and Wolfe, 1990). PGE, on I,, appears to be relatively specific since neither PGF,,, PGs alter intracellular CAMP content in superior cervical ganglia arachidonic acid, nor the lipoxygenase product LTB, enhances (Tomasi et al., 1977) neuroblastoma-glioma cells (Sharma et I,, in an analogous manner. The voltage dependence for acti- al., 1975) and bovine adrenal medulla (Negishi et al., 1989). vation of I,. does not appear to be affected by PGE, since the In addition, in animal models of pain, the enhancedsensitivity maximal value of I,, is obtained at the same voltage step for to noxious stimuli produced by PGE, may be mediatedby CAMP both the control and experimental conditions. This enhance- (Ferreira and Nakamura, 1979; Taiwo et al., 1989). In bovine ment of Ica occurred with a rapid onset, reachingmaximal levels chromaffin cells, the addition of PGE, increasesthe influx of within 2 min, but appearedto be a transient phenomenon since calcium into the cell through potentiation ofthe calcium current. the peak value of I,, had returned to nearly the control value Presumably, this effect is mediated via PG stimulation of the after about 10 min in the PGE, solution. A similar time course inositol trisphosphate (IP,) pathway wherein IP, presumably has been reported for PGE,-induced I, activation in bovine activates the calcium channels directly (Mochizuki-Oda et al., chromaffin cells (Mochizuki-Oda et al., 199I). This result sug- 1991). These resultsin bovine chromaffin cellsare, at this point, geststhat the neuron has somehowdesensitized to the activating speculativebecause the calcium currents activated by PGE, have effectsof PGE, and implies either that the transduction cascade not been shown to be the samepopulation of currents activated has decreasedits sensitivity to PGE, or that PGE,-sensitive by IP,. Further studies are warranted to determine whether 1926 Nkd et al. l PGE, Stimulates Ic. and Releases SP

PGE, activates or recruits additional calcium channelssuch as Halushka PV, Mais DE, Mayeux PR, Morinelli TA (1989) Throm- is the casefor facilitation-type calcium channels(Artalejo et al., boxane, prostaglandin and leukotriene receptors. Annu Rev Phar- macol Toxicol IO:2 13-239. 1990) or whether PGE, shifts the probability toward the open Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (198 1) Im- state for active calcium channels through modulating the activ- proved patch-clamp techniques for high-resolution current recording ity of a secondmessenger system. from cells and cell-free membrane patches. Pfluegers Arch 391:85- It is interesting to speculatethat the increase in neurotrans- 100. mitter releaseand activation of voltage-sensitive calcium chan- Handwerker HO (1976) Influences of algogenic substances and pros- taglandins on the discharges of unmyelinated cutaneous nerve fibers nels may be one mechanismto explain the sensitizingaction of identified as nociceptors. In: Advances in pain research and therapy, PGE, on sensory neurons. It is largely accepted that PGs are Vol 1 (Bonica JJ, Albe-Fessard D, eds), pp 41-45. New York: Raven. synthesizedand releasedin responseto tissueinjury, contribute H&s GA, Moncada S (1983) Interactions of arachidonate products to hyperalgesia,and are involved with the acute inflammatory with other pain mediators. In: Advances in pain research and therapy, Vol 5 (Bonica JJ, Lindblom U, Iggo A, eds), pp 6 17-626. New York: response(Ferreira, 1983; Higgs and Moncada, 1983; Payan, Raven. 1989). The hyperalgesiceffect of PGE, appearsto result from a Holz GG, Dunlap K, Kream RM (1988) Characterization of the elec- direct action on sensoryneurons becausethe onset of effect is trically evoked release of substance P from dorsal root ganglion neu- rapid (Taiwo et al., 1987) and becauseneither indomethacin rons: methods and dihydropyridine sensitivity. 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