- Email: [email protected]
Modulation of calcitonin gene-related peptide release evoked by bradykinin and electrical field stimulation in guinea-pig atria
ELSEVIER
Neuroscience Letters 170 (1994) 163 166
HEUROSClENCE LETTERS
Modulation of calcitonin gene-related peptide release evoked by bradykinin and electrical field stimulation in guinea-pig atria Elena Del Bianco a'*, Carlo Alberto Maggi b, Paolo Santicioli b, Pierangelo GeppettF "Institute of Clinical Dermatology, Florence UniversiO', Via degli Alfani 37, 50121 Florence, ltah' bPharmacology Department, Research Laboratories, Menarini Pharmaceuticals, Florence. Italy 'Institute of Internal Medicine IV, University of Florence, Florence, Italy Received 16 November 1993; Revised version received 2 February 1994; Accepted 2 February 1994
Abstract Electrical field stimulation (EFS) and bradykinin (BK) are able to activate capsaicin-sensitive sensory neurons of guinea-pig atria in a (o-conotoxin (CTX)-sensitive and Ruthenium Red (RR)-insensitive manner. The aim of this work was to study EFS and BK-induced release of calcitonin gene-related peptide (CGRP) from guinea-pig atria and in particular the action of morphine and neuropeptide Y (NPY) on this release. EFS-induced CGRP release was frequency-dependent and tetrodotoxin (TTX)-sensitive, while BK-evoked CGRP release was TTX-insensitive. On the other hand, CGRP outflow induced by either EFS or BK was similarly reduced in the presence of morphine and NPY. It is therefore hypothesized that NPY and opioids exert their inhibitory action by acting on the very terminal region of the nerve fibre. Moreover, our results show that dermorphine but not dynorphin reduced BK-evoked CGRP release, suggesting that p opioid receptors are responsible for morphine action. Studying the action of peptide YY and NPY(16 36) on BK-evoked CGRP release, we demonstrated that both had similar inhibitory effects, supporting the presence of Y2 receptors on the nerve terminal region.
Key words: CGRP; Atrium; Bradykinin; Opioid; NPY; NPY(16 36); PYY
A subpopulation of primary sensory neurons is characterized by their sensitivity to capsaicin, the pungent principle of red peppers, and by their content of neuropeptides, including calcitonin gene-related peptide (CGRP) [5]. Various stimuli, including capsaicin itself, electrical field stimulation (EFS) and bradykinin (BK), are capable of exciting the peripheral endings of these neurons, thus inducing release of CGRP. Different modes of transmitter secretion have been described [6]: the secretion of C G R P due to antidromic invasion of the nerve endings by a propagated action potential (axon reflex) is blocked by tetrodotoxin (TTX), is strongly inhibited by m-conotoxin (CTX, a selective antagonist of N-type voltage-sensitive calcium channels), but is unaffected by the inorganic dye Ruthenium Red (RR). On the contrary, neuropeptide release evoked by capsaicin is unaffected by T T X and CTX but is selectively blocked by RR [6]. Recently [3], it has been found that BK acti* Corresponding author. 0304-3940•94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD1 0304-3940(94)00112-N
vates capsaicin-sensitive sensory afferents inducing release of C G R P from guinea-pig atria by a mechanism similar to that evoked by EFS, being CTX-sensitive and RR-insensitive. In contrast to EFS, BK-evoked release of C G R P is unaffected by T T X and blocked by indomethacin, the cycloxygenate inhibitor [3]. Because of this difference we addressed the question of whether C G R P release produced by EFS or BK could be subjected to differential pharmacological modulation; in particular, we tested the actions of morphine and neuropeptide Y (NPY) which have been proposed to exert a prejunctional modulatory influence on sensory fibre activation in the guinea-pig atrium [4]. In addition, this work tried to identify the receptor types that could be involved in the modulatory actions of opioids and of NPY at the sensory nerve endings level. Male albino guinea-pigs weighing 270 340 g were stunned and bled. The hearts were rapidly removed and placed in cold oxygenated Krebs solution of the following composition (in mM): NaC1 119, NaHCO~ 25,
164
E, D e l Bianco e / a l . I Neuro,~cience Letters 17(1 ' 1994 ) 163-166
u
200
-
160
--
10 H e r t z
120
o
E 80
-
40 L)
0
1
2
3
4
5
6
FRACTION5
u L
160-
c~
120
E v
80
,
40
L~ U
Hertz
20
200
-
0
I
2
3
4
5
6
5
6
FRACTIONS
g
200 16o
f2D-
-
L m
120
E v
80
Hertz
30
-q ,
40
u
0
I
2
3
4
FRACTION5
Fig. 1. Increase in calcitonin gene-related peptide-like immunoreactiv-
ity (CGRP-LI) outflow produced by electrical field stimulation in slices of guinea-pig atria at different frequencies: 10 H z ( u p p e r panel), 20 H z (medium panel), 30 Hz (lower panel). The other electric parameters remain invariate (10 V, 50 mA/cm z, for 5 min). Each column represents a 5 min fraction. The first and the second columns represent the basal values * P < 0.05, ** P < 0.01 vs. mean basal value, n = 5.
period. In experiments in which BK was used as stimulus to induce CGRP release, two fractions before a n d t b u r during exposure to BK were collected. After a further superfusion (30 min) with Krebs solution containing the v a r i o u s tested compounds, E F S o r BK were given for a second time and samples were collected tbllowing the same protocol described above. At the end of the experiments, the tissues were blotted 2-3 times on filter paper and weighed. The superfusates were freeze-dried, reconstituted with the assay buffer (0.1 M, pH 7.4 phosphate buffer containing 0.9 NaC1, 0.01% NaN 3 and 0.t% bovine serum albumin) and CGRP was measured by radioimmunoassay as reported previously [2]. Rat ~-CGRP (Peninsula) as standard, '25I-iodohistidyl-CGRP (Amersham) and RAS 6012 anti-CGRP antiserum (Peninsula) were used. The coefficient of percentage variation was less than 10% for values between 20 and 300 pmol/1. Sensitivity of the assay was 2.5 fmol/tube. The antiserum cross-reacted 100% with rat fl-CGRP and human CGRP-I and -II. Each value in the text and figures is the mean + S.E.M. of at least 5 experiments. Total evoked release (TER) was calculated by the sum of the values observed during the stimulation period from which the mean basal value was subtracted. The ratio between TER of CGRP-LI caused by the first (S1) and the second ($2) stimulation period was calculated ($2/S1 ratio). The ratio ($2/S1) observed in control preparation was compared with the ratio ($2/ S1) measured when the $2 stimulus was given in the presence of the compounds under investigation. Statistical analysis was done by means of Student's t-test or with Dunnett's test when applicable. Bradykinin, thiorphan, captopril, neuropeptide Y (NPY), peptide YY (PYY), NPY(16-36) (Sigma); tetroBRADYKININ
1.5
KH2PO 4 1.2, MgSO4 1.5, KCI 4.7, CaC12 2.5, glucose 11. Both atria were removed from the hearts and thick (500 /IM) slices were prepared using a tissue slicer (MacIlwain). Slices were pooled, transferred (150-200 mg of tissue) in a 2 ml perfusion chamber and superfused at a rate of 0.4 ml/min with oxygenated (96% 02-4% CO2) Krebs solution, at 37°C, containing 0.1% bovine serum albumin, thiorphan (10 pM) and captopril (10 pM). A 30 min equilibration period was allowed to elapse, after which 5 rain fractions were collected in tubes containing acetic acid (final concentration 2 N). In experiments in which release was induced by EFS, electrical impulses (10 V, 50 mA/cm:, 10=30 Hz, t ms pulse duration, 10 s train every 20 s) were applied for 5 min with platinum electrodes connected to a Grass stimulator via a constant current unit (Basile). Fractions were collected for 10 min before, 5 min during and 15 min after the stimulation
T I
._o
1
1 Fig. 2. The effect of tetrodotoxin (TTX, 0.3 ~tM), c0-conotoxin qCTX. 0.1 BM), morphine (MORPH. 3 ttM) and neuropeptide Y (NPY. 0.3 BM) on the release of calcitonin gene-related peptide (CGRP) evoked by electrical field stimulation (10 V. 50 mA/cm z. 30 Hz~ from slices of guinea-pig atria. Each column represents the ratio between total evoked release of CGRP caused by the first ($1) and the second ($2) stimulation period. *P < 0.05, **P < 0.01 vs. $2/S1 ratio of controls, n = 5.
165
E. Del Bianco et al./Neuroscience Letters 170 (1994) 163 166
ELECTRICAL
FIELD
In order to characterize opioid and NPY receptor modulation of C G R P - L I release, BK (10/aM) was used as stimulus to evoke C G R P - L I release from capsaicinsensitive sensory neurons. When atrium slices were exposed to dermorphine (0.1 /aM), BK-evoked C G R P - L I release was markedly reduced (TER being 43 4- 5.8 fmol/ g/30 min, P<0.01), while dynorphine (1/aM) did not have an inhibitory effect on BK-evoked C G R P - L I release (TER being 190 + 42 fmol/g/30 min) (Fig. 4). Exposure of the same preparations to NPY(16 36) (1 /aM) and to PYY (0.3/aM) reduced BK-evoked C G R P L1 outflow in a very significant manner, T E R 35 +_ 9 and 43 4- 11 fmol/gJ25 min, respectively, versus 113 4- 13 fmol/g/25 min of control preparations (P<0.01) (Fig. 4). BK exerts a powerful excitatory action on primary sensory neurons [7]. This effect accounts for the algesic action of this autacoid and for its ability to release peptide transmitters from peripheral endings of capsaicinsensitive sensory neurons. BK-induced C G R P release from slices of guinea-pig atria was insensitive to TTX, thus indicating that BK exerts its action on the very terminal region of the neuron without the involvement of propagated action potentials (axon reflexes), but was inhibited by CTX, suggesting participation of voltagesensitive calcium channels [4,5]. Giuliani et al. [4] have shown that in guinea-pig isolated left atria EFS produces a positive inotropic response that is blocked by CTX and TTX. Responses to EFS were also inhibited by opioids and NPY [1,4]. The present findings confirm the presence of a marked release of C G R P evoked by EFS in the guinea pig atrium. Besides, we show that, irrespective of the involvement of fast Na channels in the EFS-produced release of C G R R the CTX-sensitive release evoked by BK and by EFS is similarly depressed by opioids and NPY. Therefore,
STIMULATION
3 2.5
2
.£
EI.5 ,i==
I/)
0.5
0 CONTR
TTX
CTX
MORPH
I~Y
Fig. 3. The effect of tetrodoxin (TTX, 0.3/aM), ~o-conotoxin(CTX, 0.1 /aM), morphine (MORPH, 3/aM) and neuropeptide Y (NPY, 0.3/aM) on the release of calcitonin gene-related peptide (CGRP) evoked by bradykinin ( 10/aM) from slices of guinea-pig atria. Each column represents the ratio between total evoked release of CGRP caused by the first (S1) and the second ($2) stimulation period. *P < 0.05, **P < 0.01 vs. $2/S1 ratio of controls, n = 5.
dotoxin (Sankio); o)-conotoxin fraction-GVIA (Peninsula); morphine hydrochloride, dynorphine, dermorphine (Carlo Erba). EFS produced a frequency-dependent release of CGRP-like immunoreactivity ( C G R P - L I ) from slices of guinea-pig atria: T E R at 10 Hz, 20 Hz and 30 Hz was 44 + 17, 115 + 27 and 213 + 46 fmol/g/20 min, respectively (Fig. 1). A frequency of 30 Hz was selected for further experiments. C G R P - L I outflow induced by the second application of EFS (30 Hz frequency) was higher (183 + 33 fmol/g/20 rain, P < 0.05, $2/S1 ratio 2.02 + 0.38) than that observed after the first cycle of stimulation (127 + 32 fmol/g/20 min) (Fig. 2). Exposure of guinea-pig atria to 10/aM BK produced an increase in the C G R P - L I outflow that was reproducible at the first (102 + 16 fmol/g/30 min) and the second (97 + 8 fmol/g/30 rain) exposure to the drug ($2/S1 ratio 1.14 4- 0.2) (Fig. 3). Superfusion of guinea-pig atria with Krebs solution containing 0.3/aM T T X blocked C G R P - L I release induced by EFS ($2/S1 ratio 0.06 + 0.02, P < 0.01 ), while did not significantly affect that produced by a second challenge of BK ($2/S1 ratio 0.71 + 0.18). C T X (0.1/aM) had a similar inhibitory effect on EFS ($2/S1 ratio 0.21 + 0.06, P < 0.05) and BK stimulation ($2/S1 ratio 0.15 + 0.08, P < 0.01) (Figs. 2 and 3). In the presence of morphine (3/aM), C G R P - L I release evoked both by EFS and BK was markedly reduced ($2/S1 ratio being 0.33 + 0.15 and 0.48 4- 0.09, respectively, P < 0.05). CGRP-L1 outflow, induced by either EFS or BK from slices of guinea-pig atria, was also strongly inhibited by the exposition of the preparation to N P Y (0.3 /aM) ($2/S1 ratio 0.81 + 0 . 1 4 and 0.37 + 0.15, respectively, P < 0.05) (Figs. 2 and 3).
nO Z>-
300 "2"
250 200 l--
"~
150
E 100 --I
,.t
rt0
50 0
z o (J
/
o (L Z
O.
II
Fig. 4. The effect of dynorphin {DYNOR, 1/aM), dermorphine (DERMOR, 0.1 /aM), neuropeptide Y(16 36) (NPY 16 36, 1 /aM) and peptide YY (PYY, 0.3/aM) on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) evoked by bradykinin l l0/aM) from slices of guinea-pig atria. Each column represents CGRP-LI total evoked release induced in control preparations (CONTR) and in atria exposed to various agents. *P < 0.01 xs. control preparations, n = 5.
166
E. Del Bianco et al./Neuroscience Letters 170 (1994) 163 166
since BK produces peptide release by a TTX-insensitive mechanism, it is hypothesized that NPY and opioids exert their inhibitory action on sensory neuropeptide secretion by acting on the very terminal region of the nerve fibre or, anyway, that prejunctional inhibition by these agents does not involve blockade of impulse conduction at the axonal level. Our results show that dynorphin did not reduce BKevoked release while dermorphine had a marked inhibitory effect on it. On this basis we can conclude that/2 opioid receptors are responsible for the morphine action on sensory nerve terminals and we can exclude the participation of x opioid receptor, in agreement with a previous pharmacological characterization of this response. The similar actions of NPY, PYY and NPY(16-36) on BK-evoked release support the presence of Y2 receptors on the sensory nerve terminal. [1] Amerini, S., Rubino, A., Filippi, S., Ledds, F. and Mantelli, L., Modulation by adrenergic transmitter of the efferent function of capsaicin-sensitive nerves in cardiac tissues, Neuropeptides, 20 (1991) 225-232.
[2] Geppetti, P., Frilli, S., Renzi, D., Santicioli, P., Maggi, C . A , Theodorsson, E. and Fanciullacci, M., Distribution of calcitonin gene-related peptide-like immunoreactivity in various rat tissues: correlation with substance P and other tachykinins and sensitivity to capsaicin, Regul. Pept., 23 (I988) 289-298. [3] Geppetti, P., Tramontana, M., Santicioli, P., Del Bianco, E., Giuliani, S. and Maggi, C.A., Bradykinin-induced release ofcalcitonin gene-related peptide from capsaicin-sensitive sensory nerves in guinea-pig atria: mechanism of action and calcium requirements. Neuroscience, 38 (1990) 687.692. [4] Giuliani, S., Maggi, C.A. and Meli, A., Opioid receptors and prejunctional modulation of capsaicin-sensitive sensory nerves in guinea-pig left atrium, Gen. Pharmacol., 21 (1990) 417-421. [5] Maggi, C.A. and Meli, A., The sensory-efferent function of"capsaicin-sensitive sensory neurons, Gen. Pharmacol., 19 (1988) 143. [6] Maggi, C.A., Patacchini, R., Santicioli, R, Giuliani, S., Del Bianco, E., Geppetti, P. and Meli, A., The efferent function of capsaicin sensitive nerves: ruthenium red discriminates between different mechanism of activation, Eur. J. Pharmacol., 170 (1989) 167 177. [7] Regoli, D. and Barab6, G., Pharmacology of bradykinin and related kinins, Pharmacol. Rev., 123 (1980) 61-65.
Neuroscience Letters 170 (1994) 163 166
HEUROSClENCE LETTERS
Modulation of calcitonin gene-related peptide release evoked by bradykinin and electrical field stimulation in guinea-pig atria Elena Del Bianco a'*, Carlo Alberto Maggi b, Paolo Santicioli b, Pierangelo GeppettF "Institute of Clinical Dermatology, Florence UniversiO', Via degli Alfani 37, 50121 Florence, ltah' bPharmacology Department, Research Laboratories, Menarini Pharmaceuticals, Florence. Italy 'Institute of Internal Medicine IV, University of Florence, Florence, Italy Received 16 November 1993; Revised version received 2 February 1994; Accepted 2 February 1994
Abstract Electrical field stimulation (EFS) and bradykinin (BK) are able to activate capsaicin-sensitive sensory neurons of guinea-pig atria in a (o-conotoxin (CTX)-sensitive and Ruthenium Red (RR)-insensitive manner. The aim of this work was to study EFS and BK-induced release of calcitonin gene-related peptide (CGRP) from guinea-pig atria and in particular the action of morphine and neuropeptide Y (NPY) on this release. EFS-induced CGRP release was frequency-dependent and tetrodotoxin (TTX)-sensitive, while BK-evoked CGRP release was TTX-insensitive. On the other hand, CGRP outflow induced by either EFS or BK was similarly reduced in the presence of morphine and NPY. It is therefore hypothesized that NPY and opioids exert their inhibitory action by acting on the very terminal region of the nerve fibre. Moreover, our results show that dermorphine but not dynorphin reduced BK-evoked CGRP release, suggesting that p opioid receptors are responsible for morphine action. Studying the action of peptide YY and NPY(16 36) on BK-evoked CGRP release, we demonstrated that both had similar inhibitory effects, supporting the presence of Y2 receptors on the nerve terminal region.
Key words: CGRP; Atrium; Bradykinin; Opioid; NPY; NPY(16 36); PYY
A subpopulation of primary sensory neurons is characterized by their sensitivity to capsaicin, the pungent principle of red peppers, and by their content of neuropeptides, including calcitonin gene-related peptide (CGRP) [5]. Various stimuli, including capsaicin itself, electrical field stimulation (EFS) and bradykinin (BK), are capable of exciting the peripheral endings of these neurons, thus inducing release of CGRP. Different modes of transmitter secretion have been described [6]: the secretion of C G R P due to antidromic invasion of the nerve endings by a propagated action potential (axon reflex) is blocked by tetrodotoxin (TTX), is strongly inhibited by m-conotoxin (CTX, a selective antagonist of N-type voltage-sensitive calcium channels), but is unaffected by the inorganic dye Ruthenium Red (RR). On the contrary, neuropeptide release evoked by capsaicin is unaffected by T T X and CTX but is selectively blocked by RR [6]. Recently [3], it has been found that BK acti* Corresponding author. 0304-3940•94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD1 0304-3940(94)00112-N
vates capsaicin-sensitive sensory afferents inducing release of C G R P from guinea-pig atria by a mechanism similar to that evoked by EFS, being CTX-sensitive and RR-insensitive. In contrast to EFS, BK-evoked release of C G R P is unaffected by T T X and blocked by indomethacin, the cycloxygenate inhibitor [3]. Because of this difference we addressed the question of whether C G R P release produced by EFS or BK could be subjected to differential pharmacological modulation; in particular, we tested the actions of morphine and neuropeptide Y (NPY) which have been proposed to exert a prejunctional modulatory influence on sensory fibre activation in the guinea-pig atrium [4]. In addition, this work tried to identify the receptor types that could be involved in the modulatory actions of opioids and of NPY at the sensory nerve endings level. Male albino guinea-pigs weighing 270 340 g were stunned and bled. The hearts were rapidly removed and placed in cold oxygenated Krebs solution of the following composition (in mM): NaC1 119, NaHCO~ 25,
164
E, D e l Bianco e / a l . I Neuro,~cience Letters 17(1 ' 1994 ) 163-166
u
200
-
160
--
10 H e r t z
120
o
E 80
-
40 L)
0
1
2
3
4
5
6
FRACTION5
u L
160-
c~
120
E v
80
,
40
L~ U
Hertz
20
200
-
0
I
2
3
4
5
6
5
6
FRACTIONS
g
200 16o
f2D-
-
L m
120
E v
80
Hertz
30
-q ,
40
u
0
I
2
3
4
FRACTION5
Fig. 1. Increase in calcitonin gene-related peptide-like immunoreactiv-
ity (CGRP-LI) outflow produced by electrical field stimulation in slices of guinea-pig atria at different frequencies: 10 H z ( u p p e r panel), 20 H z (medium panel), 30 Hz (lower panel). The other electric parameters remain invariate (10 V, 50 mA/cm z, for 5 min). Each column represents a 5 min fraction. The first and the second columns represent the basal values * P < 0.05, ** P < 0.01 vs. mean basal value, n = 5.
period. In experiments in which BK was used as stimulus to induce CGRP release, two fractions before a n d t b u r during exposure to BK were collected. After a further superfusion (30 min) with Krebs solution containing the v a r i o u s tested compounds, E F S o r BK were given for a second time and samples were collected tbllowing the same protocol described above. At the end of the experiments, the tissues were blotted 2-3 times on filter paper and weighed. The superfusates were freeze-dried, reconstituted with the assay buffer (0.1 M, pH 7.4 phosphate buffer containing 0.9 NaC1, 0.01% NaN 3 and 0.t% bovine serum albumin) and CGRP was measured by radioimmunoassay as reported previously [2]. Rat ~-CGRP (Peninsula) as standard, '25I-iodohistidyl-CGRP (Amersham) and RAS 6012 anti-CGRP antiserum (Peninsula) were used. The coefficient of percentage variation was less than 10% for values between 20 and 300 pmol/1. Sensitivity of the assay was 2.5 fmol/tube. The antiserum cross-reacted 100% with rat fl-CGRP and human CGRP-I and -II. Each value in the text and figures is the mean + S.E.M. of at least 5 experiments. Total evoked release (TER) was calculated by the sum of the values observed during the stimulation period from which the mean basal value was subtracted. The ratio between TER of CGRP-LI caused by the first (S1) and the second ($2) stimulation period was calculated ($2/S1 ratio). The ratio ($2/S1) observed in control preparation was compared with the ratio ($2/ S1) measured when the $2 stimulus was given in the presence of the compounds under investigation. Statistical analysis was done by means of Student's t-test or with Dunnett's test when applicable. Bradykinin, thiorphan, captopril, neuropeptide Y (NPY), peptide YY (PYY), NPY(16-36) (Sigma); tetroBRADYKININ
1.5
KH2PO 4 1.2, MgSO4 1.5, KCI 4.7, CaC12 2.5, glucose 11. Both atria were removed from the hearts and thick (500 /IM) slices were prepared using a tissue slicer (MacIlwain). Slices were pooled, transferred (150-200 mg of tissue) in a 2 ml perfusion chamber and superfused at a rate of 0.4 ml/min with oxygenated (96% 02-4% CO2) Krebs solution, at 37°C, containing 0.1% bovine serum albumin, thiorphan (10 pM) and captopril (10 pM). A 30 min equilibration period was allowed to elapse, after which 5 rain fractions were collected in tubes containing acetic acid (final concentration 2 N). In experiments in which release was induced by EFS, electrical impulses (10 V, 50 mA/cm:, 10=30 Hz, t ms pulse duration, 10 s train every 20 s) were applied for 5 min with platinum electrodes connected to a Grass stimulator via a constant current unit (Basile). Fractions were collected for 10 min before, 5 min during and 15 min after the stimulation
T I
._o
1
1 Fig. 2. The effect of tetrodotoxin (TTX, 0.3 ~tM), c0-conotoxin qCTX. 0.1 BM), morphine (MORPH. 3 ttM) and neuropeptide Y (NPY. 0.3 BM) on the release of calcitonin gene-related peptide (CGRP) evoked by electrical field stimulation (10 V. 50 mA/cm z. 30 Hz~ from slices of guinea-pig atria. Each column represents the ratio between total evoked release of CGRP caused by the first ($1) and the second ($2) stimulation period. *P < 0.05, **P < 0.01 vs. $2/S1 ratio of controls, n = 5.
165
E. Del Bianco et al./Neuroscience Letters 170 (1994) 163 166
ELECTRICAL
FIELD
In order to characterize opioid and NPY receptor modulation of C G R P - L I release, BK (10/aM) was used as stimulus to evoke C G R P - L I release from capsaicinsensitive sensory neurons. When atrium slices were exposed to dermorphine (0.1 /aM), BK-evoked C G R P - L I release was markedly reduced (TER being 43 4- 5.8 fmol/ g/30 min, P<0.01), while dynorphine (1/aM) did not have an inhibitory effect on BK-evoked C G R P - L I release (TER being 190 + 42 fmol/g/30 min) (Fig. 4). Exposure of the same preparations to NPY(16 36) (1 /aM) and to PYY (0.3/aM) reduced BK-evoked C G R P L1 outflow in a very significant manner, T E R 35 +_ 9 and 43 4- 11 fmol/gJ25 min, respectively, versus 113 4- 13 fmol/g/25 min of control preparations (P<0.01) (Fig. 4). BK exerts a powerful excitatory action on primary sensory neurons [7]. This effect accounts for the algesic action of this autacoid and for its ability to release peptide transmitters from peripheral endings of capsaicinsensitive sensory neurons. BK-induced C G R P release from slices of guinea-pig atria was insensitive to TTX, thus indicating that BK exerts its action on the very terminal region of the neuron without the involvement of propagated action potentials (axon reflexes), but was inhibited by CTX, suggesting participation of voltagesensitive calcium channels [4,5]. Giuliani et al. [4] have shown that in guinea-pig isolated left atria EFS produces a positive inotropic response that is blocked by CTX and TTX. Responses to EFS were also inhibited by opioids and NPY [1,4]. The present findings confirm the presence of a marked release of C G R P evoked by EFS in the guinea pig atrium. Besides, we show that, irrespective of the involvement of fast Na channels in the EFS-produced release of C G R R the CTX-sensitive release evoked by BK and by EFS is similarly depressed by opioids and NPY. Therefore,
STIMULATION
3 2.5
2
.£
EI.5 ,i==
I/)
0.5
0 CONTR
TTX
CTX
MORPH
I~Y
Fig. 3. The effect of tetrodoxin (TTX, 0.3/aM), ~o-conotoxin(CTX, 0.1 /aM), morphine (MORPH, 3/aM) and neuropeptide Y (NPY, 0.3/aM) on the release of calcitonin gene-related peptide (CGRP) evoked by bradykinin ( 10/aM) from slices of guinea-pig atria. Each column represents the ratio between total evoked release of CGRP caused by the first (S1) and the second ($2) stimulation period. *P < 0.05, **P < 0.01 vs. $2/S1 ratio of controls, n = 5.
dotoxin (Sankio); o)-conotoxin fraction-GVIA (Peninsula); morphine hydrochloride, dynorphine, dermorphine (Carlo Erba). EFS produced a frequency-dependent release of CGRP-like immunoreactivity ( C G R P - L I ) from slices of guinea-pig atria: T E R at 10 Hz, 20 Hz and 30 Hz was 44 + 17, 115 + 27 and 213 + 46 fmol/g/20 min, respectively (Fig. 1). A frequency of 30 Hz was selected for further experiments. C G R P - L I outflow induced by the second application of EFS (30 Hz frequency) was higher (183 + 33 fmol/g/20 rain, P < 0.05, $2/S1 ratio 2.02 + 0.38) than that observed after the first cycle of stimulation (127 + 32 fmol/g/20 min) (Fig. 2). Exposure of guinea-pig atria to 10/aM BK produced an increase in the C G R P - L I outflow that was reproducible at the first (102 + 16 fmol/g/30 min) and the second (97 + 8 fmol/g/30 rain) exposure to the drug ($2/S1 ratio 1.14 4- 0.2) (Fig. 3). Superfusion of guinea-pig atria with Krebs solution containing 0.3/aM T T X blocked C G R P - L I release induced by EFS ($2/S1 ratio 0.06 + 0.02, P < 0.01 ), while did not significantly affect that produced by a second challenge of BK ($2/S1 ratio 0.71 + 0.18). C T X (0.1/aM) had a similar inhibitory effect on EFS ($2/S1 ratio 0.21 + 0.06, P < 0.05) and BK stimulation ($2/S1 ratio 0.15 + 0.08, P < 0.01) (Figs. 2 and 3). In the presence of morphine (3/aM), C G R P - L I release evoked both by EFS and BK was markedly reduced ($2/S1 ratio being 0.33 + 0.15 and 0.48 4- 0.09, respectively, P < 0.05). CGRP-L1 outflow, induced by either EFS or BK from slices of guinea-pig atria, was also strongly inhibited by the exposition of the preparation to N P Y (0.3 /aM) ($2/S1 ratio 0.81 + 0 . 1 4 and 0.37 + 0.15, respectively, P < 0.05) (Figs. 2 and 3).
nO Z>-
300 "2"
250 200 l--
"~
150
E 100 --I
,.t
rt0
50 0
z o (J
/
o (L Z
O.
II
Fig. 4. The effect of dynorphin {DYNOR, 1/aM), dermorphine (DERMOR, 0.1 /aM), neuropeptide Y(16 36) (NPY 16 36, 1 /aM) and peptide YY (PYY, 0.3/aM) on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) evoked by bradykinin l l0/aM) from slices of guinea-pig atria. Each column represents CGRP-LI total evoked release induced in control preparations (CONTR) and in atria exposed to various agents. *P < 0.01 xs. control preparations, n = 5.
166
E. Del Bianco et al./Neuroscience Letters 170 (1994) 163 166
since BK produces peptide release by a TTX-insensitive mechanism, it is hypothesized that NPY and opioids exert their inhibitory action on sensory neuropeptide secretion by acting on the very terminal region of the nerve fibre or, anyway, that prejunctional inhibition by these agents does not involve blockade of impulse conduction at the axonal level. Our results show that dynorphin did not reduce BKevoked release while dermorphine had a marked inhibitory effect on it. On this basis we can conclude that/2 opioid receptors are responsible for the morphine action on sensory nerve terminals and we can exclude the participation of x opioid receptor, in agreement with a previous pharmacological characterization of this response. The similar actions of NPY, PYY and NPY(16-36) on BK-evoked release support the presence of Y2 receptors on the sensory nerve terminal. [1] Amerini, S., Rubino, A., Filippi, S., Ledds, F. and Mantelli, L., Modulation by adrenergic transmitter of the efferent function of capsaicin-sensitive nerves in cardiac tissues, Neuropeptides, 20 (1991) 225-232.
[2] Geppetti, P., Frilli, S., Renzi, D., Santicioli, P., Maggi, C . A , Theodorsson, E. and Fanciullacci, M., Distribution of calcitonin gene-related peptide-like immunoreactivity in various rat tissues: correlation with substance P and other tachykinins and sensitivity to capsaicin, Regul. Pept., 23 (I988) 289-298. [3] Geppetti, P., Tramontana, M., Santicioli, P., Del Bianco, E., Giuliani, S. and Maggi, C.A., Bradykinin-induced release ofcalcitonin gene-related peptide from capsaicin-sensitive sensory nerves in guinea-pig atria: mechanism of action and calcium requirements. Neuroscience, 38 (1990) 687.692. [4] Giuliani, S., Maggi, C.A. and Meli, A., Opioid receptors and prejunctional modulation of capsaicin-sensitive sensory nerves in guinea-pig left atrium, Gen. Pharmacol., 21 (1990) 417-421. [5] Maggi, C.A. and Meli, A., The sensory-efferent function of"capsaicin-sensitive sensory neurons, Gen. Pharmacol., 19 (1988) 143. [6] Maggi, C.A., Patacchini, R., Santicioli, R, Giuliani, S., Del Bianco, E., Geppetti, P. and Meli, A., The efferent function of capsaicin sensitive nerves: ruthenium red discriminates between different mechanism of activation, Eur. J. Pharmacol., 170 (1989) 167 177. [7] Regoli, D. and Barab6, G., Pharmacology of bradykinin and related kinins, Pharmacol. Rev., 123 (1980) 61-65.