Release of CGRP-Like Immunoreactivity from Cultured Sensory Neurons M. RYDH, A. FRANCO-CERECEDA, AND C.-J. DALSGAARD Departments of Anatomy and Pharmacology Karolinska Institute 104 01 Stockholm, Sweden and Astra Pain Control Sodertulje, Sweden The release of neurotransmitters from primary sensory neurons can be evoked by a variety of substances, including capsaicin, nicotine, and potassium, which all elicit a Ca2+-dependent release. Neuropeptide Y (NPY) exerts an inhibitory effect on substance P (SP) release from cultured sensory neurons.2 Ruthenium red (RR) is a blocker of transmembranal CaZs transport3 and blocks the effects evoked by c a ~ s a i c i n . ~Noradrenaline -~ (NA) blocks Ca?' -dependent SP release from chicken sensory neurons evoked by electrical stimulation. We therefore studied the effects of capsaicin, nicotine, and potassium on the calcitonin gene-related peptide-like immunoreactive (CGRP-LI) release in the presence and absence of Ca2+and the modulatory effect of RR, NPY, and NA.
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MATERIALS AND METHODS Dissociated dorsal root ganglion (DRG) cells from adult guinea pigs (250 g) were seeded out on collagen-coated 16 mm Petri dishes. The cultures were incubated in culture medium supplemented with 10% fetal calf serum, 10% horse serum, 0.9% glucose, and 10 ng/ml nerve growth factor (NGF) at 37 "C in a 5% C 0 2atmosphere. Medium was changed every second day. After 5 days the cells had developed extensive neurite formations. The medium was removed and the cultures were rinsed before exposure to Tyrode's solution for 10 minutes at 37 "C. The supernatants were collected to determine the basal CGRP secretion. The effects of potassium (60 mM), capsaicin (lov5M), and nicotine M) (all diluted in Tyrode's solution) on the outflow of CGRP were then studied in the presence and absence of Ca2+.RR M), NPY (lo-' M), and NA M) were added to study the modulatory effect of these agents. The total content of CGRP-LI was determined in six control cultures not subjected to any experimental procedures. The collected samples were desalted using SEP-PAK c18 cartridges, lyophilized, and redissolved in 0.5 ml buffer for the determination of CGRP-LI by radioimmunoassay .
RESULTS The total content in the DRG cultures was 265 2 4.5 fmol/culture ( n = 4 ) . The basal release after a 10-minute incubation with Tyrode's solution of all cultures 497
ANNALS NEW YORK ACADEMY OF SCIENCES
498
H
Control -Ca +RR
e
+NW
150
dl
c] +NA
I
9?!
125
2
100
K c)
0
.-
75
Nicotine
Capsaicin
Potassium
FIGURE 1. Effects of a 10-minute exposure to nicotine M; left). capsaicin M; middle), or potassium (60 mM; right) on the content of CGRP-LI in the medium of DRG cell cultures. The effects of incubation with Ca*+-freemedium or medium containing RR (lo-' M), NPY (lo-' M), or NA (lo-' M ) on the CGRP-LI outflow evoked by nicotine or capsaicin are also shown. Values are expressed as means SEM. Student's f test for paired samples was used for statistical evaluation; p < 0.05 was considered significant.
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was 19.6 ? 2.2 fmol/fraction. The basal level of CGRP-LI was not altered after incubation with Ca2+-freeTyrode's solution, RR, NPY, or NA. Incubation with capsaicin induced a Ca2+-dependentrelease that could be blocked by RR. NPY and NA did not change the increased outflow. Exposure to nicotine as well as potassium evoked a clearcut increase in the release of CGRP-LI, dependent on extracellular Ca2+.RR inhibited the outflow caused by nicotine, whereas the effect evoked by potassium was left unchanged. The administration of NPY prevented the release elicited by nicotine. NA caused a slight inhibition in the nicotine effect (FIG.1).
DISCUSSION AND CONCLUSIONS The present findings of the release of CGRP-LI after exposure to capsaicin, nicotine, and potassium are in line with earlier studies on C-fiber endings.' We conclude that CGRP-LI can be released from cultured DRG cells after exposure to nicotine, capsaicin, and potassium in a Ca?+-dependentway. RR can abolish the effect caused by nicotine and capsaicin. The release elicited by nicotine can also be modulated by NPY. This method represents a model in which to study the modulation of release of different transmitters from sensory neurons in uitro. REFERENCES 1. FRANCO-CERECEDA, A. 1988. Acta Physiol. Scand. 113(Suppl. 569): 1-163 2. WALKER,M. et a / . 1988. J. Neurosci 8: 2438-2446. 3. SWANSON, L . W. et a/. 1974. Biochem. Biophys. Acta 356: 174-183. 4. MAGGI,C. A. et ul. 1988. Eur. J. Pharmacol. 1 1 5 4 1-10, 5. AMANN,R. & F. LEMBECK.1988. Eur. J. Pharmacol. 160: 227-229. 6. FRANCO-CERECEDA, A. et al. 1989. Acta Physiol. Scand. 137: 457-458.
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MATERIALS AND METHODS Dissociated dorsal root ganglion (DRG) cells from adult guinea pigs (250 g) were seeded out on collagen-coated 16 mm Petri dishes. The cultures were incubated in culture medium supplemented with 10% fetal calf serum, 10% horse serum, 0.9% glucose, and 10 ng/ml nerve growth factor (NGF) at 37 "C in a 5% C 0 2atmosphere. Medium was changed every second day. After 5 days the cells had developed extensive neurite formations. The medium was removed and the cultures were rinsed before exposure to Tyrode's solution for 10 minutes at 37 "C. The supernatants were collected to determine the basal CGRP secretion. The effects of potassium (60 mM), capsaicin (lov5M), and nicotine M) (all diluted in Tyrode's solution) on the outflow of CGRP were then studied in the presence and absence of Ca2+.RR M), NPY (lo-' M), and NA M) were added to study the modulatory effect of these agents. The total content of CGRP-LI was determined in six control cultures not subjected to any experimental procedures. The collected samples were desalted using SEP-PAK c18 cartridges, lyophilized, and redissolved in 0.5 ml buffer for the determination of CGRP-LI by radioimmunoassay .
RESULTS The total content in the DRG cultures was 265 2 4.5 fmol/culture ( n = 4 ) . The basal release after a 10-minute incubation with Tyrode's solution of all cultures 497
ANNALS NEW YORK ACADEMY OF SCIENCES
498
H
Control -Ca +RR
e
+NW
150
dl
c] +NA
I
9?!
125
2
100
K c)
0
.-
75
Nicotine
Capsaicin
Potassium
FIGURE 1. Effects of a 10-minute exposure to nicotine M; left). capsaicin M; middle), or potassium (60 mM; right) on the content of CGRP-LI in the medium of DRG cell cultures. The effects of incubation with Ca*+-freemedium or medium containing RR (lo-' M), NPY (lo-' M), or NA (lo-' M ) on the CGRP-LI outflow evoked by nicotine or capsaicin are also shown. Values are expressed as means SEM. Student's f test for paired samples was used for statistical evaluation; p < 0.05 was considered significant.
*
was 19.6 ? 2.2 fmol/fraction. The basal level of CGRP-LI was not altered after incubation with Ca2+-freeTyrode's solution, RR, NPY, or NA. Incubation with capsaicin induced a Ca2+-dependentrelease that could be blocked by RR. NPY and NA did not change the increased outflow. Exposure to nicotine as well as potassium evoked a clearcut increase in the release of CGRP-LI, dependent on extracellular Ca2+.RR inhibited the outflow caused by nicotine, whereas the effect evoked by potassium was left unchanged. The administration of NPY prevented the release elicited by nicotine. NA caused a slight inhibition in the nicotine effect (FIG.1).
DISCUSSION AND CONCLUSIONS The present findings of the release of CGRP-LI after exposure to capsaicin, nicotine, and potassium are in line with earlier studies on C-fiber endings.' We conclude that CGRP-LI can be released from cultured DRG cells after exposure to nicotine, capsaicin, and potassium in a Ca?+-dependentway. RR can abolish the effect caused by nicotine and capsaicin. The release elicited by nicotine can also be modulated by NPY. This method represents a model in which to study the modulation of release of different transmitters from sensory neurons in uitro. REFERENCES 1. FRANCO-CERECEDA, A. 1988. Acta Physiol. Scand. 113(Suppl. 569): 1-163 2. WALKER,M. et a / . 1988. J. Neurosci 8: 2438-2446. 3. SWANSON, L . W. et a/. 1974. Biochem. Biophys. Acta 356: 174-183. 4. MAGGI,C. A. et ul. 1988. Eur. J. Pharmacol. 1 1 5 4 1-10, 5. AMANN,R. & F. LEMBECK.1988. Eur. J. Pharmacol. 160: 227-229. 6. FRANCO-CERECEDA, A. et al. 1989. Acta Physiol. Scand. 137: 457-458.
