87
Brain Research, 551 (1991) 87-93 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116641G BRES 16641
Ruthenium red antagonism of the effects of capsaicin mediated by extrinsic sensory nerves on myenteric plexus neurons of the isolated guinea-pig ileum Miyako Takaki 1, Akio Kikuta 2 and Sosogu Naka~cama 1 ~Department of Physiology and 2Departmentof Anatomy, Okayama University Medical School, Okayama (Japan) (Accepted 8 January 1991)
Key words: Myenteric neuron; Capsaicin; Ruthenium red; Electrical activity; Extrinsic sensory nerve (guinea-pig)
The effects of Ruthenium red and its antagonism of capsaicin-induced action on the electrophysiologicalbehavior of myenteric neurons were investigated with intracellular recording techniques in the isolated guinea-pig ileum. Ruthenium red antagonized dose-dependently (1-10/~M) a capsaicin-induced marked long-lasting slow depolarizing action associated with increased input resistance, during which the cells spiked repeatedly or displayed anodal break excitation. This action of capsaicin has been found to be mediated via a release of substance P from sensory nerve endings. The slow depolarizing response to exogenous substance P applied by pressure microejection, which mimicked the capsaicin-induced action, was not affected by Ruthenium red. Therefore, present results indicate that Ruthenium red antagonizes the specific effect of capsaicin on myenteric neurons by acting on the presynaptically located peripheral nerve terminals of sensory neurons and inhibiting the release of substance P. Electron-microscopic examination showed that the neurotoxic action of capsaicin towards extrinsic sensory nerve fibers was also dose-dependently (1-10/~M) protected by pretreatment of ruthenium red. Present results suggest that Ruthenium red inhibits a capsaicin-induced activation of cation channels at the cell membrane of sensory nerves.
INTRODUCTION It has been well-known that capsaicin evokes release of n e u r o p e p t i d e s from p e r i p h e r a l nerve terminals of prim a r y afferent neurons and the capsaicin-evoked release closely resembles the e v o k e d release of neurotransmitters at synaptic nerve terminals. Capsaicin actually induced release of n e u r o p e p t i d e s in a Ca2+-dependent m a n n e r 9' 19, but the capsaicin-induced Ca 2÷ influx in sensory neurons and the capsaicin-evoked release of n e u r o p e p tides are not r e d u c e d by b l o c k a d e of voltage sensitive Ca 2+ channels 17"21'31. Accordingly, the capsaicin-induced influx of Ca 2+ in afferent neurons is apparently not secondary to the opening of voltage-sensitive Ca 2+ channels, but is m e d i a t e d by cation channels which are activated by capsaicin 21'31. On the o t h e r hand, it has been shown that R u t h e n i u m red ( R R ) , an inorganic dye, blocks Ca 2÷ entry into neural tissues 1°'22'27 and potently inhibits the capsaicine v o k e d release of n e u r o p e p t i d e s 3-6'16'2°. O n the motility of various organs in the guinea-pig and rat, much evidence has been p r e s e n t e d to indicate R R antagonism of the specific effects of capsaicin 13-16A8'2°'24. H o w e v e r , no direct evidence of R R antagonism of the specific
effects of capsaicin on myenteric neurons, which control the motility of the various organs has b e e n presented. The aim of the p r e s e n t study was to obtain a direct evidence whether R R could antagonize the specific action of capsaicin on the m y e n t e r i c ganglion cells with intracellular recording techniques in the isolated guinea-pig ileum. In addition, we investigated w h e t h e r or not R R could antagonize the neurotoxic action of capsaicin on primary afferent nerve fibers within the myenteric plexus by electron-microscopic examination.
MATERIALS AND METHODS Segments of intestine were removed from the ileum of adult guinea-pigs (200-300 g) that had been stunned by a blow to the head and exsanguinated. Flat sheet preparations of longitudinal muscle with the myenteric plexus attached were prepared, mounted in a superfusion chamber and viewed with an inverted microscope (Olympus, P021, Tokyo, Japan), lighted by a microelectrode illumination system (Narishige, Tokyo, Japan). The preparations were immobilized for intracellular recording by pressing two L-shaped wires onto the surface of the muscle on either side of an identified myenteric ganglion. Intracellular recordings were made with glass microelectrodes (resistances of 40-80 MI2) that were filled with 3 M KCI. A high impedance preamplifier (Nihon-Kohden, Tokyo, Japan) that was used for recording membrane potentials, had negative-capacity compensations and bridge circuitry for injecting
Correspondence: M. Takaki, Dept. of Physiology, Okayama University Medical School, 700 Okayama, Japan.
88 current (200 ms in duration) through the recording micropipette. Some of the records were played back on a Recticorder (NihonKohden, Tokyo, Japan) from data previously recorded on magnetic tape. The tissues were maintained in Krebs solution23 at 35 °C and gassed with 95% 02/5% CO2. The tissue chamber was perfused at a rate of 11-12 ml/min. The perfusate completely reached the tissue chamber 3-4 rain after onset of the perfusion. Capsaicin (1-I0 ruM) and RR (1-10/~M) were applied by superfusion. Capsaicin (0.1 mM), substance P (0.1 raM) and RR (1 mM) were applied by microejection from fine-tipped pipettes (tip diameter 10-20 /~m) with nitrogen pulses of controlled amplitude (1.41 kg/cm2) and duration (100-900 ms). Drugs used were capsaicin (Sigma), ethyleneglycol-bis (fl-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA; Nakarai), Ruthenium red '(RR; Sigma), substance P (Peptide Institute) and tetrodotoxin (TTX; Sankyo). Capsaicin (10 mM) was dissolved in 100% dimethylsulfoxide (DMSO). When used, the stock solution was diluted to 100-fold (0.1 mM) with distilled water containing 0.1% Fast green for pressure microejection. For application by superfusion at concentrations of 1 ltM and 10 ,uM the stock solution was diluted to 1000- and 10,000-fold with Krebs solution containing 0.01% Fast green, respectively. Stock solution of substance P (1 raM) was prepared by dissolving substance P in 0.02 M acetic acid. Substance P (0.1 raM) was diluted with distilled water containing 0.1% Fast green. The Fast green was included so that exposure of the impaled neurons to the microejected solution could be confirmed by direct observation through the microscope and so that entering the chamber of the superfusate could be confirmed by direct observation. Fast green in concentrations up to 10 times (1%) that used in these experiments had no effect on myenteric neurons. DMSO at 1% had no effect of its own. For electron-microscopic examination, segments of intestine were removed from the ileum of adult guinea pigs that had been stunned by a blow to the head and exsanguinated. The segments were rapidly placed in an organ bath containing 15 ml of Krebs solution at 37 °C, and aerated with 5% CO 2 in 0 2. The tissues were pretreated with RR (0-10 ~M) for 25 min and then exposed to capsaicin 1 /~M for 60 min. After washout, the tissues were incubated for a further 5 min. The tissues were dissected at antimesenteric border and extended flat by pinning and fixed by immersion for 3 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The tissues were then washed in the same buffer for 6 h and postfixed for 2 h in 0.1% OsO4 in 0.1 M PB, pH 7.4, dehydrated in alcohol, and embedded in Epon. Thin sections were cut, stained with uranyl acetate and lead citrate, and observed in a Hitachi H-700 electron microscope. We examined the portions close to the mesenteric nerves in electron-microscopic study, since all of the responsive neurons to mesenteric nerve stimulation were distributed in all ganglia close to the mesenteric border with attached mesenteric nerves25. RESULTS
(1) Electrophysiological study The effects of R R and its antagonism of the capsaicininduced action on myenteric neurons were investigated in 86 myenteric neurons from 52 guinea pigs. These neurons were classified into 3 categories using criteria that have been described before 12'23"26'3°. Type 1/S cells were defined as those with a high input resistance and which spiked repeatedly during the injection of a depolarizing current and displayed anodal break excitation after the termination of hyperpolarizing current pulses. The current-voltage relationship in Type 2/AH and Type 1/S cells was iinear for small changes in m e m b r a n e potentials.
When the cells were hyperpolarized by 2{i-40 mV, anomalous rectifications were observed. Type 2/AH cells were defined as those with a low input resistance and which displayed a characteristic afterhyperpolarization (AH). During the AH, the cell was refractory to further stimuli. These cells usually did not show anodal break excitation except when activated by application of exogenous compounds. NS cells were defined as those cells that did not spike in response to ejection of depolarizing current or application of drugs. The sample studied included 33 Type 2/AH cells, 10 Type 1/s cells and 43 NS cells.
Effects of RR (1-10 /aM) on the capsaicin (10 /aM) response Capsaicin (1 /aM) applied by superfusion has been found to elicit no marked effect on the electrophysiological behavior of all of 8 myenteric neurons tested, but superfusion with capsaicin (10/aM) has been found to elicit a long-lasting (for 9.5-33 min) slow depolarization (16.8 + 3.1 mV) associated with increased input resistance (135.3 _+ 13.5%) in 3 Type 2/AH n e u r o n s of 4 myenteric neurons 26. This effect was reconfirmed in the present study. Increased (or decreased) input resistance is reflected by increased (or decreased) amplitude of electrotonic potentials that occurred in response to intraneural injection of hyperpolarizing constant current pulses. This slow depolarizing response to capsaicin is irreversibly desensitized 26. Therefore, in the present study, the effects of capsaicin were always examined after pretreatment or in the presence of RR. Superfusion of tissues with R R (1/aM) did not cause any marked effect on all of 6 myenteric neurons (3 A H and 3 NS) tested. In the presence of R R (1/aM), capsaicin (10/aM) applied by superfusion elicited a slow depolarization associated with increased input resistance in 4 ( A H cells) of 8 myenteric neurons. A n example is shown in Fig. 1. Mean peak amplitude of the depolarization was 15.1 + 1.1 mV (n = 4) and the duration of the response ranged between 3 and 31 min (n = 4). The mean percent of increase of input resistance was 119.3 +_ 8.7% of the control (n = 4). Therefore, superfusion with R R (1/aM) did not significantly affect the mean amplitude, mean percent increase of input resistance and duration of the slow depolarizing response to capsaicin (10/aM) in 50% of neurons tested. In the remaining 4 myenteric neurons, superfusion with capsaicin (10/aM) did not elicit any effect in the presence of R R (1/aM) (see Fig. 3). Superfusion with R R ( t 0 / a M ) did not elicit any effect in 6 (1 S, 2 A H and 3 NS) of 9 myenteric neurons. Of the remaining 3 myenteric neurons, R R (10 /aM) evoked anodal break excitation and/or spontaneous spike potentials with no change of m e m b r a n e potential in two Type 1/S cells and evoked a slow depolarization associated with
89 Ruthenium red 1 uM
1 min
(3) 20-
V
o
-IO
I
(4)
0 [, o
g
~
lO-
Capsalcin I0 pM Fig. l.Effect of superfusion with capsaicin (10/aM) on electrophysiological behavior of a myenteric Type 2/AH cell in the presence of ruthenium red (RR; 1/aM). RR by itself did not elicit any effect. After pretreatment of RR ( 1/aM) for 5 min, the superfusate was substituted by the solution containing RR (1/aM) and capsaicin (10 MM). Constant hyperpolarizing current pulses were intracellularly injected to measure input resistance. Capsaicin (10/aM) still caused a slow depolarizing response associated with increased input resistance, during which anodal break excitation could be seen. During the ongoing response, at the arrow, the injection current was increased and then the full spike potential was evoked. Resting membrane potential was -46 mV.
increased input resistance in one Type 2/AH cell. In the presence of RR (10 #M), superfusion with capsaicin (10 MM) did not cause any marked effect in 5 (2 AH, 1 S and 2 NS) (71%) of 7 myenteric neurons. An example is shown in Fig. 2. In this Type 2/AH neuron, RR (10 MM) selectively blocked the effect of capsaicin (10 MM), although a slight depolarization ( < 3 mV) associated with a slight increase of input resistance (112.5%) remained. In the remaining two Type 1/S cells, capsaicin did not elicit the slow depolarizing response, although anodal break excitation and/or spontaneous action potentials were exerted. Therefore, the slow depolarizing response associated with increased input resistance elicited by capsaicin (10/~M) was blocked by RR (10/~M) in all myenteric neurons tested (Fig. 3).
o L-
0 Cap I0
pM
RR 1 pM
RR I0 pM
i
I +
Cap 10 ~M Fig. 3. Ruthenium red (RR; 1-10/aM) antagonism of the capsaicin (Cap; 10/aM)-induced slow depolarization of myenteric neurons. Numbers in parentheses denote number of responsive neurons. Blank column, mean amplitude of depolarization induced by capsaicin in responsive neurons (mV). Dotted column, number of neurons did not respond to capsaicin.
Effects of rnicroejected RR (1 mM) on the microejected capsaicin (0.1 raM) response In 31 (3 AH and 28 NS) of 63 myenteric neurons, no
J SP 1pulse
Rutheniumred-pretreated ~
20 mV
Ist Capsaicin 10 pM
~ SPlp
2nd Capsaicin 10 pM
Ruthenium red 10 pM A
c
1
I 40 mV
••••••••••••••••••••••••••••••••••••••••u••••••1•
~
SP Ip
10 s
Capsalcln 10 uM B
SP 1pulse
I
__
SP 1pulse
~11111111111111111111111111t1111111111111111111__111111
capsaic4n
Fig. 2. Ruthenium red (RR; 10/aM) specific antagonism of capsaicin (10/aM)-induced action on a myenteric Type 2/AH cell. A: after superfusion with RR (10/aM) for 5 min, which did not cause any effect, superfusion with capsaicin (10 /aM) did not cause a characteristic slow depolarizing response. Only a small depolarization associated with a slight increase of input resistance remained. B: 8 min after onset of superfusion with capsaicin (10 /aM), exogenous substance P applied by pressure microejection (SP; 0.1 mM; 1 pulse; at the arrow) elicited a slow depolarizing action associated with increased input resistance. C: 5 min after washout of capsaicin, SP produced a similar response to that in B. Resting membrane potential was -40 mV.
1 mln
Fig. 4. Antagonism of the effect of capsaicin (10 /aM) and no antagonism of the effect of substance P (0.1 mM) by pressure microejection of ruthenium red (RR; 1 mM; 900 ms; 30 pulses) in a Type 2/AH neuron. RR by itself did not cause any effect. A: 5 min after pretreatment of RR, superfusion with capsaicin (10/aM) did not cause a characteristic slow depolarization, but a small depolarization associated with slightly increased input resistance remained. Substance P (SP; 0.1 mM; 900 ms; 1 pulse; at the arrow) applied by pressure microejection caused a pronounced depolarizing response associated with increased input resistance. B: 17 min after the first application of capsaicin (10/aM), the second application of capsaicin elicited no response. SP (1 pulse; 1 p) caused a similar response to that in A. C: after 6-min washout of capsaicin, SP (1 pulse) caused a similar response to that in B. Resting membrane potential was -63 mV.
90
91
Fig. 5. Electron micrographs of myenteric plexus in the guinea-pig ileum, showing dose-dependent (1-10/~M) protective effects of RR from capsaicin-induced neurotoxic actions toward nerve fibers. A: control nerve fibers in the myenteric plexus treated with a vehicle (0.01% DMSO); the ultrastructure of the nerve fibers, terminals and somas in the myenteric plexus was preserved intact. B: nerve fibers in the myenteric plexus incubated in Krebs solution to which capsaicin (1 #M) was added for 60 rain. Many nerve fibers showed massive degenerative changes, i.e. many nerve fibers became slightly expanded and mitochondria were grossly swollen. C: nerve fibers in the myenteric plexus incubated in Krebs solution to which RR (1/~M) was added for 25 min and then capsaicin (1 #M) was added for 60 rain. Cytoplasmic organelles of the nerve fibers and ganglion cells were kept intact in their ultrastructure. However, the mitochondria in many nerve fibers and cells condensed. D: nerve fibers in myenteric plexus incubated in Krebs solution to which RR (10/~M) was added for 25 min and then capsaicin (1/~M) was added for 60 min. No obvious ultrastructural damages occurred in the somas of ganglion neurons, nerve terminals and nerve fibers. In particular the mitochondria have not been expanded. Bar = 1/~m.
response was evoked by RR (1 mM) applied by pressure microejection (1-30 pulses; 100-900 ms). In the remaining 32 (18 AH, 8 S and 6 NS) myenteric neurons, RR (1 mM) applied by pressure microejection (100-900 ms; 1-10 pulses) elicited a long-lasting (for 3-60.3 min) slow depolarization associated with increased (n = 14; 8 AH and 6 NS cells) or decreased input resistance (n = 12; 6 AH and 6 S cells), or increased neural discharges without any change of membrane potential (n = 6; 4 A H and 2 S cells). In Ca2+-free Krebs solution containing 0.1 mM EGTA, RR did not cause any effect in 9 (7 AH and 2 NS) of 10 myenteric neurons. In only one Type 2/AH neuron, RR applied by pressure microejection produced a slow depolarization associated with increased input resistance and evoked action potentials. In the previous paper z6, capsaicin (0.1 mM) applied by pressure microejection close to the cells has been found to cause a long-lasting slow depolarization associated with increased input resistance in 7 of 13 Type 2/AH neurons. After microejection of an approximately threshold pulse of capsaicin, the mean peak amplitude was 21.4 _+ 2.5 mV and the mean duration of this response was 6.9 _+ 2.6 min (n -- 7). The mean percent of increase of input resistance was 222.7 _+ 40.5% of the control (n -- 6). This effect was reconfirmed in the present study. After sufficient volume of RR (1 mM; 1-30 pulses) was microejected, pressure microejection of capsaicin (0.1 mM) had no effect on 11 (2 S and 9 NS) (about 70%) of 16 myenteric neurons. Of the 5 remaining neurons, microejected capsaicin caused a slow depolarization associated with increased input resistance in one NS cell and decreased input resistance in two (1 AH and 1 NS) cells, respectively. In two Type 1/S cells, RR dit not block the capsaicin-induced anodal break excitation and spontaneous action potentials. This effect of capsaicin has been demonstrated as a repeatable non-specific effect of capsaicin 26.
Effect of RR on the substance P response RR (1 mM) by pressure microejection or superfusion with RR (10/~M) did not affect the slow depolarizing response to exogenous substance P (0.1 mM) by pressure
microejection in all of 5 (4 AH and 1 NS) myenteric neurons, in which the depolarizing response to capsaicin (10/~M) was blocked by RR (Figs. 2 and 4). As shown in Fig. 4, pressure microejection of RR (1 raM; 30 pulses) attenuated a characteristic slow depolarizing response to capsaicin (10 ~M); the amplitude reduced to 8 mV; percent increase of input resistance reduced to 107%. However, an intense slow depolarization (23.5 mV) associated with increased input resistance (167% of the control) for 4.3 rain evoked by exogenous substance P was not affected by RR.
(2) Electron-microscopic examination In control treated with only vehicle, intense damage in the intestinal epithelial cells was caused, while the ultrastructure of the nerve fibers, terminals and somas in the myenteric plexus was preserved intact (Fig. 5A). After treatment of the myenteric plexus with capsaicin (1 ktM), many nerve fibers became slightly expanded with swollen mitochondria, showing conspicuous degenerative changes (Fig. 5B). Such changes thus occurred at the same concentration of capsaicin at which specific action on the extrinsic sensory nerve fibers could be detected in p h y s i o l o g i c a l s t u d i e s 24'26. In the 1-/~M RR-pretreated, capsaicin-treated plexus, cytoplasmic organelles of the nerve fibers and ganglion cells were kept intact in their ultrastructure. However, the mitochondria in many nerve fibers and cells were strikingly condensed. No obvious ultrastructural changes occurred in the somas of ganglionic neurons, nerve terminals and nerve fibers in the 10-/~M RR-pretreated, capsaicin-treated plexus. DISCUSSION
Present results showed that RR dose-dependently (1-10/~M) antagonized capsaicin-induced slow depolarization associated with an increase in input resistance that is due to a decrease in calcium-mediated potassium conductance in Type 2/AH cells. Type 2/AH cells of the small intestine have an on-going calcium-mediated potassium conductance that contributes to the relatively
92 negative resting membrane potential of these cells ~. Reduction of this conductance in Type 2/AH cells results in a depolarization of the membrane with an associated increase in input resistance. This slow depolarizing response to capsaicin of myenteric neurons has been found to be due to endogenous neuropeptides, such as substance P released from capsaicin-sensitive sensory nerve endings z6. The enhancement of neural discharges, unaccompanied with slow depolarization could be evoked by capsaicin. This effect was often seen in Type 1/S cells. This effect of capsaicin has been found to be repeatable and non-specific26. RR did not block this non-specific effect of capsaicin. When RR blocked the slow depolarization induced by capsaicin, the slow depolarizing response evoked by exogenous substance P, which obviously mimicked the response induced by capsaicin, was unaffected by RR. In addition, much evidence has been presented that RR specifically antagonizes a neuropeptide release induced by capsaicin 3-6'16"2°. Therefore, it is considered that RR does not act postsynaptically on myenteric neurons but specifically acts on presynaptically located capsaicin-sensitive extrinsic sensory nerve terminals, where RR inhibits the release of substance P. By electron-microscopic examination, neurotoxic action of capsaicin (1/~M) on nerve fibers in myenteric plexus was found to be dose-dependently (1-10 #M) protected by RR, although the effects of capsaicin appear not to be specifically restricted towards extrinsic sensory nerve fibers. According to the report by Marsh et al. 21, clear expansion of mitochondria and accompanying changes were visible after only 5 min in capsaicin (1-10 ~M) in vagal sensory neurons. In the present study, the tissues were incubated for 60 min in capsaicin (1 ~M). Thus, long incubation periods appeared to be the reason of more extended changes than expected. Therefore, the results from electron-microscopic observation supported the postulate based on the electrophysiological study that RR acts on presynaptically located capsaicin-sensitive sensory nerve terminals. Although the specific mechanism by which RR antagonizes the capsaicin-induced excitatory and neurotoxic effect is not clearly established by the present study, a possible mechanism can be proposed. It has been
REFERENCES 1 Adams, D.J., Takeda, K. and Umbach, J.A., Inhibition of calcium buffering depresses evoked transmitter release at the squid giant synapse, J. Physiol., 369 (1985) 145-149. 2 Alnaes, E. and Rahamimoff, R., On the role of mitochondria in transmitter release from motor nerve terminals, J. Physiol., 248 (1975) 285-306. 3 Amann, R., Donnerer, J. and Lembeck, E, Ruthenium red selectively inhibits capsaicin-induced release of calcitonin generelated peptide from the isolated perfused guinea-pig lung,
suggested that RR inhibits the release process at a mitochondrial site of action, where RR blocks Ca 2+ sequestration ~'s. However, RR has a generally low permeability to cells 7. Direct inhibition of Ca 2÷ influxes at the cell membrane by RR has been suggested to be the mechanism by which RR inhibits evoked release 2-2°'28. Recently, it has been reported that low concentrations of RR preferentially inhibit capsaicin-evoked neuropeptide release probably by interfering with the capsaicin-induced activation of cation channels at the cell membrane of sensory nerves in the isolated rat urinary bladder 6. Therefore, it might be possible that RR preferentially inhibits the capsaicin-induced activation of cation channels at the cell membrane of sensory nerves within myenteric plexus of the isolated guinea pig ileum as suggested by Amann et al. 6. Application of RR by superfusion at 1 /~M did not cause any effect on myenteric neurons but at 10 /~M elicited a slow depolarization associated with increased input resistance or an enhancement of neural discharges in about 33% of neurons tested. Pressure microejection of high concentration (1 mM) of RR caused a long-lasting slow depolarization associated with increased or decreased input resistance and/or generated spike potentials in about 51% of myenteric neurons tested. It has been suggested that RR can act through direct interaction with extracellularly localized sialic acid residues which are somehow involved in the calcium binding and blocks Ca2+-entry into the nerve tissues 29. Therefore, RR at high concentrations could act not only indirectly but also directly on the myenteric neurons. This direct action is considered to be CaZ+-dependent. From the present results, it is concluded that RR at low concentrations selectively antagonizes a specific effect of capsaicin on myenteric neurons by inhibiting a neuropeptide release from sensory nerve terminals evoked by capsaicin. However, high concentrations of RR act directly on myenteric neurons by some other mechanism. RR at low concentrations is a promising tool for investigation of the action of capsaicin, and elucidation of the mechanism of interaction of these two substances should add to our knowledge of sensory neuron function.
Neurosci. Lett., 101 (1989)311-315. 4 Amann, R., Donnerer, J. and Lembeck, E, Capsaicin-induced stimulation of polymodal nociceptors is antagonized by ruthenium red independently of extracellular calcium, Neurosci, 32 (1989) 255-259. 5 Amann, R., Donnerer, J. and Lembeck, F., Activation of primary afferent neurons by thermal stimulation. Influence of ruthenium red, Naunyn-Schrnied. Arch. Pharmacol., 341 (1990) 108-113. 6 Amann, R., Maggi, C.A., Giuliani, S., Donnerer, J. and Lembeck, F., Effects of carbonyl cyanide p-trichloro-methoxy-
93
7
8
9 10
11
12
13
14
15
16
17
18
phenylhydrazone(CCCP) and of ruthenium red (RR) on capsaicin-evoked neuropeptide release from peripheral terminals of primary afferent neurones, Naunyn-Schmied. Arch. Pharmacol., 341 (1990) 534-537. Babai, F., Ultrastructural method for the study of cell viability using ruthenium red. In: 9th International Congress on Electron Microscopy, 11, 1978, pp. 304-305. Bernath, S. and Vizi, E.S., Inhibitory effect of ionized free calcium enhanced by ruthenium red and m-chlorocarbonylcyanide phenylhydrazon on the evoked release of acetylcholine, Biochem. Pharmacol., 38 (1987) 179-226. Gamse, R., Molnar, A. and Lembeck, E, Substance P release from spinal cord slices by capsaicin, Life Sci., 25 (1979) 629-636. Goddard, Q.A. and Robinson, J.D., Uptake and release of calcium by rat brain synaptosomes, Brain Research, 110 (1976) 331-350. Grafe, P., Mayer, C.J. and Wood, J.D., Synaptic modulation of calcium-dependent potassium conductance in myenteric neurones in the guinea-pig, J. Physiol., 305 (1980) 235-248. Hirst, G.D.S., Holman, M.E. and Spence, I., Two types of neurones in the myenteric plexus of duodenum in the guinea-pig, J. Physiol., 236 (1974) 303-326. Jin, J.-G., Takaki, M. and Nakayama, S., Ruthenium red prevents capsaicin-induced neurotoxic action on sensory fibers of the guinea pig ileum, Neurosci. Lea., 106 (1989) 152-156. Jin, J.-G., Takaki, M. and Nakayama, S., Inhibitory effect of capsaicin on the ascending pathway of the guinea-pig ileum and antagonism of this effect by ruthenium red, Eur. J. Pharmacol., 180 (1990) 13-19. Maggi, C.A., Giuliani, S. and Meli, A., Effect of ruthenium red on responses mediated by activation of capsaicin-sensitive nerves of the rat urinary bladder, Naunyn-Schmied. Arch. Pharrnacol., 340 (1989) 541-546. Maggi, C.A., Patacchini, R., Santicioli, P., Giuliani, S., Bianco, E.D., Geppetti, P. and Meli, A., The 'efferent' function of capsaicin-sensitive nerves: Ruthenium Red discriminates between different mechanisms of activation, Eur. J. Pharmacol., 170 (1989) 167-177. Maggi, C.A., Patacchini, R., Giuliani, S., Santicioli, P. and Meli, A., Evidence for two independent modes of activation of the 'efferent' function mediated by peripheral terminals of capsaicin-sensitive sensory neurons by the use of omega conotoxin GVIA, a peptide modulator of neuronal calcium channels, Eur. J. Pharmacol., 156 (1988) 367-375. Maggi, C.A., Patacchini, R., Santicioli, P., Giuliani, S., Geppetti, P. and Meli, A., Protective action of Ruthenium Red toward capsaicin desensitization of sensory fibers, Neurosci. Lea., 88 (1988) 201-205.
19 Maggi, C.A., Santicioli, P., Geppetti, P., Parlani, M., Astolfi, M., DelBianco, E., Patacchini, R., Giuliani, S. and Meli, A., The effect of calcium free medium and nifedipine on the release of substance P-like immunoreactivity and contractions induced by capsaicin in the isolated guinea-pig bladder, Gen. Pharmacol., 40 (1989) 445-456. 20 Maggi, C.A., Santicioli, P., Geppetti, P., Parlani, M., Astolfi, M., PradeUes, P., Patacchini, R. and Meli, A., The antagonism induced by Ruthenium Red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies, Eur. J. Pharmacol., 154 (1988) 1-10. 21 Marsh, S.J., Stansfeld, C.E., Brown, D.A., Davey, R. and McCarthy, D., The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro, Neuroscience, 23 (1987) 275-289. 22 Swanson, P.D., Anderson, L. and Stahl, W.L., Uptake of calcium ions by synaptosomes from rat brain, Biochim. Biophys. Acta, 356 (1974) 174-183. 23 Takaki, M., Branchek, T., Tamir, H. and Gershon, M.D., Specific antagonism of enteric neural serotonin receptors by dipeptides of 5-hydroxytryptophan: evidence that serotonin is a mediator of slow synaptic excitation in the myenteric plexus, J. Neurosci., 5 (1985) 1769-1780. 24 Takaki, M., Jin, J.-G. and Nakayama, S., Ruthenium red antagonism of the effects of capsaicin on the motility of the isolated guinea-pig ileum, Eur. J. Pharmacol., 174 (1989) 57-62. 25 Takaki, M. and Nakayama, S., Effects of mesenteric nerve stimulation on the electrical activity of myenteric neurons in the guinea pig ileum, Brain Research, 442 (1988) 351-353. 26 Takaki, M. and Nakayama, S., Effects of capsaicin on myenteric neurons of the guinea pig ileum, Neurosci. Lett., 105 (1989) 125-130. 27 Tapia, R., Arias, C. and Morales, E., Binding of lanthanum ions and ruthenium red to synaptosomes and its effects on neurotransmitter response, J. Neurochem., 45 (1985) 1464-1473. 28 Tapia, R. and Meza-Ruiz, G., Inhibition by ruthenium red of the calcium-dependent release of 3H-GABA in synaptosomal fractions, Brain Research, 126 (1977) 160-166. 29 Wieraszko, A., Evidence that ruthenium red disturbs the synaptic transmission in the rat hippocampus slices through interacting with sialic acid residues, Brain Research, 378 (1986) 120-126. 30 Wood, J.D., Physiology of the enteric nervous system. In L.R. Johnson (Ed.), Physiology of the Gastrointestinal Tract, Vol. 1, 2nd edn., Raven Press, New York, 1987, pp. 67-109. 31 Wood, J.N., Winter, J., James, I.F., Rang, H.P., Yeats, J. and Bevan, S., Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture, J. Neurosci., 8 (1988) 3208-3220.
Brain Research, 551 (1991) 87-93 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116641G BRES 16641
Ruthenium red antagonism of the effects of capsaicin mediated by extrinsic sensory nerves on myenteric plexus neurons of the isolated guinea-pig ileum Miyako Takaki 1, Akio Kikuta 2 and Sosogu Naka~cama 1 ~Department of Physiology and 2Departmentof Anatomy, Okayama University Medical School, Okayama (Japan) (Accepted 8 January 1991)
Key words: Myenteric neuron; Capsaicin; Ruthenium red; Electrical activity; Extrinsic sensory nerve (guinea-pig)
The effects of Ruthenium red and its antagonism of capsaicin-induced action on the electrophysiologicalbehavior of myenteric neurons were investigated with intracellular recording techniques in the isolated guinea-pig ileum. Ruthenium red antagonized dose-dependently (1-10/~M) a capsaicin-induced marked long-lasting slow depolarizing action associated with increased input resistance, during which the cells spiked repeatedly or displayed anodal break excitation. This action of capsaicin has been found to be mediated via a release of substance P from sensory nerve endings. The slow depolarizing response to exogenous substance P applied by pressure microejection, which mimicked the capsaicin-induced action, was not affected by Ruthenium red. Therefore, present results indicate that Ruthenium red antagonizes the specific effect of capsaicin on myenteric neurons by acting on the presynaptically located peripheral nerve terminals of sensory neurons and inhibiting the release of substance P. Electron-microscopic examination showed that the neurotoxic action of capsaicin towards extrinsic sensory nerve fibers was also dose-dependently (1-10/~M) protected by pretreatment of ruthenium red. Present results suggest that Ruthenium red inhibits a capsaicin-induced activation of cation channels at the cell membrane of sensory nerves.
INTRODUCTION It has been well-known that capsaicin evokes release of n e u r o p e p t i d e s from p e r i p h e r a l nerve terminals of prim a r y afferent neurons and the capsaicin-evoked release closely resembles the e v o k e d release of neurotransmitters at synaptic nerve terminals. Capsaicin actually induced release of n e u r o p e p t i d e s in a Ca2+-dependent m a n n e r 9' 19, but the capsaicin-induced Ca 2÷ influx in sensory neurons and the capsaicin-evoked release of n e u r o p e p tides are not r e d u c e d by b l o c k a d e of voltage sensitive Ca 2+ channels 17"21'31. Accordingly, the capsaicin-induced influx of Ca 2+ in afferent neurons is apparently not secondary to the opening of voltage-sensitive Ca 2+ channels, but is m e d i a t e d by cation channels which are activated by capsaicin 21'31. On the o t h e r hand, it has been shown that R u t h e n i u m red ( R R ) , an inorganic dye, blocks Ca 2÷ entry into neural tissues 1°'22'27 and potently inhibits the capsaicine v o k e d release of n e u r o p e p t i d e s 3-6'16'2°. O n the motility of various organs in the guinea-pig and rat, much evidence has been p r e s e n t e d to indicate R R antagonism of the specific effects of capsaicin 13-16A8'2°'24. H o w e v e r , no direct evidence of R R antagonism of the specific
effects of capsaicin on myenteric neurons, which control the motility of the various organs has b e e n presented. The aim of the p r e s e n t study was to obtain a direct evidence whether R R could antagonize the specific action of capsaicin on the m y e n t e r i c ganglion cells with intracellular recording techniques in the isolated guinea-pig ileum. In addition, we investigated w h e t h e r or not R R could antagonize the neurotoxic action of capsaicin on primary afferent nerve fibers within the myenteric plexus by electron-microscopic examination.
MATERIALS AND METHODS Segments of intestine were removed from the ileum of adult guinea-pigs (200-300 g) that had been stunned by a blow to the head and exsanguinated. Flat sheet preparations of longitudinal muscle with the myenteric plexus attached were prepared, mounted in a superfusion chamber and viewed with an inverted microscope (Olympus, P021, Tokyo, Japan), lighted by a microelectrode illumination system (Narishige, Tokyo, Japan). The preparations were immobilized for intracellular recording by pressing two L-shaped wires onto the surface of the muscle on either side of an identified myenteric ganglion. Intracellular recordings were made with glass microelectrodes (resistances of 40-80 MI2) that were filled with 3 M KCI. A high impedance preamplifier (Nihon-Kohden, Tokyo, Japan) that was used for recording membrane potentials, had negative-capacity compensations and bridge circuitry for injecting
Correspondence: M. Takaki, Dept. of Physiology, Okayama University Medical School, 700 Okayama, Japan.
88 current (200 ms in duration) through the recording micropipette. Some of the records were played back on a Recticorder (NihonKohden, Tokyo, Japan) from data previously recorded on magnetic tape. The tissues were maintained in Krebs solution23 at 35 °C and gassed with 95% 02/5% CO2. The tissue chamber was perfused at a rate of 11-12 ml/min. The perfusate completely reached the tissue chamber 3-4 rain after onset of the perfusion. Capsaicin (1-I0 ruM) and RR (1-10/~M) were applied by superfusion. Capsaicin (0.1 mM), substance P (0.1 raM) and RR (1 mM) were applied by microejection from fine-tipped pipettes (tip diameter 10-20 /~m) with nitrogen pulses of controlled amplitude (1.41 kg/cm2) and duration (100-900 ms). Drugs used were capsaicin (Sigma), ethyleneglycol-bis (fl-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA; Nakarai), Ruthenium red '(RR; Sigma), substance P (Peptide Institute) and tetrodotoxin (TTX; Sankyo). Capsaicin (10 mM) was dissolved in 100% dimethylsulfoxide (DMSO). When used, the stock solution was diluted to 100-fold (0.1 mM) with distilled water containing 0.1% Fast green for pressure microejection. For application by superfusion at concentrations of 1 ltM and 10 ,uM the stock solution was diluted to 1000- and 10,000-fold with Krebs solution containing 0.01% Fast green, respectively. Stock solution of substance P (1 raM) was prepared by dissolving substance P in 0.02 M acetic acid. Substance P (0.1 raM) was diluted with distilled water containing 0.1% Fast green. The Fast green was included so that exposure of the impaled neurons to the microejected solution could be confirmed by direct observation through the microscope and so that entering the chamber of the superfusate could be confirmed by direct observation. Fast green in concentrations up to 10 times (1%) that used in these experiments had no effect on myenteric neurons. DMSO at 1% had no effect of its own. For electron-microscopic examination, segments of intestine were removed from the ileum of adult guinea pigs that had been stunned by a blow to the head and exsanguinated. The segments were rapidly placed in an organ bath containing 15 ml of Krebs solution at 37 °C, and aerated with 5% CO 2 in 0 2. The tissues were pretreated with RR (0-10 ~M) for 25 min and then exposed to capsaicin 1 /~M for 60 min. After washout, the tissues were incubated for a further 5 min. The tissues were dissected at antimesenteric border and extended flat by pinning and fixed by immersion for 3 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The tissues were then washed in the same buffer for 6 h and postfixed for 2 h in 0.1% OsO4 in 0.1 M PB, pH 7.4, dehydrated in alcohol, and embedded in Epon. Thin sections were cut, stained with uranyl acetate and lead citrate, and observed in a Hitachi H-700 electron microscope. We examined the portions close to the mesenteric nerves in electron-microscopic study, since all of the responsive neurons to mesenteric nerve stimulation were distributed in all ganglia close to the mesenteric border with attached mesenteric nerves25. RESULTS
(1) Electrophysiological study The effects of R R and its antagonism of the capsaicininduced action on myenteric neurons were investigated in 86 myenteric neurons from 52 guinea pigs. These neurons were classified into 3 categories using criteria that have been described before 12'23"26'3°. Type 1/S cells were defined as those with a high input resistance and which spiked repeatedly during the injection of a depolarizing current and displayed anodal break excitation after the termination of hyperpolarizing current pulses. The current-voltage relationship in Type 2/AH and Type 1/S cells was iinear for small changes in m e m b r a n e potentials.
When the cells were hyperpolarized by 2{i-40 mV, anomalous rectifications were observed. Type 2/AH cells were defined as those with a low input resistance and which displayed a characteristic afterhyperpolarization (AH). During the AH, the cell was refractory to further stimuli. These cells usually did not show anodal break excitation except when activated by application of exogenous compounds. NS cells were defined as those cells that did not spike in response to ejection of depolarizing current or application of drugs. The sample studied included 33 Type 2/AH cells, 10 Type 1/s cells and 43 NS cells.
Effects of RR (1-10 /aM) on the capsaicin (10 /aM) response Capsaicin (1 /aM) applied by superfusion has been found to elicit no marked effect on the electrophysiological behavior of all of 8 myenteric neurons tested, but superfusion with capsaicin (10/aM) has been found to elicit a long-lasting (for 9.5-33 min) slow depolarization (16.8 + 3.1 mV) associated with increased input resistance (135.3 _+ 13.5%) in 3 Type 2/AH n e u r o n s of 4 myenteric neurons 26. This effect was reconfirmed in the present study. Increased (or decreased) input resistance is reflected by increased (or decreased) amplitude of electrotonic potentials that occurred in response to intraneural injection of hyperpolarizing constant current pulses. This slow depolarizing response to capsaicin is irreversibly desensitized 26. Therefore, in the present study, the effects of capsaicin were always examined after pretreatment or in the presence of RR. Superfusion of tissues with R R (1/aM) did not cause any marked effect on all of 6 myenteric neurons (3 A H and 3 NS) tested. In the presence of R R (1/aM), capsaicin (10/aM) applied by superfusion elicited a slow depolarization associated with increased input resistance in 4 ( A H cells) of 8 myenteric neurons. A n example is shown in Fig. 1. Mean peak amplitude of the depolarization was 15.1 + 1.1 mV (n = 4) and the duration of the response ranged between 3 and 31 min (n = 4). The mean percent of increase of input resistance was 119.3 +_ 8.7% of the control (n = 4). Therefore, superfusion with R R (1/aM) did not significantly affect the mean amplitude, mean percent increase of input resistance and duration of the slow depolarizing response to capsaicin (10/aM) in 50% of neurons tested. In the remaining 4 myenteric neurons, superfusion with capsaicin (10/aM) did not elicit any effect in the presence of R R (1/aM) (see Fig. 3). Superfusion with R R ( t 0 / a M ) did not elicit any effect in 6 (1 S, 2 A H and 3 NS) of 9 myenteric neurons. Of the remaining 3 myenteric neurons, R R (10 /aM) evoked anodal break excitation and/or spontaneous spike potentials with no change of m e m b r a n e potential in two Type 1/S cells and evoked a slow depolarization associated with
89 Ruthenium red 1 uM
1 min
(3) 20-
V
o
-IO
I
(4)
0 [, o
g
~
lO-
Capsalcin I0 pM Fig. l.Effect of superfusion with capsaicin (10/aM) on electrophysiological behavior of a myenteric Type 2/AH cell in the presence of ruthenium red (RR; 1/aM). RR by itself did not elicit any effect. After pretreatment of RR ( 1/aM) for 5 min, the superfusate was substituted by the solution containing RR (1/aM) and capsaicin (10 MM). Constant hyperpolarizing current pulses were intracellularly injected to measure input resistance. Capsaicin (10/aM) still caused a slow depolarizing response associated with increased input resistance, during which anodal break excitation could be seen. During the ongoing response, at the arrow, the injection current was increased and then the full spike potential was evoked. Resting membrane potential was -46 mV.
increased input resistance in one Type 2/AH cell. In the presence of RR (10 #M), superfusion with capsaicin (10 MM) did not cause any marked effect in 5 (2 AH, 1 S and 2 NS) (71%) of 7 myenteric neurons. An example is shown in Fig. 2. In this Type 2/AH neuron, RR (10 MM) selectively blocked the effect of capsaicin (10 MM), although a slight depolarization ( < 3 mV) associated with a slight increase of input resistance (112.5%) remained. In the remaining two Type 1/S cells, capsaicin did not elicit the slow depolarizing response, although anodal break excitation and/or spontaneous action potentials were exerted. Therefore, the slow depolarizing response associated with increased input resistance elicited by capsaicin (10/~M) was blocked by RR (10/~M) in all myenteric neurons tested (Fig. 3).
o L-
0 Cap I0
pM
RR 1 pM
RR I0 pM
i
I +
Cap 10 ~M Fig. 3. Ruthenium red (RR; 1-10/aM) antagonism of the capsaicin (Cap; 10/aM)-induced slow depolarization of myenteric neurons. Numbers in parentheses denote number of responsive neurons. Blank column, mean amplitude of depolarization induced by capsaicin in responsive neurons (mV). Dotted column, number of neurons did not respond to capsaicin.
Effects of rnicroejected RR (1 mM) on the microejected capsaicin (0.1 raM) response In 31 (3 AH and 28 NS) of 63 myenteric neurons, no
J SP 1pulse
Rutheniumred-pretreated ~
20 mV
Ist Capsaicin 10 pM
~ SPlp
2nd Capsaicin 10 pM
Ruthenium red 10 pM A
c
1
I 40 mV
••••••••••••••••••••••••••••••••••••••••u••••••1•
~
SP Ip
10 s
Capsalcln 10 uM B
SP 1pulse
I
__
SP 1pulse
~11111111111111111111111111t1111111111111111111__111111
capsaic4n
Fig. 2. Ruthenium red (RR; 10/aM) specific antagonism of capsaicin (10/aM)-induced action on a myenteric Type 2/AH cell. A: after superfusion with RR (10/aM) for 5 min, which did not cause any effect, superfusion with capsaicin (10 /aM) did not cause a characteristic slow depolarizing response. Only a small depolarization associated with a slight increase of input resistance remained. B: 8 min after onset of superfusion with capsaicin (10 /aM), exogenous substance P applied by pressure microejection (SP; 0.1 mM; 1 pulse; at the arrow) elicited a slow depolarizing action associated with increased input resistance. C: 5 min after washout of capsaicin, SP produced a similar response to that in B. Resting membrane potential was -40 mV.
1 mln
Fig. 4. Antagonism of the effect of capsaicin (10 /aM) and no antagonism of the effect of substance P (0.1 mM) by pressure microejection of ruthenium red (RR; 1 mM; 900 ms; 30 pulses) in a Type 2/AH neuron. RR by itself did not cause any effect. A: 5 min after pretreatment of RR, superfusion with capsaicin (10/aM) did not cause a characteristic slow depolarization, but a small depolarization associated with slightly increased input resistance remained. Substance P (SP; 0.1 mM; 900 ms; 1 pulse; at the arrow) applied by pressure microejection caused a pronounced depolarizing response associated with increased input resistance. B: 17 min after the first application of capsaicin (10/aM), the second application of capsaicin elicited no response. SP (1 pulse; 1 p) caused a similar response to that in A. C: after 6-min washout of capsaicin, SP (1 pulse) caused a similar response to that in B. Resting membrane potential was -63 mV.
90
91
Fig. 5. Electron micrographs of myenteric plexus in the guinea-pig ileum, showing dose-dependent (1-10/~M) protective effects of RR from capsaicin-induced neurotoxic actions toward nerve fibers. A: control nerve fibers in the myenteric plexus treated with a vehicle (0.01% DMSO); the ultrastructure of the nerve fibers, terminals and somas in the myenteric plexus was preserved intact. B: nerve fibers in the myenteric plexus incubated in Krebs solution to which capsaicin (1 #M) was added for 60 rain. Many nerve fibers showed massive degenerative changes, i.e. many nerve fibers became slightly expanded and mitochondria were grossly swollen. C: nerve fibers in the myenteric plexus incubated in Krebs solution to which RR (1/~M) was added for 25 min and then capsaicin (1 #M) was added for 60 rain. Cytoplasmic organelles of the nerve fibers and ganglion cells were kept intact in their ultrastructure. However, the mitochondria in many nerve fibers and cells condensed. D: nerve fibers in myenteric plexus incubated in Krebs solution to which RR (10/~M) was added for 25 min and then capsaicin (1/~M) was added for 60 min. No obvious ultrastructural damages occurred in the somas of ganglion neurons, nerve terminals and nerve fibers. In particular the mitochondria have not been expanded. Bar = 1/~m.
response was evoked by RR (1 mM) applied by pressure microejection (1-30 pulses; 100-900 ms). In the remaining 32 (18 AH, 8 S and 6 NS) myenteric neurons, RR (1 mM) applied by pressure microejection (100-900 ms; 1-10 pulses) elicited a long-lasting (for 3-60.3 min) slow depolarization associated with increased (n = 14; 8 AH and 6 NS cells) or decreased input resistance (n = 12; 6 AH and 6 S cells), or increased neural discharges without any change of membrane potential (n = 6; 4 A H and 2 S cells). In Ca2+-free Krebs solution containing 0.1 mM EGTA, RR did not cause any effect in 9 (7 AH and 2 NS) of 10 myenteric neurons. In only one Type 2/AH neuron, RR applied by pressure microejection produced a slow depolarization associated with increased input resistance and evoked action potentials. In the previous paper z6, capsaicin (0.1 mM) applied by pressure microejection close to the cells has been found to cause a long-lasting slow depolarization associated with increased input resistance in 7 of 13 Type 2/AH neurons. After microejection of an approximately threshold pulse of capsaicin, the mean peak amplitude was 21.4 _+ 2.5 mV and the mean duration of this response was 6.9 _+ 2.6 min (n -- 7). The mean percent of increase of input resistance was 222.7 _+ 40.5% of the control (n -- 6). This effect was reconfirmed in the present study. After sufficient volume of RR (1 mM; 1-30 pulses) was microejected, pressure microejection of capsaicin (0.1 mM) had no effect on 11 (2 S and 9 NS) (about 70%) of 16 myenteric neurons. Of the 5 remaining neurons, microejected capsaicin caused a slow depolarization associated with increased input resistance in one NS cell and decreased input resistance in two (1 AH and 1 NS) cells, respectively. In two Type 1/S cells, RR dit not block the capsaicin-induced anodal break excitation and spontaneous action potentials. This effect of capsaicin has been demonstrated as a repeatable non-specific effect of capsaicin 26.
Effect of RR on the substance P response RR (1 mM) by pressure microejection or superfusion with RR (10/~M) did not affect the slow depolarizing response to exogenous substance P (0.1 mM) by pressure
microejection in all of 5 (4 AH and 1 NS) myenteric neurons, in which the depolarizing response to capsaicin (10/~M) was blocked by RR (Figs. 2 and 4). As shown in Fig. 4, pressure microejection of RR (1 raM; 30 pulses) attenuated a characteristic slow depolarizing response to capsaicin (10 ~M); the amplitude reduced to 8 mV; percent increase of input resistance reduced to 107%. However, an intense slow depolarization (23.5 mV) associated with increased input resistance (167% of the control) for 4.3 rain evoked by exogenous substance P was not affected by RR.
(2) Electron-microscopic examination In control treated with only vehicle, intense damage in the intestinal epithelial cells was caused, while the ultrastructure of the nerve fibers, terminals and somas in the myenteric plexus was preserved intact (Fig. 5A). After treatment of the myenteric plexus with capsaicin (1 ktM), many nerve fibers became slightly expanded with swollen mitochondria, showing conspicuous degenerative changes (Fig. 5B). Such changes thus occurred at the same concentration of capsaicin at which specific action on the extrinsic sensory nerve fibers could be detected in p h y s i o l o g i c a l s t u d i e s 24'26. In the 1-/~M RR-pretreated, capsaicin-treated plexus, cytoplasmic organelles of the nerve fibers and ganglion cells were kept intact in their ultrastructure. However, the mitochondria in many nerve fibers and cells were strikingly condensed. No obvious ultrastructural changes occurred in the somas of ganglionic neurons, nerve terminals and nerve fibers in the 10-/~M RR-pretreated, capsaicin-treated plexus. DISCUSSION
Present results showed that RR dose-dependently (1-10/~M) antagonized capsaicin-induced slow depolarization associated with an increase in input resistance that is due to a decrease in calcium-mediated potassium conductance in Type 2/AH cells. Type 2/AH cells of the small intestine have an on-going calcium-mediated potassium conductance that contributes to the relatively
92 negative resting membrane potential of these cells ~. Reduction of this conductance in Type 2/AH cells results in a depolarization of the membrane with an associated increase in input resistance. This slow depolarizing response to capsaicin of myenteric neurons has been found to be due to endogenous neuropeptides, such as substance P released from capsaicin-sensitive sensory nerve endings z6. The enhancement of neural discharges, unaccompanied with slow depolarization could be evoked by capsaicin. This effect was often seen in Type 1/S cells. This effect of capsaicin has been found to be repeatable and non-specific26. RR did not block this non-specific effect of capsaicin. When RR blocked the slow depolarization induced by capsaicin, the slow depolarizing response evoked by exogenous substance P, which obviously mimicked the response induced by capsaicin, was unaffected by RR. In addition, much evidence has been presented that RR specifically antagonizes a neuropeptide release induced by capsaicin 3-6'16"2°. Therefore, it is considered that RR does not act postsynaptically on myenteric neurons but specifically acts on presynaptically located capsaicin-sensitive extrinsic sensory nerve terminals, where RR inhibits the release of substance P. By electron-microscopic examination, neurotoxic action of capsaicin (1/~M) on nerve fibers in myenteric plexus was found to be dose-dependently (1-10 #M) protected by RR, although the effects of capsaicin appear not to be specifically restricted towards extrinsic sensory nerve fibers. According to the report by Marsh et al. 21, clear expansion of mitochondria and accompanying changes were visible after only 5 min in capsaicin (1-10 ~M) in vagal sensory neurons. In the present study, the tissues were incubated for 60 min in capsaicin (1 ~M). Thus, long incubation periods appeared to be the reason of more extended changes than expected. Therefore, the results from electron-microscopic observation supported the postulate based on the electrophysiological study that RR acts on presynaptically located capsaicin-sensitive sensory nerve terminals. Although the specific mechanism by which RR antagonizes the capsaicin-induced excitatory and neurotoxic effect is not clearly established by the present study, a possible mechanism can be proposed. It has been
REFERENCES 1 Adams, D.J., Takeda, K. and Umbach, J.A., Inhibition of calcium buffering depresses evoked transmitter release at the squid giant synapse, J. Physiol., 369 (1985) 145-149. 2 Alnaes, E. and Rahamimoff, R., On the role of mitochondria in transmitter release from motor nerve terminals, J. Physiol., 248 (1975) 285-306. 3 Amann, R., Donnerer, J. and Lembeck, E, Ruthenium red selectively inhibits capsaicin-induced release of calcitonin generelated peptide from the isolated perfused guinea-pig lung,
suggested that RR inhibits the release process at a mitochondrial site of action, where RR blocks Ca 2+ sequestration ~'s. However, RR has a generally low permeability to cells 7. Direct inhibition of Ca 2÷ influxes at the cell membrane by RR has been suggested to be the mechanism by which RR inhibits evoked release 2-2°'28. Recently, it has been reported that low concentrations of RR preferentially inhibit capsaicin-evoked neuropeptide release probably by interfering with the capsaicin-induced activation of cation channels at the cell membrane of sensory nerves in the isolated rat urinary bladder 6. Therefore, it might be possible that RR preferentially inhibits the capsaicin-induced activation of cation channels at the cell membrane of sensory nerves within myenteric plexus of the isolated guinea pig ileum as suggested by Amann et al. 6. Application of RR by superfusion at 1 /~M did not cause any effect on myenteric neurons but at 10 /~M elicited a slow depolarization associated with increased input resistance or an enhancement of neural discharges in about 33% of neurons tested. Pressure microejection of high concentration (1 mM) of RR caused a long-lasting slow depolarization associated with increased or decreased input resistance and/or generated spike potentials in about 51% of myenteric neurons tested. It has been suggested that RR can act through direct interaction with extracellularly localized sialic acid residues which are somehow involved in the calcium binding and blocks Ca2+-entry into the nerve tissues 29. Therefore, RR at high concentrations could act not only indirectly but also directly on the myenteric neurons. This direct action is considered to be CaZ+-dependent. From the present results, it is concluded that RR at low concentrations selectively antagonizes a specific effect of capsaicin on myenteric neurons by inhibiting a neuropeptide release from sensory nerve terminals evoked by capsaicin. However, high concentrations of RR act directly on myenteric neurons by some other mechanism. RR at low concentrations is a promising tool for investigation of the action of capsaicin, and elucidation of the mechanism of interaction of these two substances should add to our knowledge of sensory neuron function.
Neurosci. Lett., 101 (1989)311-315. 4 Amann, R., Donnerer, J. and Lembeck, E, Capsaicin-induced stimulation of polymodal nociceptors is antagonized by ruthenium red independently of extracellular calcium, Neurosci, 32 (1989) 255-259. 5 Amann, R., Donnerer, J. and Lembeck, F., Activation of primary afferent neurons by thermal stimulation. Influence of ruthenium red, Naunyn-Schrnied. Arch. Pharmacol., 341 (1990) 108-113. 6 Amann, R., Maggi, C.A., Giuliani, S., Donnerer, J. and Lembeck, F., Effects of carbonyl cyanide p-trichloro-methoxy-
93
7
8
9 10
11
12
13
14
15
16
17
18
phenylhydrazone(CCCP) and of ruthenium red (RR) on capsaicin-evoked neuropeptide release from peripheral terminals of primary afferent neurones, Naunyn-Schmied. Arch. Pharmacol., 341 (1990) 534-537. Babai, F., Ultrastructural method for the study of cell viability using ruthenium red. In: 9th International Congress on Electron Microscopy, 11, 1978, pp. 304-305. Bernath, S. and Vizi, E.S., Inhibitory effect of ionized free calcium enhanced by ruthenium red and m-chlorocarbonylcyanide phenylhydrazon on the evoked release of acetylcholine, Biochem. Pharmacol., 38 (1987) 179-226. Gamse, R., Molnar, A. and Lembeck, E, Substance P release from spinal cord slices by capsaicin, Life Sci., 25 (1979) 629-636. Goddard, Q.A. and Robinson, J.D., Uptake and release of calcium by rat brain synaptosomes, Brain Research, 110 (1976) 331-350. Grafe, P., Mayer, C.J. and Wood, J.D., Synaptic modulation of calcium-dependent potassium conductance in myenteric neurones in the guinea-pig, J. Physiol., 305 (1980) 235-248. Hirst, G.D.S., Holman, M.E. and Spence, I., Two types of neurones in the myenteric plexus of duodenum in the guinea-pig, J. Physiol., 236 (1974) 303-326. Jin, J.-G., Takaki, M. and Nakayama, S., Ruthenium red prevents capsaicin-induced neurotoxic action on sensory fibers of the guinea pig ileum, Neurosci. Lea., 106 (1989) 152-156. Jin, J.-G., Takaki, M. and Nakayama, S., Inhibitory effect of capsaicin on the ascending pathway of the guinea-pig ileum and antagonism of this effect by ruthenium red, Eur. J. Pharmacol., 180 (1990) 13-19. Maggi, C.A., Giuliani, S. and Meli, A., Effect of ruthenium red on responses mediated by activation of capsaicin-sensitive nerves of the rat urinary bladder, Naunyn-Schmied. Arch. Pharrnacol., 340 (1989) 541-546. Maggi, C.A., Patacchini, R., Santicioli, P., Giuliani, S., Bianco, E.D., Geppetti, P. and Meli, A., The 'efferent' function of capsaicin-sensitive nerves: Ruthenium Red discriminates between different mechanisms of activation, Eur. J. Pharmacol., 170 (1989) 167-177. Maggi, C.A., Patacchini, R., Giuliani, S., Santicioli, P. and Meli, A., Evidence for two independent modes of activation of the 'efferent' function mediated by peripheral terminals of capsaicin-sensitive sensory neurons by the use of omega conotoxin GVIA, a peptide modulator of neuronal calcium channels, Eur. J. Pharmacol., 156 (1988) 367-375. Maggi, C.A., Patacchini, R., Santicioli, P., Giuliani, S., Geppetti, P. and Meli, A., Protective action of Ruthenium Red toward capsaicin desensitization of sensory fibers, Neurosci. Lea., 88 (1988) 201-205.
19 Maggi, C.A., Santicioli, P., Geppetti, P., Parlani, M., Astolfi, M., DelBianco, E., Patacchini, R., Giuliani, S. and Meli, A., The effect of calcium free medium and nifedipine on the release of substance P-like immunoreactivity and contractions induced by capsaicin in the isolated guinea-pig bladder, Gen. Pharmacol., 40 (1989) 445-456. 20 Maggi, C.A., Santicioli, P., Geppetti, P., Parlani, M., Astolfi, M., PradeUes, P., Patacchini, R. and Meli, A., The antagonism induced by Ruthenium Red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies, Eur. J. Pharmacol., 154 (1988) 1-10. 21 Marsh, S.J., Stansfeld, C.E., Brown, D.A., Davey, R. and McCarthy, D., The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro, Neuroscience, 23 (1987) 275-289. 22 Swanson, P.D., Anderson, L. and Stahl, W.L., Uptake of calcium ions by synaptosomes from rat brain, Biochim. Biophys. Acta, 356 (1974) 174-183. 23 Takaki, M., Branchek, T., Tamir, H. and Gershon, M.D., Specific antagonism of enteric neural serotonin receptors by dipeptides of 5-hydroxytryptophan: evidence that serotonin is a mediator of slow synaptic excitation in the myenteric plexus, J. Neurosci., 5 (1985) 1769-1780. 24 Takaki, M., Jin, J.-G. and Nakayama, S., Ruthenium red antagonism of the effects of capsaicin on the motility of the isolated guinea-pig ileum, Eur. J. Pharmacol., 174 (1989) 57-62. 25 Takaki, M. and Nakayama, S., Effects of mesenteric nerve stimulation on the electrical activity of myenteric neurons in the guinea pig ileum, Brain Research, 442 (1988) 351-353. 26 Takaki, M. and Nakayama, S., Effects of capsaicin on myenteric neurons of the guinea pig ileum, Neurosci. Lett., 105 (1989) 125-130. 27 Tapia, R., Arias, C. and Morales, E., Binding of lanthanum ions and ruthenium red to synaptosomes and its effects on neurotransmitter response, J. Neurochem., 45 (1985) 1464-1473. 28 Tapia, R. and Meza-Ruiz, G., Inhibition by ruthenium red of the calcium-dependent release of 3H-GABA in synaptosomal fractions, Brain Research, 126 (1977) 160-166. 29 Wieraszko, A., Evidence that ruthenium red disturbs the synaptic transmission in the rat hippocampus slices through interacting with sialic acid residues, Brain Research, 378 (1986) 120-126. 30 Wood, J.D., Physiology of the enteric nervous system. In L.R. Johnson (Ed.), Physiology of the Gastrointestinal Tract, Vol. 1, 2nd edn., Raven Press, New York, 1987, pp. 67-109. 31 Wood, J.N., Winter, J., James, I.F., Rang, H.P., Yeats, J. and Bevan, S., Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture, J. Neurosci., 8 (1988) 3208-3220.
