European Journal of Pharmacology, 187 (1990) 291-292
291
Elsevier EJP 0276R
Rapid communication
Resiniferatoxin-evoked CGRP release and bronchoconstriction in the guinea-pig lung are inhibited by ruthenium red A n d e r s F r a n c o - C e r e c e d a , Y a - P i n g L o u a n d J a n M. L u n d b e r g Department of Pharmacology, Karolinska Institute, Box 60 400, S-104 01 Stockholm, Sweden
Received 13 September1990, accepted 14 September1990
Activation of capsaicin-sensitive sensory nerves not only elicits a centrally directed impulse but also results in local release of multiple neuropeptides stored in the peripheral nerve terminal region. Thus, abundant evidence suggests that the co-stored substance P, neurokinin A and calcitonin gene-related peptide (CGRP) are released in response to excitation of capsaicin-sensitive afferents in a variety of organs, resulting in various local biological effects (see Holzer, 1988). The recent demonstration of binding sites for resiniferatoxin (RTX), a phorbol-related diterpene with a capsaicin-like structure (Szallasi and Blumberg, 1989) indicated that capsaicin interacts with a specific receptor present on sensory ganglia. To evaluate this hypothesis we studied whether RTX activates peripheral sensory terminals in the lung via a mechanism similar to that for capsaicin, i.e. ruthenium red-sensitive C G R P release and bronchoconstriction (Maggi et al., 1988). Guinea-pigs (weight 250-350 g) of either sex were anesthetized with Mebumal (pentobarbital, 60 mg × kg - I i.p.) and a tracheal cannula connected to a respirator (Harvard, mod. 68) was used to ventilate the lungs at a rate of 70 breaths x min-1 with a tidal volume of 3 ml. After opening of the chest, the lungs were perfused through the pulmonary artery with Krebs solution of the
Correspondence to: A. Franco-Cereceda, Department of Pharmacology, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden.
following composition (mM): NaC1 118, CaC12 2.5, MgSO 4 1.2, N a H C O 3 24.9, K H 2 P O a 1.2, KC1 4.7, glucose 5.6 and Hepes 12.6 and aerated with 4% CO 2 in O 2 (for further details see Kr011 et al., 1990). The lungs were perfused with RTX (3 x 10 -1° M; Bio-Zac AB, Sweden; dissolved in absolute ethanol and further diluted in buffer) through a second perfusion solution chamber. The ganglionic blocking agent, chlorisondamine, (10 -6 M; CIBA, Switzerland) was added in all experiments to inhibit parasympathetic nerve activation; terbutaline (10 -7 M, Draco, Sweden) was also added in order to obtain reversible functional effects. Perfusate fractions (3 min) were collected through a right atrial catheter into a beaker placed on ice and containing acetic acid to give a final concentration of 0.2 M. The perfusate samples were desalted using SEP-PAK Cls cartridges, lyophilized and redissolved in buffer before determination of CGRP-LI by radioimmunoassay (RIA) with an antiserum raised against human C G R P alpha (RAS 6009, Peninsula, USA). RTX perfusion caused a slowly developping bronchoconstriction (increase in lung resistance, RE, by 689 _+ 150%; fig. 1). There was a parallel, significant release of CGRP-LI from the RTXperfused lungs (fig. 1). Thus, the outflow of CGRP-LI increased from 16.4 -t- 2.5 to 42.1 _+ 3.6 fmol X fraction -1. Perfusion with ruthenium red (RR) (5 X 10 -6 M for 30 rain; Serva, F R G ) did not per se influence basal lung performance or CGRP-LI outflow. However, R R completely in-
0014-2999/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
292 A
i
,oo -= 4so t 300 ] 1
O"
250 ]
OOl
T
I.i
g OOl
:
-RTX 0.3 nM
O 0
0
:
tive increase in cation conductance (March et al., 1987). In conclusion, in the guinea-pig lung, RTX may be considered a potent analogue of capsaicin with regard to functional effects and release of peptides from peripheral terminals of C-fibre afferents.
--rRTX 0.3 nM
Fig. 1. Effects of resiniferatoxin (RTX) perfusion (3 × 10-10 M for 3 min) on the bronchoconstriction (increased pulmonary resistance, RL) and outflow of CGRP-LI (% increase) in control experiments (open bars) and in experiments performed in the presence of ruthenium red (RR; 5 × 10 -6 M perfused for 30 rain; filled bars). The values (n = 4 in each group) are means_+ S.E.M. and are given as percentage changes compared to a control period immediately before perfusion with RTX. * * P < 0.01, Student's t-test.
hibited by RTX-induced bronchoconstriction and CGRP release from the isolated lung preparations (fig. 1). The present data demonstrate that perfusion of isolated guinea-pig lungs with RTX in a very low concentration is associated with non-cholinergic bronchoconstriction and release of CGRP-LI from presumably C-fibre afferents, thereby resembling the effects of capsaicin. It also seems clear that the mechanisms of activation of these nerves by capsaicin and RTX are similar as regards the inhibitory effects of RR. Thus, RR, a blocker of transmembrane Ca 2+ fluxes evoked by capsaicin, inhibited the bronchoconstriction and CGRP-LI release induced by the low concentration of RTX used (3 × 10 -1° M). This implies that the RTX-evoked excitation of sensory nerves may be caused by interaction with the proposed 'capsaicin-receptor' (Szallasi and Blumberg, 1989) and effected through a RR-sensi-
Acknowledgements The present study was supported by the Swedish HeartLung Foundation, the Laerdal Foundation, M. Bervall Foundation, T. Nilsson Foundation, the Lars Hierta Foundation, the Swedish Society of Medicine, the Swedish Tobacco Company, the American Tobacco Council, the Swedish Work and Environmental Fund, the Swedish Medical Research Council (14X-6554), the Swedish National Environmental Protection board and funds from the Karolinksa Institute.
References Holzer, P., 1988, Local effector function of capsaicin-sensitive sensory nerve endings: involvement of tachykinins and calcitonin gene-related peptide and other neuropeptides, Neuroscience 24, 739. Kr/511, F., J.-A. Karlsson, J.M. Lundberg and C.G.A. Persson, 1990, Capsaicin-induced bronchoconstriction and neuropeptide release in guinea-pig perfused lung: evidence for a local axon reflex. J. Appl. Physiol. 68, 1679. Maggi, C.A., R. Patacchini, P. Santicioli, S. Giuliani, P. Gepetti and A. Meli, 1988, Protective action of Ruthenium Red toward capsaicin desensitization of sensory nerves, Neurosci. Lett. 88, 201. March, S.J., D.A. Stansfeld, R. Brown, R. Davey and D. McCarthy, 1987, The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro, Neuroscience 23, 275. Szallasi, A. and P. Blumberg, 1989, Specific binding of resiniferatoxin, an ultrapotent capsaicin analogue to dorsal root ganglia membranes, The Pharmacologist 31, 183.
291
Elsevier EJP 0276R
Rapid communication
Resiniferatoxin-evoked CGRP release and bronchoconstriction in the guinea-pig lung are inhibited by ruthenium red A n d e r s F r a n c o - C e r e c e d a , Y a - P i n g L o u a n d J a n M. L u n d b e r g Department of Pharmacology, Karolinska Institute, Box 60 400, S-104 01 Stockholm, Sweden
Received 13 September1990, accepted 14 September1990
Activation of capsaicin-sensitive sensory nerves not only elicits a centrally directed impulse but also results in local release of multiple neuropeptides stored in the peripheral nerve terminal region. Thus, abundant evidence suggests that the co-stored substance P, neurokinin A and calcitonin gene-related peptide (CGRP) are released in response to excitation of capsaicin-sensitive afferents in a variety of organs, resulting in various local biological effects (see Holzer, 1988). The recent demonstration of binding sites for resiniferatoxin (RTX), a phorbol-related diterpene with a capsaicin-like structure (Szallasi and Blumberg, 1989) indicated that capsaicin interacts with a specific receptor present on sensory ganglia. To evaluate this hypothesis we studied whether RTX activates peripheral sensory terminals in the lung via a mechanism similar to that for capsaicin, i.e. ruthenium red-sensitive C G R P release and bronchoconstriction (Maggi et al., 1988). Guinea-pigs (weight 250-350 g) of either sex were anesthetized with Mebumal (pentobarbital, 60 mg × kg - I i.p.) and a tracheal cannula connected to a respirator (Harvard, mod. 68) was used to ventilate the lungs at a rate of 70 breaths x min-1 with a tidal volume of 3 ml. After opening of the chest, the lungs were perfused through the pulmonary artery with Krebs solution of the
Correspondence to: A. Franco-Cereceda, Department of Pharmacology, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden.
following composition (mM): NaC1 118, CaC12 2.5, MgSO 4 1.2, N a H C O 3 24.9, K H 2 P O a 1.2, KC1 4.7, glucose 5.6 and Hepes 12.6 and aerated with 4% CO 2 in O 2 (for further details see Kr011 et al., 1990). The lungs were perfused with RTX (3 x 10 -1° M; Bio-Zac AB, Sweden; dissolved in absolute ethanol and further diluted in buffer) through a second perfusion solution chamber. The ganglionic blocking agent, chlorisondamine, (10 -6 M; CIBA, Switzerland) was added in all experiments to inhibit parasympathetic nerve activation; terbutaline (10 -7 M, Draco, Sweden) was also added in order to obtain reversible functional effects. Perfusate fractions (3 min) were collected through a right atrial catheter into a beaker placed on ice and containing acetic acid to give a final concentration of 0.2 M. The perfusate samples were desalted using SEP-PAK Cls cartridges, lyophilized and redissolved in buffer before determination of CGRP-LI by radioimmunoassay (RIA) with an antiserum raised against human C G R P alpha (RAS 6009, Peninsula, USA). RTX perfusion caused a slowly developping bronchoconstriction (increase in lung resistance, RE, by 689 _+ 150%; fig. 1). There was a parallel, significant release of CGRP-LI from the RTXperfused lungs (fig. 1). Thus, the outflow of CGRP-LI increased from 16.4 -t- 2.5 to 42.1 _+ 3.6 fmol X fraction -1. Perfusion with ruthenium red (RR) (5 X 10 -6 M for 30 rain; Serva, F R G ) did not per se influence basal lung performance or CGRP-LI outflow. However, R R completely in-
0014-2999/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
292 A
i
,oo -= 4so t 300 ] 1
O"
250 ]
OOl
T
I.i
g OOl
:
-RTX 0.3 nM
O 0
0
:
tive increase in cation conductance (March et al., 1987). In conclusion, in the guinea-pig lung, RTX may be considered a potent analogue of capsaicin with regard to functional effects and release of peptides from peripheral terminals of C-fibre afferents.
--rRTX 0.3 nM
Fig. 1. Effects of resiniferatoxin (RTX) perfusion (3 × 10-10 M for 3 min) on the bronchoconstriction (increased pulmonary resistance, RL) and outflow of CGRP-LI (% increase) in control experiments (open bars) and in experiments performed in the presence of ruthenium red (RR; 5 × 10 -6 M perfused for 30 rain; filled bars). The values (n = 4 in each group) are means_+ S.E.M. and are given as percentage changes compared to a control period immediately before perfusion with RTX. * * P < 0.01, Student's t-test.
hibited by RTX-induced bronchoconstriction and CGRP release from the isolated lung preparations (fig. 1). The present data demonstrate that perfusion of isolated guinea-pig lungs with RTX in a very low concentration is associated with non-cholinergic bronchoconstriction and release of CGRP-LI from presumably C-fibre afferents, thereby resembling the effects of capsaicin. It also seems clear that the mechanisms of activation of these nerves by capsaicin and RTX are similar as regards the inhibitory effects of RR. Thus, RR, a blocker of transmembrane Ca 2+ fluxes evoked by capsaicin, inhibited the bronchoconstriction and CGRP-LI release induced by the low concentration of RTX used (3 × 10 -1° M). This implies that the RTX-evoked excitation of sensory nerves may be caused by interaction with the proposed 'capsaicin-receptor' (Szallasi and Blumberg, 1989) and effected through a RR-sensi-
Acknowledgements The present study was supported by the Swedish HeartLung Foundation, the Laerdal Foundation, M. Bervall Foundation, T. Nilsson Foundation, the Lars Hierta Foundation, the Swedish Society of Medicine, the Swedish Tobacco Company, the American Tobacco Council, the Swedish Work and Environmental Fund, the Swedish Medical Research Council (14X-6554), the Swedish National Environmental Protection board and funds from the Karolinksa Institute.
References Holzer, P., 1988, Local effector function of capsaicin-sensitive sensory nerve endings: involvement of tachykinins and calcitonin gene-related peptide and other neuropeptides, Neuroscience 24, 739. Kr/511, F., J.-A. Karlsson, J.M. Lundberg and C.G.A. Persson, 1990, Capsaicin-induced bronchoconstriction and neuropeptide release in guinea-pig perfused lung: evidence for a local axon reflex. J. Appl. Physiol. 68, 1679. Maggi, C.A., R. Patacchini, P. Santicioli, S. Giuliani, P. Gepetti and A. Meli, 1988, Protective action of Ruthenium Red toward capsaicin desensitization of sensory nerves, Neurosci. Lett. 88, 201. March, S.J., D.A. Stansfeld, R. Brown, R. Davey and D. McCarthy, 1987, The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro, Neuroscience 23, 275. Szallasi, A. and P. Blumberg, 1989, Specific binding of resiniferatoxin, an ultrapotent capsaicin analogue to dorsal root ganglia membranes, The Pharmacologist 31, 183.
