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Substance P potentiates ATP-activated currents in rat primary sensory neurons
BRAIN RESEARCH Brain Research 739 (1996) 163-168
Research report
Substance P potentates ATP-activated currents in rat primary sensory neurons Hong-Zhen Hu, Zhi-Wang Li * Research Center of Experimental Medicine, Tongji Medical University, 13 Hangkong Road, Wuhan 430030, People’s Republic of China Accepted 25 June 1996
Abstract The aim of this study was to explore whether substance P could modulate the response mediated by ATP receptor. Experiments were carried out on rat dorsal root ganglion (DRG) neurons isolated acutely with enzymatic and mechanical treatment. The ATP-activated
inward currents were recorded using the whole-cell patch-clamp technique. The majority of the neurons examined (82/85, 96.5%) were sensitive to ATP (1– 1000 p,M). Application of substance P (0.01– 100 pM) also caused an inward current. Several differences between these two kinds of currents were observed. 0.01, 0.1 and 1 p,M substance P increased the ATP (10 pM)-activated current to 113.7 ~ 3.1%, (n = 8); 127.2 ~ 6.7%, (n = 12) and 154.7 + 14.4% (n = 6) (means ~ S.E.M.), respectively. This potentiating effect can be blocked by spantide, an NK1 receptor antagonist, and intracellular application of H7 (which is a potent inhibitor of PKC) could also block this kind of potentiation of SP on ATP-activated current. Since the substance P receptor and ATP receptor can coexist in rat DRG neurons and activation of substance P receptor can modulate the response mediated by ATP receptor, it suggests that they may cooperate with each other in activating peripheral nociceptive endings of sensory neurons, especially during tissue damage and/or inflammation. Keywords; Substance P; Dorsal root ganglion; ATP receptor; Whole-cell patch-clamp technique; Modulation
1. Introduction
It is generally
agreed
that adenosine-5’-triphosphate
(ATP) plays a role of extracellular chemical messenger, either as a neurotransmitter or a co-transmitter acting on peripheral neurons including primary sensory, sympathetic and parasympathetic neurons and on central neurons in a variety of brain areas [1,10]. The receptor for ATP is referred to as P2 purinoceptor which is further classified into PZXand P2Ysubtypes pharmacologically according to the potency order of the agonists [5]. The activation of ATP receptor which belongs predominantly to the superfamily of ligand-gated ion channel receptors results in the opening of nonselective cationic channels, thereby depolarizing its target cells. Growing evidence indicates that ATP exerts an excitatory effect on effecter cells innervated by purinergic nerves such as the cardiac and smooth muscle and exocrinic glands while it may also mediate the fast and slow transmission in neuro-neuronal synapses both in the peripheral and central nervous systems [2–4]. Since the evidence for the existence of ATP receptor in
* Corresponding author. Fax: +86 (27) 5858920.
the membrane of sensory neurons including dorsal root ganglion (DRG) neurons first proved by Krishtal et al. [11], a number of works have been carried out in order to elucidate the characteristics, pharmacology and kinetics for ATP receptor. It is well known that many other ligand-gated ion channel receptors such as nAChR, GABA~R, NMDAR, 5-HT~R, etc., could be modulated by a number of agents [20] and it has been identified in recent year-s that ATP-activated inward current could be potentiated by micromolar concentration of Zn2+ and inhibited by ETOH [12-14]. Recently, we found that some neurotransmitters can also exert modulatory effects on ATP-activated currents [6]. In the present work the effect of peptide neurotransmitter substance P (SP) on the ATP-mediated current was studied. A preliminary report has been published elsewhere [8].
2. Materials and methods 2.1. Isolation of DRG neurons 2–3-week-old Sprague–Dawley rats,irrespective of sex, were decapitated, and the thoracic and lumbar segments of
0006-8993/96/$15.00 Copyright CI 1996 Elsevier Science B.V. All rights reserved. PH S0006-8993(96)00821 -9
164
H.-Z. Hu, Z.-W. Li/Brain Research 739 (1996) 163-168
vertebrate column were dissected and longitudinally divided into two halves along the median lines on both dorsal and ventral sides. The DRGs, together with dorsal and ventral roots and attached spinal nerves, were taken out from the inner side of each half of the dissected vertebrate and transferred immediately into Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) at pH = 7.4, 340 mOsm/kg. After the removal of attached nerves and surrounding connective tissues the DRGs were minced with iridectomy scissors and incubated with enzymes including trypsin (type III, Sigma) 0.5 mg/ml, collagenase (type IA, Sigma) 1 mg/ml and DNase (type IV, Sigma) 0.1 mg/ml in 5 ml DMEM at 35°C in a shaking bath for 40 min. To stop the enzymatic digestion 1.25 mg/ml soybean trypsin inhibitor (type II-S 1, Sigma) was added. The isolated neurons were transferred into a 35-mm culture dish and kept still for at least 30 min. Experiments were performed at room temperature (20–30”C) [13,21]. 2.2. Recording Whole-cell patch-clamp recordings were carried out using a PC-II patch clamp amplifier (Huazhong University of Science and Technology, Wuhan, People’s Republic of China). The micropipette was filled with internal solution, the composition of which was as follows (in mM): KC1 140, MgClz 2.5, HEPES 10, EGTA 11, ATP 5, its osmolarity was adjusted to 310 mOsm with sucrose and pH was adjusted to 7.4 with KOH. The external solution contained (in mM) NaCl 150, KC1 5, CaCl, 2.5, MgCl, 2, HEPES 10, D-glucose 10, its osmolarity was adjusted to 340 mOsm with sucrose and pH was adjusted to 7.4 with NaOH. The resistance of recording electrodes were in the range of 1–4 MQ. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1–10 GQ) and then a more negative pressure was applied to rupture it, thus a whole-cell mode was established. The adjustment of series resistance and capacitance compensation was done before the start of experiments. Membrane currents were filtered at 10 HZ ( – 3 dB). Data were stored and analyzed on a super 386 computer with a data acquisition software and hardware system (Huazhong University of Science and Technology, Wuhan) or recorded by a pen recorder. Experiments were carried out at holding potential (HP) = –60 mV. 2.3. Drugs Drugs used in the experiments were substance P (SP; Sigma), spantide (Sigma), ATP (Boehringer Mannheim), l-(5 -isoquinolinesulfony l)-2-methylpiperazine (H7) (RBI). The drugs were dissolved in external solution and applied by gravity flow from a row of tubules (OD/ID = 500 pm/200 pm), which were connected to a series of independent reservoirs. The distance from the mouth of tubule to the cell examined was approx. 100 pm. This rapid
solution exchange system was manipulated by shifting the tubules horizontally with a micromanipulator. 2.4. Stati~tics Values of ATP-activated currents were reported as means t S.E.M. The Student’s t-test was used.
3. Results 3.1. ATP- and SP-actiuated currents in the membrane of DRG neurons
In the present study, the majority of the cells examined responded to externally applied ATP (1– 1000 ~M) in a concentration-dependent manner (82/85, 96.5%); many of them were also sensitive to substance P. In other words, there was a coexistence of ATP and SP receptor in the membrane of these neurons. The neurons chosen for this experiment were requested to be sensitive both to ATP and SP, the size distribution of them modified from Scroggs et al. [19] were as follows: large cells ( >45 p,m) 4; intermediate cells (30–45 pm) 54 and small cells ( <30 pm) 27. Fig. 1 shows the ATP- and SP-activated currents recorded from one DRG neuron. The amplitude of ATP-activated current is larger than that of SP while the desensitization in the latter is less apparent than that in the former; it takes 4 min to get a full recovery of ATP (10 pM)-activated currents. 3.2. Ionic mechanism of SP-euoked current The I–V relation for the SP-evoked current was studied and the reversal potential was found to be around +35 mV at normal external sodium concentration ([Na+]O= 150 mM) (n = 8). When INa+]O decreased step-by-step from 150 mM to 1.5 mM by replacing sodium chloride with choline chloride the SP-evoked currents decreased gradually and the reversal potential shifted to be more and more negative. Application of Cdz+ (0.05 KM) containing external solution also suppressed the SP-activated currents to a smaller but detectable extent [9]. From the results mentioned above it is assumed that conductance changes of nonselective cations, especially sodium and/or calcium ions might be involved. Fig. 2 shows an example in which the Z–V relation from a single cell is illustrated. 3.3. SP potentates ATP-activated currents Fig. 3 illustrates the increase in the amplitude of ATP (10 KM)-activated currents by the preapplication of SP (0.001-10 ~M). If the concentration of SP applied was too low, i.e. 0.001 p.M or less, no effect could be detected. When the concentration of SP increased beyond 0.01 FM the potentiating effect of SP on the 10 PM ATP-activated
H.-Z. Hu, Z.-W. Li/Brain Research 739 (1996) 163-168
165
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Fig. 2. Z–V relation for SP-activated current obtained from a single neuron. The current traces in the top panel show SP-evoked currents at different levels of clamped voltage. The Z–V relation below show a linear relation and the reversal potential was found to be arround +30 mV at normal external sodium concentration (150 mM) which is close to the mean vatue of +35 mV obtained from other 8 neurons.
currents increased gradually with the increase of SP concentration. However, when concentration of SP increased to 10 p,M, its potentiating effect decreased markedly as compared with that of 1 PM SP. In these neurons 0.01, 0.1 and 1 p,M substance P can increase the ATP (10 ~M)activated current to 113.7 t 3.1YO(n = 8); 127.2 + 6.790 (n= 12) and 154.7 & 14.4% (n= 16) (means+ S.E.M.), respectively. 3.4. Dose–response curve of ATP-activated currents with or without pretreatment of SP Fig. 4 shows the concentration–response curve of ATP-activated current with or without substance P pretreatment. From the concentration–response relationship for ATP alone it shows that the value of threshold, EC~O and maximum concentration were 1 PM, 30 FM and 500 pM respectively. As compared with which, the concentration response curve for ATP pretreated with SP shifts to the left apparently. The threshold value in both curves are very close, the EC~Ovalue in the dose–response curve for ATP is lower than that of ATP pretreated with SP (15 KM) while the response of maximum concentration in the later is higher than that in the former.
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Fig, 3. Potentiation of SP on the ATP-activated current. Records in the upper row illustrate the current activzted by 10 PM ATP with pretreatment of different concentration of SP (0.001–10 p,M). Records are sequential (from left to right) current traces obtained from a single DRG neuron (HP = —60 mV). The graph below shows the dose–response curve for SP potentiation on the ATP (10 p,M)-activated current. Each point represents the means f S.E.M. of’ 8–16 DRG nerumns voltageclamped at –60 mV. It is obvious that the ATP-activated current was potentiated step-by-step by SP from O.001 to I p,M. However, when the concentration of SP increased to 10 PM, the potentiation disappeared.
Fig. 4. Concentration–response relationships for ATP with and without preapplication of SP. In the concentration–response curve for ATP eacb point represents the means + S.E.M. of ATP-activated current of 8–16 neurons. The curve shown is the best fit of the data to the logistic equation Y= Em:,X/[1 + ( K6 / c)rr], where C is the concentration of ATP, 2’ is the fraction of the maximum value, and K~, the dissociation constant of the ATP receptor, is 30 KM. The curve was drawn according to tbe equation described above assuming Hill coefficients ( n = 0.72). As compared with concentration–response curve for ATP the dose-response curve for ATP with pretreatment of SP shifted to the left obviously. ‘ P <0,05, “ “ P <0.01, compared with the ATP-activated current without preapplication of SP.
3.5. Time-course of the potentiation qf ATP-actiuated current by SP Fig. 5 shows the time-course of the potentiation of ATP-activated current by SP (lo PM). The potentiation of ATP-activated current appeared at 30– 120 s after preapplication of SP and reached its peak at about 6 min. It achieved a full recovery after 10 min when the concentration used was O.01 pM; it took 20 min to recover if the concentration used was 1 wM. However, if the period for preapplication of SP lasted less than 30 s no effects could be observed.
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3.6. Inuo[vemerrtof second messenger system Spantide, the SP antagonist could suppress, and block in two neurons, SP-activated current (n = 8) (data not shown). It also abolished the potentiating effect of substance P on ATP-evoked response in DRG neurons as shown in Fig. 6a (n = 6). In order to explore whether this facilitator effect is mediated through NK1 receptor-induced intracellular transduction, such as activating protein kinase C (PKC) we used 1-(5-isoquinolinesulfony l)-2-methyl-piperazine dihydrochloride (H7) (200 p,M) [7, 15], the potent inhibitor of
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Fig. 5. Time-course of SP (0.01 wM) potentiation nt ATP (10 wM)activated current. Records in the upper row illustrate tbe current activated by 10 WM ATP witb pretreatment of SP (0.01 wM). Records are sequential (from left to right) current traces obtained frnm a single DRG neuron (HP = —60 mV). The curve below shows that the potentiation begins at about 2 min after application of SP and reachs its peak at 6 min. It takes about 10 min to get a full recovery.
H.-Z. Hu, Z.-W. Li/Brain Research 739 (1996) 163-168
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Fig. 6. Blockade of the potentiating effect of SP on ATP-activated current by coapplication of spantide or internal H7. The current traces in (a) show that SP (0.1 KM) can potentate ATP (10 pM)-activated current (upper row) and spantide can block this potentiating effect in the same cell (lower row). (b) shows current traces obtained from a neuron in which SP (1 PM) induced a small inward current and potentiated ATP (10 ~M)-activated current at 2 and 6 tin after SP application (upper row) and the same neuron in (b) was repatched with a pipette which contained H7 (200 PM) 30 min later. Although SP-induced current remained, the potentiating effect of SP was blocked (lower row). The bar chart in (c) shows the effect of intracellular application of PKC inhibitor, H7 on the potentiating effect of SP on ATP-activated current. Inclusion of H7 (200 wM) in the recording pipette was effective in reversing the potentiation of ATP (10 WM)-activatedcurrent by SP. Under this condition 1 I.LMSP increase 7.8 + 2.5% (n = 10), compared 55.2 + 12.3% (n = 14) witi normal internal solutiori (t = 3.10, P < 0.01).
PKC in the internal solution and the potentiating effect was suppressed significantly (P < 0.01) as shown in Fig. 6b,c.
4. Discussion For investigating the modulatory effect of SP on ATPactivated currents it was required that the experiments were carried out on neurons endowed with these two (SP and ATP) receptors. It is practicable because there are a large number of DRG cells in response to SP and ATP according to the data obtained from our previous work, in which the cells sensitive to SP and ATP are 90.6% [18] and 94.5Y0[6] respectively. It is evident from Fig. 3 that the enhancement of amplitude of ATP-activated currents increases gradually with the increase of the concentration of SP from 0.01 to 1 PM. However, when the concentration of SP increased to 10 pM the modulatory effect of SP on ATP-activated currents did not increase further. On the contrary, it reduced dramatically as compared with the effect of SP in 1 PM. We have also observed this anomalous phenomenon of SP modulation in another work (unpublished observation) that the inhibition of SP on GABA (100 KM)-
activated current increased with the increase of SP concentration from 0.0001 to 1 p,M, but was abolished once the SP concentration increased to 10 I.LM;in fact, a facilitator effect on GABA-activated currents occurred. We think this might be a non-specific action of agonist emerged at such considerable high concentration. It is of interest that the enhancement of ATP-activated currents by SP was mainly due to the increase in peak value (Ip) while the increase in steady state (1ss) was relatively small. This is apparent in the records demonstrated in Fig. 3. It is considered that this enhancement may result from an increase in the fast component of the desensitizing current other than the slow component. Comparison between the dose–response curves for ATP-activated currents, with and without preapplication of SP in Fig. 4, indicates that the potentiation of ATPactivated current by preapplication of SP is evident from the fact that the curve appears to shift to the left. Since the K~ value in the curve for ATP-activated current pretreated with SP decreased and the response in the maximum concentration increased. This obviously reflects the increase in affinity for the ATP receptor, which may result from the phosphorylation of ATP receptor-ion channel complex following the activation of SP receptor. In the present study the time-course of the potentiation
168
H.-Z. Hu, Z.-W, Li/Brain Research 739 (1996) 163-168
of ATP-activated current by SP took about 6 min to reach the peak, and the full recovery needed 10 to 20 rein, which was dependent on the concentration of SP preapplied. It is worth noting that the onset of the potentiation needed at least 30–120 s after the preapplication of SP. This implies that the modulation must take place through a pathway of complex intracellular transduction while the direct allosteric modulatory effect of SP on its receptor might be excluded. It is known that SP receptor belongs to the superfamily of G protein-coupled receptors [16]. SP binds to NK1 receptor couples via G protein to PLC, which, in turn, breaks down PIP2 into IP3 and DAG, the latter activates PKC and may result in phosphorylation of ion channel in ATP receptor. In the experiment we found that spantide, the NKI receptor antagonist, could block the SP-evoked membrane-response and the facilitator effect of SP on ATP-activated current. Furthermore, H7, which is a potent inhibitor of PKC, could suppress the effect of SP on ATP-activated current, also supporting this hypothesis. What is the physiological significance of the result obtained in the present investigation? It is now open for discussion. In this work we use the cell body of DRG neuron as a simple and accessible model for studying the characteristics in the terminals of axonal membrane, either peripheral or central. As we know that the nerve endings of the peripheral axon of DRG cell are sensitive to many chemical substances, especially those produced in tissue damage and inflammation, of which ATP and SP are two potent stimulating agents, the receptors for them coexist in the membrane of nerve endings [17]. In the case of inflammation or tissue damage these two substances exert a stimulating effect on the nerve endings not only separately, but also in cooperation. Furthermore, since a number of SP released from the terminals can act on the SP autoreceptors [9] in the membrane of the terminal, it may favour the modulatory effect as mentioned above. Acknowledgements
We are grateful to Mrs. Y.Z. Fan for preparing isolated neurons, Dr. W.Z. Wei for technical help, and Prof. X.R. Jin for reading the manuscript. This work was supported by Grant 39270244 from the National Natural Science Foundation of China and a fund from the Ministry of Health of China. References [1] Bean, B.P. and Friel, D.D., ATP-activated channels in excitable cells. In T. Namhashi (Ed.), Ion Channels, Vol. 2, Plenum Press, 1990, pp. 501–519.
[2] Dubyak, G.R. and E1-Moatassim, C., Signal transduction via P2purinergic receptors for extracellular ATP and other nucleotides, Am. J. Physiol,, 265 (1993) 557-606. [3] Edwards, F.A., Gibb, A,J. and Colquhoun, D,, ATP receptor-mediated synaptic currents in the nervous system, Narure, 359 (1992) 144-147. [4] Evans, R,, Derkach,V. and Surprenant, A,, ATP mediates fast synaptic transmission in mammalian neurons, Nature, 357 (1992) 503-505. [5] Fredholm, B.B., Abbracchio, M.P., Burnstock, G., Daly, J.W., Harden, T.K., Jaccobson, K.A., Leff,P. and Williams, M., Nomenclature and classification of purinoceptors, Pharmacol. Reo., 46 (1994) 143-156. [61 Gu, Q.H., Li, Z.W. and Fan, Y.Z., Enhancement of dopamine on ATP-activated currents in the neurons acutely isolated from rat DRG, Chin. J. Neurosci., (1995) 32. [7] Hidaka, H. and Hagiwam, M., Pharmacology of the isoquinoline sulfonamide protein kinase C inhibitors, Trends Pharrrracol.,Sci,, 8 (1987) 162-164. [8] Hu, HZ,, Li, Z.W. and Fan, Y.Z., Substance P potentates ATPactivated currents rat in primary sensory neurons, Chin. J Nearosci., (1995) p. 35. [9] Hu, HZ., Li, Z.W. and Si, J.Q., Evidence for the existance of substance P autoreceptor in the membrane of rat DRG neurons, Neuroscience, (1996) in press. [10] Illes, P. and Noernberg, W., Neuronal ATP receptors and their mechanism of action, Trends Pharmacol. Sci., 14 (1993) 50–54. [11] Krishtal, O.A., Marchenko, S.M. and Pidoplichko, V.L., Receptor for ATP in the membrane of mammalian sensory neurons, Neurosci. Left., 35 (1983) 41-45. [12] Li, C., Aguayo, L., Peoples, R.W, and Weight, F.F., Ethanol inhibits a neuronal ATP-gated ion channel, Mol. Pharmacol., 44 (1993) 871-875. [13] Li, C., Peoples, R.W., Li, Z.W, and Weight, F.F., Zn2+ potentates excitatory action of ATP on mammalian neurons, Proc. Natl. Acad. ~Ci. USA, 90 (1993) 8261-8267. [14] Li, C., Peoples, R.W. and Weight, F.F., Alcohol action on a neuronal membrane receptor: evidence for a direct interaction with the receptor protein, Proc. Natl. Acad. Sci. USA, 94 ( 1994) 8200– 8204. [15] Nishikawa, M., Uemura, Y., Hidaka, H. and Shirakawa, S., l-(5 -lsoquinolinesulfonyl)-2-ethylpiperazine (H7), a potent inhibitor of protein kinase, inhibits the differentiation of HL-60 cells induced by phorbol diester, .L~eSci., 39 (1986) 110I–1 107. [16] Otsuka, M. and Yoshioka, K., Neurotransmitter functions of mammalian tachykinins, Physiol. Re(?.,73 (1993) 229–308. [17] Rong, H.P., Bevan, S. and Dray, A., Chemical activation of nonciceptive peripheral neurons, Br. Med. Bull., 47 (1991) 534–548. [18] Si, J.Q, and Li, Z.W., Actions of substance P on the somatic membrane of rat DRG neurons, Acta Physiol. Sinica, 48 (1996) 8-14. [19] Scroggs, R.S. and Fox, A.P., Calcium current variation between acutely isolated adult rat dorsal root ganglion neurones of different size, .J. Physiol., 445 (1992) 639–658. [20] Swope, S.L., Moss, S.J., Blackstone, C.D. and Huganir, R.L., Phosphorylation of Iigand-gated ion channels: a possible mode of synaptic plasticity, FASEB J., 6 (1992) 2514–2523. [21] Wu, X.P., Li, Z.W. and Fan, Y.Z., Inhibitory effect of substance P on GABA-activated currents in neurons acutely isolated from rat dorsal root ganglion, Chin. J. Physiol. Sci., 10 (1994) 371-375.
Research report
Substance P potentates ATP-activated currents in rat primary sensory neurons Hong-Zhen Hu, Zhi-Wang Li * Research Center of Experimental Medicine, Tongji Medical University, 13 Hangkong Road, Wuhan 430030, People’s Republic of China Accepted 25 June 1996
Abstract The aim of this study was to explore whether substance P could modulate the response mediated by ATP receptor. Experiments were carried out on rat dorsal root ganglion (DRG) neurons isolated acutely with enzymatic and mechanical treatment. The ATP-activated
inward currents were recorded using the whole-cell patch-clamp technique. The majority of the neurons examined (82/85, 96.5%) were sensitive to ATP (1– 1000 p,M). Application of substance P (0.01– 100 pM) also caused an inward current. Several differences between these two kinds of currents were observed. 0.01, 0.1 and 1 p,M substance P increased the ATP (10 pM)-activated current to 113.7 ~ 3.1%, (n = 8); 127.2 ~ 6.7%, (n = 12) and 154.7 + 14.4% (n = 6) (means ~ S.E.M.), respectively. This potentiating effect can be blocked by spantide, an NK1 receptor antagonist, and intracellular application of H7 (which is a potent inhibitor of PKC) could also block this kind of potentiation of SP on ATP-activated current. Since the substance P receptor and ATP receptor can coexist in rat DRG neurons and activation of substance P receptor can modulate the response mediated by ATP receptor, it suggests that they may cooperate with each other in activating peripheral nociceptive endings of sensory neurons, especially during tissue damage and/or inflammation. Keywords; Substance P; Dorsal root ganglion; ATP receptor; Whole-cell patch-clamp technique; Modulation
1. Introduction
It is generally
agreed
that adenosine-5’-triphosphate
(ATP) plays a role of extracellular chemical messenger, either as a neurotransmitter or a co-transmitter acting on peripheral neurons including primary sensory, sympathetic and parasympathetic neurons and on central neurons in a variety of brain areas [1,10]. The receptor for ATP is referred to as P2 purinoceptor which is further classified into PZXand P2Ysubtypes pharmacologically according to the potency order of the agonists [5]. The activation of ATP receptor which belongs predominantly to the superfamily of ligand-gated ion channel receptors results in the opening of nonselective cationic channels, thereby depolarizing its target cells. Growing evidence indicates that ATP exerts an excitatory effect on effecter cells innervated by purinergic nerves such as the cardiac and smooth muscle and exocrinic glands while it may also mediate the fast and slow transmission in neuro-neuronal synapses both in the peripheral and central nervous systems [2–4]. Since the evidence for the existence of ATP receptor in
* Corresponding author. Fax: +86 (27) 5858920.
the membrane of sensory neurons including dorsal root ganglion (DRG) neurons first proved by Krishtal et al. [11], a number of works have been carried out in order to elucidate the characteristics, pharmacology and kinetics for ATP receptor. It is well known that many other ligand-gated ion channel receptors such as nAChR, GABA~R, NMDAR, 5-HT~R, etc., could be modulated by a number of agents [20] and it has been identified in recent year-s that ATP-activated inward current could be potentiated by micromolar concentration of Zn2+ and inhibited by ETOH [12-14]. Recently, we found that some neurotransmitters can also exert modulatory effects on ATP-activated currents [6]. In the present work the effect of peptide neurotransmitter substance P (SP) on the ATP-mediated current was studied. A preliminary report has been published elsewhere [8].
2. Materials and methods 2.1. Isolation of DRG neurons 2–3-week-old Sprague–Dawley rats,irrespective of sex, were decapitated, and the thoracic and lumbar segments of
0006-8993/96/$15.00 Copyright CI 1996 Elsevier Science B.V. All rights reserved. PH S0006-8993(96)00821 -9
164
H.-Z. Hu, Z.-W. Li/Brain Research 739 (1996) 163-168
vertebrate column were dissected and longitudinally divided into two halves along the median lines on both dorsal and ventral sides. The DRGs, together with dorsal and ventral roots and attached spinal nerves, were taken out from the inner side of each half of the dissected vertebrate and transferred immediately into Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) at pH = 7.4, 340 mOsm/kg. After the removal of attached nerves and surrounding connective tissues the DRGs were minced with iridectomy scissors and incubated with enzymes including trypsin (type III, Sigma) 0.5 mg/ml, collagenase (type IA, Sigma) 1 mg/ml and DNase (type IV, Sigma) 0.1 mg/ml in 5 ml DMEM at 35°C in a shaking bath for 40 min. To stop the enzymatic digestion 1.25 mg/ml soybean trypsin inhibitor (type II-S 1, Sigma) was added. The isolated neurons were transferred into a 35-mm culture dish and kept still for at least 30 min. Experiments were performed at room temperature (20–30”C) [13,21]. 2.2. Recording Whole-cell patch-clamp recordings were carried out using a PC-II patch clamp amplifier (Huazhong University of Science and Technology, Wuhan, People’s Republic of China). The micropipette was filled with internal solution, the composition of which was as follows (in mM): KC1 140, MgClz 2.5, HEPES 10, EGTA 11, ATP 5, its osmolarity was adjusted to 310 mOsm with sucrose and pH was adjusted to 7.4 with KOH. The external solution contained (in mM) NaCl 150, KC1 5, CaCl, 2.5, MgCl, 2, HEPES 10, D-glucose 10, its osmolarity was adjusted to 340 mOsm with sucrose and pH was adjusted to 7.4 with NaOH. The resistance of recording electrodes were in the range of 1–4 MQ. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1–10 GQ) and then a more negative pressure was applied to rupture it, thus a whole-cell mode was established. The adjustment of series resistance and capacitance compensation was done before the start of experiments. Membrane currents were filtered at 10 HZ ( – 3 dB). Data were stored and analyzed on a super 386 computer with a data acquisition software and hardware system (Huazhong University of Science and Technology, Wuhan) or recorded by a pen recorder. Experiments were carried out at holding potential (HP) = –60 mV. 2.3. Drugs Drugs used in the experiments were substance P (SP; Sigma), spantide (Sigma), ATP (Boehringer Mannheim), l-(5 -isoquinolinesulfony l)-2-methylpiperazine (H7) (RBI). The drugs were dissolved in external solution and applied by gravity flow from a row of tubules (OD/ID = 500 pm/200 pm), which were connected to a series of independent reservoirs. The distance from the mouth of tubule to the cell examined was approx. 100 pm. This rapid
solution exchange system was manipulated by shifting the tubules horizontally with a micromanipulator. 2.4. Stati~tics Values of ATP-activated currents were reported as means t S.E.M. The Student’s t-test was used.
3. Results 3.1. ATP- and SP-actiuated currents in the membrane of DRG neurons
In the present study, the majority of the cells examined responded to externally applied ATP (1– 1000 ~M) in a concentration-dependent manner (82/85, 96.5%); many of them were also sensitive to substance P. In other words, there was a coexistence of ATP and SP receptor in the membrane of these neurons. The neurons chosen for this experiment were requested to be sensitive both to ATP and SP, the size distribution of them modified from Scroggs et al. [19] were as follows: large cells ( >45 p,m) 4; intermediate cells (30–45 pm) 54 and small cells ( <30 pm) 27. Fig. 1 shows the ATP- and SP-activated currents recorded from one DRG neuron. The amplitude of ATP-activated current is larger than that of SP while the desensitization in the latter is less apparent than that in the former; it takes 4 min to get a full recovery of ATP (10 pM)-activated currents. 3.2. Ionic mechanism of SP-euoked current The I–V relation for the SP-evoked current was studied and the reversal potential was found to be around +35 mV at normal external sodium concentration ([Na+]O= 150 mM) (n = 8). When INa+]O decreased step-by-step from 150 mM to 1.5 mM by replacing sodium chloride with choline chloride the SP-evoked currents decreased gradually and the reversal potential shifted to be more and more negative. Application of Cdz+ (0.05 KM) containing external solution also suppressed the SP-activated currents to a smaller but detectable extent [9]. From the results mentioned above it is assumed that conductance changes of nonselective cations, especially sodium and/or calcium ions might be involved. Fig. 2 shows an example in which the Z–V relation from a single cell is illustrated. 3.3. SP potentates ATP-activated currents Fig. 3 illustrates the increase in the amplitude of ATP (10 KM)-activated currents by the preapplication of SP (0.001-10 ~M). If the concentration of SP applied was too low, i.e. 0.001 p.M or less, no effect could be detected. When the concentration of SP increased beyond 0.01 FM the potentiating effect of SP on the 10 PM ATP-activated
H.-Z. Hu, Z.-W. Li/Brain Research 739 (1996) 163-168
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currents increased gradually with the increase of SP concentration. However, when concentration of SP increased to 10 p,M, its potentiating effect decreased markedly as compared with that of 1 PM SP. In these neurons 0.01, 0.1 and 1 p,M substance P can increase the ATP (10 ~M)activated current to 113.7 t 3.1YO(n = 8); 127.2 + 6.790 (n= 12) and 154.7 & 14.4% (n= 16) (means+ S.E.M.), respectively. 3.4. Dose–response curve of ATP-activated currents with or without pretreatment of SP Fig. 4 shows the concentration–response curve of ATP-activated current with or without substance P pretreatment. From the concentration–response relationship for ATP alone it shows that the value of threshold, EC~O and maximum concentration were 1 PM, 30 FM and 500 pM respectively. As compared with which, the concentration response curve for ATP pretreated with SP shifts to the left apparently. The threshold value in both curves are very close, the EC~Ovalue in the dose–response curve for ATP is lower than that of ATP pretreated with SP (15 KM) while the response of maximum concentration in the later is higher than that in the former.
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Fig. 4. Concentration–response relationships for ATP with and without preapplication of SP. In the concentration–response curve for ATP eacb point represents the means + S.E.M. of ATP-activated current of 8–16 neurons. The curve shown is the best fit of the data to the logistic equation Y= Em:,X/[1 + ( K6 / c)rr], where C is the concentration of ATP, 2’ is the fraction of the maximum value, and K~, the dissociation constant of the ATP receptor, is 30 KM. The curve was drawn according to tbe equation described above assuming Hill coefficients ( n = 0.72). As compared with concentration–response curve for ATP the dose-response curve for ATP with pretreatment of SP shifted to the left obviously. ‘ P <0,05, “ “ P <0.01, compared with the ATP-activated current without preapplication of SP.
3.5. Time-course of the potentiation qf ATP-actiuated current by SP Fig. 5 shows the time-course of the potentiation of ATP-activated current by SP (lo PM). The potentiation of ATP-activated current appeared at 30– 120 s after preapplication of SP and reached its peak at about 6 min. It achieved a full recovery after 10 min when the concentration used was O.01 pM; it took 20 min to recover if the concentration used was 1 wM. However, if the period for preapplication of SP lasted less than 30 s no effects could be observed.
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H.-Z. Hu, Z.-W. Li/Brain Research 739 (1996) 163-168
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PKC in the internal solution and the potentiating effect was suppressed significantly (P < 0.01) as shown in Fig. 6b,c.
4. Discussion For investigating the modulatory effect of SP on ATPactivated currents it was required that the experiments were carried out on neurons endowed with these two (SP and ATP) receptors. It is practicable because there are a large number of DRG cells in response to SP and ATP according to the data obtained from our previous work, in which the cells sensitive to SP and ATP are 90.6% [18] and 94.5Y0[6] respectively. It is evident from Fig. 3 that the enhancement of amplitude of ATP-activated currents increases gradually with the increase of the concentration of SP from 0.01 to 1 PM. However, when the concentration of SP increased to 10 pM the modulatory effect of SP on ATP-activated currents did not increase further. On the contrary, it reduced dramatically as compared with the effect of SP in 1 PM. We have also observed this anomalous phenomenon of SP modulation in another work (unpublished observation) that the inhibition of SP on GABA (100 KM)-
activated current increased with the increase of SP concentration from 0.0001 to 1 p,M, but was abolished once the SP concentration increased to 10 I.LM;in fact, a facilitator effect on GABA-activated currents occurred. We think this might be a non-specific action of agonist emerged at such considerable high concentration. It is of interest that the enhancement of ATP-activated currents by SP was mainly due to the increase in peak value (Ip) while the increase in steady state (1ss) was relatively small. This is apparent in the records demonstrated in Fig. 3. It is considered that this enhancement may result from an increase in the fast component of the desensitizing current other than the slow component. Comparison between the dose–response curves for ATP-activated currents, with and without preapplication of SP in Fig. 4, indicates that the potentiation of ATPactivated current by preapplication of SP is evident from the fact that the curve appears to shift to the left. Since the K~ value in the curve for ATP-activated current pretreated with SP decreased and the response in the maximum concentration increased. This obviously reflects the increase in affinity for the ATP receptor, which may result from the phosphorylation of ATP receptor-ion channel complex following the activation of SP receptor. In the present study the time-course of the potentiation
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of ATP-activated current by SP took about 6 min to reach the peak, and the full recovery needed 10 to 20 rein, which was dependent on the concentration of SP preapplied. It is worth noting that the onset of the potentiation needed at least 30–120 s after the preapplication of SP. This implies that the modulation must take place through a pathway of complex intracellular transduction while the direct allosteric modulatory effect of SP on its receptor might be excluded. It is known that SP receptor belongs to the superfamily of G protein-coupled receptors [16]. SP binds to NK1 receptor couples via G protein to PLC, which, in turn, breaks down PIP2 into IP3 and DAG, the latter activates PKC and may result in phosphorylation of ion channel in ATP receptor. In the experiment we found that spantide, the NKI receptor antagonist, could block the SP-evoked membrane-response and the facilitator effect of SP on ATP-activated current. Furthermore, H7, which is a potent inhibitor of PKC, could suppress the effect of SP on ATP-activated current, also supporting this hypothesis. What is the physiological significance of the result obtained in the present investigation? It is now open for discussion. In this work we use the cell body of DRG neuron as a simple and accessible model for studying the characteristics in the terminals of axonal membrane, either peripheral or central. As we know that the nerve endings of the peripheral axon of DRG cell are sensitive to many chemical substances, especially those produced in tissue damage and inflammation, of which ATP and SP are two potent stimulating agents, the receptors for them coexist in the membrane of nerve endings [17]. In the case of inflammation or tissue damage these two substances exert a stimulating effect on the nerve endings not only separately, but also in cooperation. Furthermore, since a number of SP released from the terminals can act on the SP autoreceptors [9] in the membrane of the terminal, it may favour the modulatory effect as mentioned above. Acknowledgements
We are grateful to Mrs. Y.Z. Fan for preparing isolated neurons, Dr. W.Z. Wei for technical help, and Prof. X.R. Jin for reading the manuscript. This work was supported by Grant 39270244 from the National Natural Science Foundation of China and a fund from the Ministry of Health of China. References [1] Bean, B.P. and Friel, D.D., ATP-activated channels in excitable cells. In T. Namhashi (Ed.), Ion Channels, Vol. 2, Plenum Press, 1990, pp. 501–519.
[2] Dubyak, G.R. and E1-Moatassim, C., Signal transduction via P2purinergic receptors for extracellular ATP and other nucleotides, Am. J. Physiol,, 265 (1993) 557-606. [3] Edwards, F.A., Gibb, A,J. and Colquhoun, D,, ATP receptor-mediated synaptic currents in the nervous system, Narure, 359 (1992) 144-147. [4] Evans, R,, Derkach,V. and Surprenant, A,, ATP mediates fast synaptic transmission in mammalian neurons, Nature, 357 (1992) 503-505. [5] Fredholm, B.B., Abbracchio, M.P., Burnstock, G., Daly, J.W., Harden, T.K., Jaccobson, K.A., Leff,P. and Williams, M., Nomenclature and classification of purinoceptors, Pharmacol. Reo., 46 (1994) 143-156. [61 Gu, Q.H., Li, Z.W. and Fan, Y.Z., Enhancement of dopamine on ATP-activated currents in the neurons acutely isolated from rat DRG, Chin. J. Neurosci., (1995) 32. [7] Hidaka, H. and Hagiwam, M., Pharmacology of the isoquinoline sulfonamide protein kinase C inhibitors, Trends Pharrrracol.,Sci,, 8 (1987) 162-164. [8] Hu, HZ,, Li, Z.W. and Fan, Y.Z., Substance P potentates ATPactivated currents rat in primary sensory neurons, Chin. J Nearosci., (1995) p. 35. [9] Hu, HZ., Li, Z.W. and Si, J.Q., Evidence for the existance of substance P autoreceptor in the membrane of rat DRG neurons, Neuroscience, (1996) in press. [10] Illes, P. and Noernberg, W., Neuronal ATP receptors and their mechanism of action, Trends Pharmacol. Sci., 14 (1993) 50–54. [11] Krishtal, O.A., Marchenko, S.M. and Pidoplichko, V.L., Receptor for ATP in the membrane of mammalian sensory neurons, Neurosci. Left., 35 (1983) 41-45. [12] Li, C., Aguayo, L., Peoples, R.W, and Weight, F.F., Ethanol inhibits a neuronal ATP-gated ion channel, Mol. Pharmacol., 44 (1993) 871-875. [13] Li, C., Peoples, R.W., Li, Z.W, and Weight, F.F., Zn2+ potentates excitatory action of ATP on mammalian neurons, Proc. Natl. Acad. ~Ci. USA, 90 (1993) 8261-8267. [14] Li, C., Peoples, R.W. and Weight, F.F., Alcohol action on a neuronal membrane receptor: evidence for a direct interaction with the receptor protein, Proc. Natl. Acad. Sci. USA, 94 ( 1994) 8200– 8204. [15] Nishikawa, M., Uemura, Y., Hidaka, H. and Shirakawa, S., l-(5 -lsoquinolinesulfonyl)-2-ethylpiperazine (H7), a potent inhibitor of protein kinase, inhibits the differentiation of HL-60 cells induced by phorbol diester, .L~eSci., 39 (1986) 110I–1 107. [16] Otsuka, M. and Yoshioka, K., Neurotransmitter functions of mammalian tachykinins, Physiol. Re(?.,73 (1993) 229–308. [17] Rong, H.P., Bevan, S. and Dray, A., Chemical activation of nonciceptive peripheral neurons, Br. Med. Bull., 47 (1991) 534–548. [18] Si, J.Q, and Li, Z.W., Actions of substance P on the somatic membrane of rat DRG neurons, Acta Physiol. Sinica, 48 (1996) 8-14. [19] Scroggs, R.S. and Fox, A.P., Calcium current variation between acutely isolated adult rat dorsal root ganglion neurones of different size, .J. Physiol., 445 (1992) 639–658. [20] Swope, S.L., Moss, S.J., Blackstone, C.D. and Huganir, R.L., Phosphorylation of Iigand-gated ion channels: a possible mode of synaptic plasticity, FASEB J., 6 (1992) 2514–2523. [21] Wu, X.P., Li, Z.W. and Fan, Y.Z., Inhibitory effect of substance P on GABA-activated currents in neurons acutely isolated from rat dorsal root ganglion, Chin. J. Physiol. Sci., 10 (1994) 371-375.