72
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I .'7 i iV'#2* 7 : ' 4
1992 Elsevier Scientific Publishers h'eland Ltd. A.II rights rcscr~cd i)304-3940 92 $ 0"~.I)lJ NSL 08463
Nicotine- and capsaicin-, but not potassium-evoked CGP-release from cultured guinea-pig spinal ganglia is inhibited by Ruthenium red Anders Franco-Cereceda a, Malin Rydh b and Carl-Johan Dalsgaard c Departments of~Pharmaeology and bAnatomy. Karolinska Institute, Stockholm (Sweden) and 'Astra Pain Control. SOdertdlje (Sweden) (Received 28 May 1991: Revised version received 2 December 1991: Accepted 11 December 199t)
Key words: Calcitonin gene-related peptide: Capsaicin: Nicotine; Ruthenium red: Sensory nerve In the present study we have investigated the effects of nicotine, capsaicin, potassium, glutamate and aspartate on release of calcitonin gene-related peptide (CGRP)-like immunoreactivity (-L1) from guinea-pig dorsal root ganglion (DRG) cultures. In addition the possible influence of Ruthenium red (RR), neuropeptide Y (NPY) and noradrenaline (NA) on the CGRP-LI outflow has been evaluated. Nicotine, capsaicin and potassium, but not glutamate or aspartate, evoked a Ca>-dependent increase in the culture medium, suggesting release of CGRP-LI. RR inhibited the effect of both capsaicin and nicotine but did not influence potassium-induced CGRP-LI release. Furthermore, the nicotine- but not capsaicin-evoked CGRP-LI release was inhibited by NPY. It is concluded that DRG cultures represent an experimental model where regulation of CGRP-LI release can be studied. The ability of RR to inhibit not only capsaicin but also nicotine effects indicate that the proposed selectivity of RR may depend on the agent used to evoke peptide release and/or concentrations used.
Immunohistochemical and radioimmunological data has shown that calcitonin gene-related peptide (CGRP)like immunoreactivity (-LI) is present in a variety of peripheral organs in a population of C-fibre afferents originating in primary afferent neurons in dorsal root ganglia (DRG) (see ref. 7). Activation of these peripheral afferent nerves by a variety of agents including capsaicin, nicotine and potassium evokes a Ca>-dependent release of CGRP-LI with subsequent potent biological effects [3]. Recently Ruthenium red (RR), a blocker of transmembrane Ca2+-fluxes [18] and mitochondrial Ca >uptake [15], has been proposed to selectively block capsaicin-induced peripheral nociceptor stimulation [1, 4, 12]. Furthermore, capsaicin application to DRG cultures evokes a rapid RR-sensitive increase in accumulation of intracellular Ca 2+ [20]. We have therefore studied the effects of RR on CGRP-LI release from guinea-pig DRG cultures evoked by various agents. In addition, since neuropeptide Y (NPY) has been reported to modulate transmitter release from DRG cultures [19], we have evaluated the effects of NPY on CGRP-LI release. Adult guinea-pigs (b. wt. 250-350 g, n=26) were killed by cervical dislocation. DRG (n--390) were dissected under sterile conditions, carefully freed from connective tissue and roots and collected in Leibowit's L-15 Correspondence. A. Franco-Cereceda, Department of Pharmacology, Karolinska Institute, Box 60 400, S-104 01 Stockholm. Sweden.
medium. The specimens were cut with a pair of small scissors and subsequently incubated in collagenase (157 U/ml) in 37°C for 1 h and collagenase and trypsin (0.25%; l:l) for 30 min. After centrifugation and removal of the supernatant, the DRG were washed in culture medium, Eagle's modification of minimal essential medium (E-MEM) containing glutamine and supplemented with 10% fetal calf serum, 10% horse serum, 0.9% glucose and nerve growth factor l0 ng/ml (Sigma, St. Louis, MO, USA) and mechanically dissociated using a Pasteur pipette with flame-restricted opening. The cells were counted in a Btirker-chamber and 4 × 104 cells were seeded out on collagen-coated microscope glass cover Petri dishes, diameter 16 mm. The cultures (n=36) were then incubated at 37°C in 95% air, 5% CO 2 athmosphere for 4-5 days. Medium was changed every second day. The medium was removed from the cultures and the cultures were exposed to Tyrode's solution (composition in mM: NaC1 137, NaHCO3 11.9, KCl 2.7, MgC12 1.05, NaH2PO4 0.42, CaC12 1.8 and glucose 5.6 bubbled with 95% 02 and 5% CO2 giving a pH of 7.4) for l0 rain. Thereafter, the effects of potassium (60 mM), capsaicin (10 -s M; Fluka, Basel, Switzerland; dissolved in 60% ethanol in a stock solution of l0 mg/ml and further diluted in Tyrode's solution) or nicotine (t 0 -s M; Sigma) on the outflow of CGRP-LI were studied under control conditions as well as in Ca>-free Tyrode's solution containing 0.1 mM EGTA and after l0 min of incubation
73
200 ~
]
•
[]
175 1 +
Control
-Ca
[] +RR ,e
[] +NPY []
125]
+NA
lOO] 75Nicotine
Capsaicin
Potassium
Fig, I. Effects of 10 rain exposure to nicotine (10 ' M; left), capsaicin ( 10-s M: middle) or potassium (60 raM; right) on the content of CGRPL1 in the medium of D R G cell cultures. The effects of incubation in Ca>-free medium or medium containing RR (10 + M), NPY (10 7 M), or NA (10 +' M) on the CGRP-LI outflow evoked by nicotine or capsaicin are also shown. Values are expressed as percentage of a 10 rain contrcd period and given as means _+ S.E.M., n=4 8 of each. +t'
\'c~.'.~a.mc
t.~,',,~.
I .'7 i iV'#2* 7 : ' 4
1992 Elsevier Scientific Publishers h'eland Ltd. A.II rights rcscr~cd i)304-3940 92 $ 0"~.I)lJ NSL 08463
Nicotine- and capsaicin-, but not potassium-evoked CGP-release from cultured guinea-pig spinal ganglia is inhibited by Ruthenium red Anders Franco-Cereceda a, Malin Rydh b and Carl-Johan Dalsgaard c Departments of~Pharmaeology and bAnatomy. Karolinska Institute, Stockholm (Sweden) and 'Astra Pain Control. SOdertdlje (Sweden) (Received 28 May 1991: Revised version received 2 December 1991: Accepted 11 December 199t)
Key words: Calcitonin gene-related peptide: Capsaicin: Nicotine; Ruthenium red: Sensory nerve In the present study we have investigated the effects of nicotine, capsaicin, potassium, glutamate and aspartate on release of calcitonin gene-related peptide (CGRP)-like immunoreactivity (-L1) from guinea-pig dorsal root ganglion (DRG) cultures. In addition the possible influence of Ruthenium red (RR), neuropeptide Y (NPY) and noradrenaline (NA) on the CGRP-LI outflow has been evaluated. Nicotine, capsaicin and potassium, but not glutamate or aspartate, evoked a Ca>-dependent increase in the culture medium, suggesting release of CGRP-LI. RR inhibited the effect of both capsaicin and nicotine but did not influence potassium-induced CGRP-LI release. Furthermore, the nicotine- but not capsaicin-evoked CGRP-LI release was inhibited by NPY. It is concluded that DRG cultures represent an experimental model where regulation of CGRP-LI release can be studied. The ability of RR to inhibit not only capsaicin but also nicotine effects indicate that the proposed selectivity of RR may depend on the agent used to evoke peptide release and/or concentrations used.
Immunohistochemical and radioimmunological data has shown that calcitonin gene-related peptide (CGRP)like immunoreactivity (-LI) is present in a variety of peripheral organs in a population of C-fibre afferents originating in primary afferent neurons in dorsal root ganglia (DRG) (see ref. 7). Activation of these peripheral afferent nerves by a variety of agents including capsaicin, nicotine and potassium evokes a Ca>-dependent release of CGRP-LI with subsequent potent biological effects [3]. Recently Ruthenium red (RR), a blocker of transmembrane Ca2+-fluxes [18] and mitochondrial Ca >uptake [15], has been proposed to selectively block capsaicin-induced peripheral nociceptor stimulation [1, 4, 12]. Furthermore, capsaicin application to DRG cultures evokes a rapid RR-sensitive increase in accumulation of intracellular Ca 2+ [20]. We have therefore studied the effects of RR on CGRP-LI release from guinea-pig DRG cultures evoked by various agents. In addition, since neuropeptide Y (NPY) has been reported to modulate transmitter release from DRG cultures [19], we have evaluated the effects of NPY on CGRP-LI release. Adult guinea-pigs (b. wt. 250-350 g, n=26) were killed by cervical dislocation. DRG (n--390) were dissected under sterile conditions, carefully freed from connective tissue and roots and collected in Leibowit's L-15 Correspondence. A. Franco-Cereceda, Department of Pharmacology, Karolinska Institute, Box 60 400, S-104 01 Stockholm. Sweden.
medium. The specimens were cut with a pair of small scissors and subsequently incubated in collagenase (157 U/ml) in 37°C for 1 h and collagenase and trypsin (0.25%; l:l) for 30 min. After centrifugation and removal of the supernatant, the DRG were washed in culture medium, Eagle's modification of minimal essential medium (E-MEM) containing glutamine and supplemented with 10% fetal calf serum, 10% horse serum, 0.9% glucose and nerve growth factor l0 ng/ml (Sigma, St. Louis, MO, USA) and mechanically dissociated using a Pasteur pipette with flame-restricted opening. The cells were counted in a Btirker-chamber and 4 × 104 cells were seeded out on collagen-coated microscope glass cover Petri dishes, diameter 16 mm. The cultures (n=36) were then incubated at 37°C in 95% air, 5% CO 2 athmosphere for 4-5 days. Medium was changed every second day. The medium was removed from the cultures and the cultures were exposed to Tyrode's solution (composition in mM: NaC1 137, NaHCO3 11.9, KCl 2.7, MgC12 1.05, NaH2PO4 0.42, CaC12 1.8 and glucose 5.6 bubbled with 95% 02 and 5% CO2 giving a pH of 7.4) for l0 rain. Thereafter, the effects of potassium (60 mM), capsaicin (10 -s M; Fluka, Basel, Switzerland; dissolved in 60% ethanol in a stock solution of l0 mg/ml and further diluted in Tyrode's solution) or nicotine (t 0 -s M; Sigma) on the outflow of CGRP-LI were studied under control conditions as well as in Ca>-free Tyrode's solution containing 0.1 mM EGTA and after l0 min of incubation
73
200 ~
]
•
[]
175 1 +
Control
-Ca
[] +RR ,e
[] +NPY []
125]
+NA
lOO] 75Nicotine
Capsaicin
Potassium
Fig, I. Effects of 10 rain exposure to nicotine (10 ' M; left), capsaicin ( 10-s M: middle) or potassium (60 raM; right) on the content of CGRPL1 in the medium of D R G cell cultures. The effects of incubation in Ca>-free medium or medium containing RR (10 + M), NPY (10 7 M), or NA (10 +' M) on the CGRP-LI outflow evoked by nicotine or capsaicin are also shown. Values are expressed as percentage of a 10 rain contrcd period and given as means _+ S.E.M., n=4 8 of each. +t'
