0013-7227/98/$03.00/0
Endocrinology
Copyright © 1998 by The Endocrine Society
Vol. 139, No. 8
Printed in U.S.A.
Pharmacological and Functional Characterization of
Muscarinic Receptors in the Frog Pars Intermedia*
MARIANNE GARNIER†, MAREK LAMACZ, LUDOVIC GALAS†,
SEBASTIEN LENGLET, MARIE-CHRISTINE TONON, AND HUBERT VAUDRY
European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular
Neuroendocrinology, INSERM U-413, Unité Affiliée au Centre National de la Recherche Scientifique,
University of Rouen, 76821 Mont-Saint-Aignan, France
ABSTRACT
The secretion of aMSH from the intermediate lobe of the frog
pituitary is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In particular, acetylcholine (ACh),
acting via muscarinic receptors, stimulates aMSH release from frog
neurointermediate lobes (NILs) in vitro. The aim of the present study
was to characterize the type of receptor and the transduction pathways involved in the mechanism of action of ACh on frog melanotrope
cells. The nonselective muscarinic receptor agonists muscarine and
carbachol both stimulated aMSH release from perifused frog NILs,
whereas the M1-selective muscarinic agonist McN-A-343 was virtually devoid of effect. Both the M1.M3 antagonist pirenzepine and the
M3.M1 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibited muscarine-induced aMSH release. Administration of a
brief pulse of muscarine in the vicinity of cultured melanotrope cells
provoked a 4-fold increase in the cytosolic calcium concentration
([Ca21]i). Suppression of Ca21 in the culture medium or addition of 3
mM Ni21 abrogated the stimulatory effect of muscarine on [Ca21]i and
aMSH release. In contrast, v-conotoxin GVIA and nifedipine did not
I
N AMPHIBIANS, the melanotropic peptide aMSH, secreted by melanotrope cells of the pars intermedia, plays
a pivotal role in the process of skin color adaptation (1). This
camouflage aptitude allows the animals to escape to their
predators and thus is essential for the survival of endangered
amphibian species. The neuroendocrine mechanisms regulating the activity of melanotrope cells in amphibians has
been mainly studied in two representative species, the African clawed toad Xenopus laevis (2, 3) and the European
green frog Rana ridibunda (4, 5). Using these animal models,
it has been demonstrated that aMSH secretion is controlled
by multiple factors, including classical neurotransmitters
and neuropeptides. In the frog Rana ridibunda, the activity of
melanotrope cells is inhibited by dopamine (6), g-aminobutyric acid (7, 8), serotonin (9), adenosine (10, 11), a-adrenergic
Received February 6, 1998.
Address all correspondence and requests for reprints to: Dr. H.
Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM
U-413, Unité Affiliée au Centre National de la Recherche Scientifique,
University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail:
[email protected].
* This work was supported by grants from INSERM U-413, the
European Union (Human Capital and Mobility Program, Grant
ERBCHRXCT920017), and the Conseil Régional de Haute-Normandie.
† Recipient of a fellowship from the Ministère de l’Education
Nationale, de l’Enseignement Supérieur, et de la Recherche.
significantly reduce the stimulatory effect of muscarine on [Ca21]i and
aMSH secretion. Exposure of NILs to muscarine provoked an increase
in inositol phosphate formation, and this effect was dependent on
extracellular Ca21. The inhibitor of polyphosphoinositide turnover
neomycin significantly attenuated the muscarine-evoked aMSH release. Similarly, pretreatment of frog NILs with phorbol ester markedly reduced the secretory response to muscarine. In contrast, the
stimulatory effect of muscarine on aMSH release was not affected by
the phospholipase A2 inhibitor dimethyl eicosadienoic acid or by the
tyrosine kinase inhibitors lavendustin A, genistein, and tyrphostin
25. Muscarine at a high concentration (1024 M) only produced a 40%
increase in cAMP formation. Preincubation of frog NILs with pertussis toxin did not significantly affect the muscarine-induced stimulation of aMSH release. These results indicate that frog melanotrope
cells express a muscarinic receptor subtype pharmacologically related
to the mammalian M3 receptor. Activation of this receptor causes
calcium influx through Ni21-sensitive Ca21 channels and subsequent
activation of the phopholipase C/protein kinase C transduction pathway. (Endocrinology 139: 3525–3533, 1998)
agonists (12), and neuropeptide Y (13, 14) and is stimulated
by b-adrenergic agonists (12) and TRH (5, 15–17).
The intermediate lobe of the mammalian pituitary contains both melanotrope cells and corticotrope cells (18). The
pars intermedia of amphibians, which is composed of a homogeneous population of melanotrope cells (19), represents
a very appropriate model in which to investigate the transduction pathways involved in the mechanism of action of the
neuroendocrine messengers regulating aMSH secretion.
Acetylcholine (ACh) is recognized as an important modulator of the activity of various types of pituitary cells
(20 –24). In particular, ACh stimulates the activity of melanotrope cells in mammals (25, 26) and amphibians (27–29).
In both toads and frogs, the effect of ACh on aMSH secretion
is mediated by muscarinic receptors (26, 28). Local synthesis
of ACh has been demonstrated in the porcine (30) and toad
(29) pars intermedia, indicating that ACh may exert an autocrine control on the activity of melanotrope cells.
The aim of the present study was to characterize the type
of muscarinic receptor and the transduction mechanisms
mediating the action of ACh in melanotrope cells of the frog
pituitary.
Materials and Methods
Animals
Adult male frogs (Rana ridibunda) of about 30 g body weight were
purchased from a commercial supplier (Couétard, St. Hilaire de Riez,
3525
3526
MUSCARINIC CONTROL OF aMSH SECRETION IN FROG
France). The animals were maintained under artificial illumination
(lights on from 0600 –1800 h) in a temperature-controlled room
(8 6 0.5 C). The animals were killed by decapitation, and the neurointermediate lobes (NILs) were immediately dissected under a microscope. All animal manipulations were performed according to the recommendations of the French ethical committee and under the
supervision of authorized investigators.
Reagents
Carbachol, muscarine, pirenzepine, isoproterenol, genistein, forskolin, isobutylmethylxanthine, pertussis toxin (PTX), nifedipine, v-conotoxin GVIA (v-CgTx), 7,79-dimethyl eicosadienoic acid, EGTA, HEPES,
Leibovitz culture medium (L15), collagenase type IA, and phorbol 12myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO).
4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and
McN-A-343 were supplied by Research Biochemicals International
(Natick, MA). Indo-1 acetoxymethylester was obtained from Molecular
Probes (Eugene, OR). Bio-Gel P-2 and the anion exchange resin AG1-X8
(100 –200 mesh; formate form) were obtained from Bio-Rad Laboratories
(Hercules, CA). BSA (fraction V) was purchased from Boehringer Mannheim (Paris, France). Myo-[3H]inositol was obtained from Amersham
(Aylesbury, UK). Tyrphostin 25 (Tyr-A25, AG 82) was obtained from
Calbiochem (San Diego, CA). Lavendustin A was obtained from ICN
Pharmaceuticals (Costa Mesa, CA). Kanamycin was purchased from Life
Technologies (Grand Island, NY). The antibiotic-antimycotic solution
and FBS were obtained from BioWhittaker (Gagny, France). Other chemicals were purchased from Sigma.
Perifusion technique
The perifusion system used in this study has been previously described in detail (15). Briefly, NILs were incubated for 15 min in a
Ringer’s solution consisting of 15 mm HEPES, 112 mm NaCl, 2 mm KCl,
and 2 mm CaCl2 supplemented with 2 g/liter glucose and 0.3 g/liter
BSA. The solution was gassed with O2-CO2, (95:5, vol/vol) before use,
and the pH was adjusted to 7.35. Then NILs were layered between two
beds of Bio-Gel P2 into a plastic column (id, 0.9 cm) delimited by two
Teflon pestles (four NILs per perifusion chamber). The tissues were
perifused with the Ringer’s solution at a constant flow rate (0.25 ml/min)
and temperature (24 C). The effluent perifusate was collected as 7.5-min
fractions during stabilization periods and 2.5-min fractions during infusion of the secretagogues. The fractions were immediately chilled at
4 C, and the concentration of aMSH was measured in each fraction on
the same day as the perifusion experiment using a double antibody RIA
procedure (31). The perifusion profiles are expressed as percentages of
the basal secretory level, calculated as the mean of four consecutive
fractions collected just before the infusion of the secretagogue.
Cell culture
NILs were collected in Ca21-free Ringer’s solution (15 mm HEPES,
112 mm NaCl, 2 mm KCl, 1 mm EGTA, 2 g/liter glucose, and 0.3 g/liter
BSA). The tissues were enzymatically dispersed at 22 C for 20 min with
collagenase (1.5 mg/ml) in the same solution. Nondissociated neural
lobes were allowed to settle, and the supernatant containing dissociated
pars intermedia tissue was sampled and centrifuged (30 3 g, 5 min).
After three rinses with Ca21-free Ringer’s solution, the cells were dispersed by gentle aspiration through a siliconized Pasteur pipette with
a flame-polished tip. The cells were resuspended in L15 culture medium
adjusted to frog osmolality (L15/water 5 1:0.4) supplemented with 0.2
g/liter glucose, 63 mg/liter CaCl2, and 1% of the kanamycin and antibiotic-antimycotic solutions (fL15). Finally, cells were plated on 35-mm
glass coverslips previously coated with poly-l-lysine (10 mg/ml) at a
density of 15,000 cells/coverslip. When the cells had settled, coverslips
were covered with 2 ml fL15 medium supplemented with 10% FBS. Cells
were cultured in a humid atmosphere incubator at 24 C, and the culture
medium was renewed every 48 h. Microfluorometric measurements
were performed on 3- to 5-day-old cultured cells.
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Intracellular calcium measurements
Cultured melanotrope cells were incubated at room temperature with
5 mm indo-1/acetoxymethylester in fL15 medium for 30 min and washed
twice with Ringer’s solution. The cytosolic calcium concentration
([Ca21]i) was monitored by a dual emission microfluorometric system
constructed from a Nikon Diaphot inverted microscope equipped for
epifluorescence with an oil immersion objective (3100; CF Fluor series;
numerical aperture, 1.3) as previously described (32). The fluorescence
emission of indo-1, induced by excitation at 355 nm, was recorded at two
wavelengths (405 nm, corresponding to the complexed form, and 480
nm, corresponding to the free form) by separate photometers (P1; Nikon,
Melville, NY). The 405/480 ratio (R) was determined using an AS1-type
acquisition card (Notocord Systems, Croissy-sur-Seine, France). All
three signals (405 nm, 480 nm, and R) were continuously recorded with
the JAD-FLUO program (version 1.2; Notocord Systems, Croissy-surSeine, France). [Ca21]i was calculated according to the formula established by Grynkiewicz et al. (33): [Ca21]i 5 Kd 3 b (R 2 Rmin)/(Rmax 2
R), where Rmin represents the minimum fluorescence ratio obtained after
incubation of cells in Ringer’s solution containing 10 mm EGTA and 10
mm ionomycin, Rmax is the maximum fluorescence ratio obtained after
incubation of cells in Ringer’s solution containing 10 mm CaCl2 and 10
mm ionomycin, and b is the ratio of fluorescence yield from the Ca21min/
Ca21max indicator at 480 nm. The values obtained for Rmin, Rmax, and b
were 0.16, 1.82, and 1.62, respectively. The dissociation constant for
indo-1 (Kd 5 250 nm) has been previously determined (34).
Inositol phospholipid turnover
Measurement of membrane phospholipid metabolites was performed as previously described (16). Whole NILs were incubated in fL15
medium with myo-[3H]inositol (100 mCi/ml) for 18 h. The pulse medium
was then discarded, and the NILs were washed six times with Ringer’s
solution supplemented with 1 mm inositol. The NILs were preincubated
for 10 min with 10 mm LiCl and exposed for various durations to
muscarine in the presence of 10 mm LiCl. The reaction was stopped by
addition of ice-cold 20% trichloroacetic acid. The NILs were homogenized, and the membrane fraction was removed by centrifugation. Inositol phosphates contained in the supernatant were analyzed by anion
exchange chromatography on AG1-X8 resin, as previously described
(16). Free [3H]inositol was eluted by water, whereas inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3)
were sequentially eluted by a step gradient of ammonium formate (0.2,
0.45, and 0.8 m, respectively) in 0.1 m formic acid. For each sample, 38
fractions (4 ml each) were collected, and the radioactivity was determined in a 1217 Rackbeta counter (Wallac, Eury, France).
cAMP measurement
Whole NILs were preincubated for 30 min at room temperature with
0.1 mm isobuthylmethylxanthine. The NILs were then incubated for 20
min with muscarine, isoproterenol, or forskolin. The reaction was
stopped by addition of ice-cold 20% trichloroacetic acid. NILs were
homogenized and centrifuged (10,000 3 g for 10 min). Trichloroacetic
acid was eliminated from the supernatant by three successive rinses with
1 ml water-saturated diethyl ether. After evaporation of the ether phase,
the supernatant was lyophilized, and the cAMP content in the dried
extract was measured by RIA, following the procedure recommended
in the cAMP RIA kit (Amersham).
Statistical analysis
Values are expressed as the mean 6 sem. Statistical comparisons
between groups were made using ANOVA, followed by Student’s t test.
Differences were taken to be statistically significant at P , 0.05.
Results
Pharmacological characterization of muscarinic receptors
Administration of graded concentrations of muscarine to
perifused frog NILs induced a dose-related stimulation of
aMSH release (Fig. 1, A and B). The minimum effective
MUSCARINIC CONTROL OF aMSH SECRETION IN FROG
FIG. 1. Effect of muscarinic receptor agonists on aMSH secretion
from perifused frog NILs. A, Perifusion profiles showing effect of
graded concentrations of muscarine (Musc; 10 min) on aMSH release.
Profiles represent the mean (6SEM) secretion pattern of four to seven
independent perifusion experiments. The mean basal level of aMSH
release (100% basal level) was calculated as the mean aMSH concentration in the four consecutive fractions (30 min; E) collected
before administration of the muscarinic agonist. B, Semilogarithmic
plot showing the effects of increasing concentrations of the nonselective muscarinic receptor agonists muscarine (Musc) and carbachol
(Carb) and the M1-selective agonist McN-A-343 (McN) on aMSH release from perifused frog NILs. All experimental values were calculated from data similar to those presented in A. Each point represents
the mean (6SEM) of three to seven experiments. The mean basal level
of aMSH release in these experiments was 77 6 11 pg/minzNIL.
concentration was 3 3 1026 m. At a concentration of 1024 m,
muscarine caused a 3-fold increase in aMSH secretion (Fig.
1A). The dose-response curves obtained with various muscarinic agonists are compared in Fig. 1B. The nonselective
muscarinic receptor agonists muscarine and carbachol both
stimulated aMSH release in a concentration-dependent manner, and the ED50 values were, respectively, 1.2 3 1025 and
3.2 3 1026 m. In contrast, the M1-selective muscarinic agonist
McN-A-343 was virtually devoid of effect on aMSH release
from perifused frog NILs (Fig. 1B).
Administration of the muscarinic receptor antagonist
4-DAMP (1026 m) totally abolished the stimulatory effect of
muscarine (1024 m) on aMSH release (Fig. 2A). A series of
experiments similar to those presented in Fig. 2A was conducted with different concentrations of muscarinic antagonists. Both the M1.M3 antagonist pirenzepine and the
M3.M1 antagonist 4-DAMP inhibited the muscarine-induced aMSH release in a dose-dependent manner (Fig. 2B).
The respective ED50 were 1.1 3 1027 and 2.1 3 1028 m.
3527
FIG. 2. Effect of muscarine in the absence or presence of muscarinic
receptor antagonists on aMSH release from perifused frog NILs. A,
Perifusion profiles showing effect of muscarine (Musc; 1024 M; 10 min)
on aMSH release in control conditions (left panel) or during prolonged
administration of the M3.M1 antagonist 4-DAMP (1026 M; right panel). The antagonist was administered 45 min before the pulse of
muscarine. Profiles represent the mean (6SEM) secretion pattern of
four independent perifusion experiments. See Fig. 1 for other designations. B, Semilogarithmic plot showing the effects of increasing
concentrations of the M1.M3 antagonist pirenzepine (Pir) and the
M3.M1 antagonist 4-DAMP on aMSH release induced by muscarine
(1024 M). All experimental values were calculated from data similar
to those presented in A. Each point represents the mean (6SEM) of
three or four independent experiments. Results are expressed as a
percentage of the response induced by muscarine in the absence of
antagonist. The mean basal level of aMSH release in these experiments was 200 6 19 pg/minzNIL. **, P , 0.01; ***, P , 0.001.
Effect of muscarine on Ca21 mobilization
Under resting conditions, the mean [Ca21]i in cultured frog
melanotrope cells was 33 6 4 nm (n 5 26). Ejection of muscarine (1024 m; 10 sec) in the vicinity of the cells elicited a
rapid and significant (P , 0.001) increase in [Ca21]i to 120 6
11 nm (Fig. 3, trace A). Thereafter, [Ca21]i returned gradually
to the basal level within 1.5 min. When the cells were incubated in calcium-free medium supplemented with 3 mm
EGTA, the effect of muscarine on [Ca21]i was totally abolished (Fig. 3, trace B).
In the presence of 2 mm Ca21, the mean rate of secretion
of aMSH from perifused frog NILs was 69 6 10 pg/minzNIL.
Suppression of calcium in the perifusion medium markedly
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FIG. 3. Effect of muscarine (1024 M; 10 sec) on [Ca21]i in cultured frog
melanotrope cells. A, In the presence of 2 mM Ca21, muscarine induced an immediate increase in [Ca21]i. The profile represents the
mean (6SEM) response of 26 cells. B, In calcium-free medium containing 3 mM EGTA, muscarine did not modify [Ca21]i. The profile
represents the mean (6SEM) response of 17 cells. The arrow indicates
the onset of muscarine administration.
reduced the basal rate of aMSH release and completely abolished the stimulatory effect of muscarine on aMSH secretion
(Fig. 4A). Similarly, addition of NiCl2 (3 mm) to a Ringer’s
buffer containing 2 mm Ca21 inhibited both the spontaneous
and the muscarine-evoked aMSH secretion (Fig. 4B).
Preincubation of the cells for 20 – 60 min with the N-type
calcium channel blocker v-CgTx (1026 m) did not significantly modify the [Ca21]i rise evoked by 1024 m muscarine
(Fig. 5A). Likewise, addition of the L-type calcium channel
blocker nifedipine (1025 m) to the incubation medium did
not significantly affect the [Ca21]i response to muscarine
(Fig. 5B).
Prolonged exposure of perifused frog NILs to v-CgTx
(1026 m) did not impair the stimulatory effect of muscarine
(1024 m) on aMSH secretion (Fig. 6, A and B). Similarly,
perifusion of NILs in the presence of nifedipine (up to 1024
m) did not significantly reduce the muscarine-evoked aMSH
release (Fig. 6, A, C, and D).
Effect of muscarine on inositol phospholipid turnover
The effect of muscarine (1024 m) on phosphoinositide (PI)
hydrolysis was investigated by measuring inositol phosphate levels after incorporation of myo-[3H]inositol by frog
NILs. Exposure of NILs to 1024 m muscarine provoked a
modest, but significant, increase in IP3 formation. The enhancement of IP3 production occurred within the first minute
of administration of muscarine and reached a maximum at
5 min. Thereafter, the IP3 level gradually declined and returned to basal values 15 min after the onset of muscarine
administration (Fig. 7). Muscarine also caused a marked increase in IP2 levels; the maximum effect was observed after
10 min of incubation with muscarine. A significant increase
in IP1 was also observed after 5 min of incubation with
muscarine.
Incubation of NILs with HEPES buffer containing 6 mm
EGTA for 20 min did not affect the basal production of total
inositol phosphates, but totally abolished the stimulatory
effect of muscarine (1024 m; 20 min) on inositol phosphate
formation (Fig. 8).
FIG. 4. Effect of calcium suppression (Ca21, 0; EGTA, 3 mM; A) or
perifusion with NiCl2 (Ca21, 2 mM; Ni21, 3 mM; B) on basal and
muscarine-induced aMSH release. The pulses of muscarine (Musc;
1024 M; 10 min) were administered 45 min after the onset of perifusion
with Ca21-free or Ni21-containing medium. Profiles represent the
mean (6SEM) secretion pattern of 4 (A) and 5 (B) independent perifusion experiments. The mean basal level of aMSH release in these
experiments was 69 6 10 pg/minzNIL. See Fig. 1 for other designations.
Prolonged administration of the PI turnover blocker neomycin (3 mm; 110 min) to perifused frog NILs significantly
(P , 0.01) inhibited the stimulatory effect of muscarine (1024
m; 10 min) on aMSH release (Fig. 9). In addition, pretreatment of frog NILs with PMA (1026 m; 24 h) markedly reduced
the effect of muscarine on aMSH secretion (Fig. 10). In contrast, the phospholipase A2 (PLA2) inhibitor 7,79-dimethyl
eicosadienoic acid did not affect muscarine-induced aMSH
release (data not shown).
Effect of muscarine on cAMP formation
Incubation of frog NILs with muscarine (1024 m) produced
an elevation of the cAMP content in the tissue (140%;
P , 0.01; Fig. 11). However, this effect was modest compared
with those of the b-adrenergic agonist isoproterenol (1025 m)
and forskolin [5 3 1025 m; 185% (P , 0.001) and 1260%
(P , 0.001), respectively].
MUSCARINIC CONTROL OF aMSH SECRETION IN FROG
FIG. 5. Effect of muscarine (1024 M; 10 sec) on [Ca21]i in cultured frog
melanotrope cells in the presence of calcium channel blockers. A,
Effect of muscarine on [Ca21]i in the presence of the L-type calcium
channel blocker, nifedipine (1025 M). B, Effect of muscarine on [Ca21]i
in the presence of the N-type calcium channel blocker, v-conotoxin
GVIA (1026 M). Cells were incubated with nifedipine or v-conotoxin
GVIA for 20 – 60 min before the administration of the pulse of muscarine. Profiles represent the mean (6SEM) responses of 14 (A) and 15
cells (B). The arrow indicates the onset of muscarine administration.
Effect of PTX on muscarine-induced aMSH secretion
To test the possible involvement of a PTX-sensitive G
protein in the mechanism of action of muscarine, frog NILs
were incubated with PTX (1 mg/ml) for 18 h. Pretreatment
of the tissue with PTX did not inhibit the stimulatory effect
of muscarine (1024 m; 10 min) on aMSH release (Fig. 12).
Effect of tyrosine kinase inhibitors on muscarine-induced
aMSH secretion
The effect of a series of tyrosine kinase inhibitors on the
secretory response of frog NILs to muscarine was investigated (Fig. 13). Lavendustin A (1027 m; Fig. 13, A and B),
genistein (1025 m; Fig. 13, A and C), and Tyr-A25 (1024 m; Fig.
13, A and D) did not inhibit muscarine-induced aMSH
release.
3529
FIG. 6. Effect of muscarine in the absence or presence of calcium
channel blockers on aMSH release from perifused frog NILs. A single
pulse of muscarine (Musc; 1024 M; 10 min) was administered under
control conditions (A) or during prolonged infusion of 1 mM v-CgTx (B),
10 mM nifedipine (C), or 100 mM nifedipine (D). Profiles represent the
mean (6SEM) secretion pattern of three to six independent perifusion
experiments. The mean basal level of aMSH release in these experiments was 126 6 22 pg/minzNIL. See Fig. 1 for other designations.
Discussion
Pharmacological profile of muscarinic receptors in frog
melanotrope cells
Previous studies have shown that ACh stimulates the electrical (28) and secretory (27) activities of frog melanotrope
cells through activation of muscarinic receptors. As the effect
of ACh was blocked by the M1-preferring muscarinic receptor antagonist pirenzepine, but not by the selective M2 receptor antagonist gallamine, it was suggested that the action
of ACh was mediated through an M1-like muscarinic
receptor.
However, five subtypes of muscarinic receptors have now
been identified (35), allowing a more rigorous pharmacological characterization of the receptor subtypes. In particular, it was found that pirenzepine does not clearly discriminate between the M1 and M3 receptor subtypes (36).
The present study has shown that the nonspecific muscarinic agonists muscarine and carbachol both induced a
dose-dependent stimulation of aMSH release, whereas the
selective M1-receptor agonist McN-A-343 did not affect the
secretory activity of frog NILs, indicating that M1 receptors
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FIG. 8. Effect of EGTA on muscarine-induced inositol phosphate (IPx)
formation. Myo-[3H]inositol-labeled NILs were preincubated for 20
min in HEPES buffer in the absence or presence of 6 mM EGTA. NILs
were then incubated for 20 min in the same medium in the absence
or presence of muscarine (1024 M). Results are expressed as a percentage of the IPx level in the absence of EGTA and muscarine. Data
are the mean (6SEM) values from five independent experiments.
*, P , 0.05; **, P , 0.01.
FIG. 7. Effects of muscarine on IP3, IP2, and IP1 formation in myo[3H]inositol-prelabeled frog NILs. After a 10-min preincubation with
10 mM LiCl, NILs were incubated in the presence of 1024 M muscarine
for the times indicated. Results are expressed as a percentage of the
muscarine-induced inositol phosphate (IPx) level in the control. Each
value represents the mean (6SEM) of 5–14 independent experiments.
Mean inositol phosphate levels in controls were 292 6 26, 1,160 6 104,
and 16,332 6 1,490 cpm/NIL, respectively. *, P , 0.05; **, P , 0.01;
***, P , 0.001.
are not involved in the stimulatory effect of ACh on aMSH
secretion. The fact that the M3.M1 antagonist 4-DAMP was
5 times more potent than the M1.M3 antagonist pirenzepine
in inhibiting the muscarine-evoked stimulation of aMSH
secretion suggests that the effect of ACh on frog NIL is
mediated by M3, rather than M1, receptors. Consistent with
this observation, it has been reported that M3 receptors are
expressed in various types of endocrine cells (37–39).
Transduction mechanisms associated with activation of
muscarinic receptors in frog melanotrope cells
Stimulation of M1, M3, and M5 muscarinic receptors can
activate a number of signaling pathways (see Ref. 40 for
review). Although these receptor subtypes are generally associated with activation of polyphosphoinositide turnover,
the involvement of PLA2 and phospholipase D, adenylyl
cyclase, and tyrosine kinases has also been described. In the
present study, we have investigated the transduction mech-
FIG. 9. Effect of muscarine in the absence or presence of the phosphatidylinositol turnover blocker neomycin on aMSH release from
perifused frog NILs. A single pulse of muscarine (Musc; 1024 M; 10
min) was administered in control conditions (left panel) or during
prolonged infusion of neomycin (3 mM; 110 min; right panel). Profiles
represent the mean (6SEM) secretion pattern of six independent perifusion experiments. The mean basal level of aMSH release in these
experiments was 119 6 7 pg/minzNIL. See Fig. 1 for other designations.
anisms involved in the muscarine-induced stimulation of
aMSH release in frog NILs.
Administration of muscarine to cultured frog melanotrope
cells provoked a rapid, monophasic elevation of [Ca21]i.
Muscarinic agonists usually induce an immediate transient
[Ca21]i rise due to mobilization of intracellular Ca21 stores,
followed by a sustained plateau phase resulting from Ca21
influx (36). When frog melanotrope cells were incubated
under Ca21-free conditions, the [Ca21]i increase evoked by
muscarine was totally abolished. Similarly, the stimulatory
effect of muscarine on aMSH release was abrogated when
frog NILs were perifused with Ca21-free or Ni21-supplemented medium. These results indicate that the response of
melanotrope cells to muscarine requires calcium influx.
However, neither the L-type Ca21 channel blocker nifedipine
MUSCARINIC CONTROL OF aMSH SECRETION IN FROG
FIG. 10. Effect of long term treatment with PMA on muscarineinduced aMSH release from perifused frog NILs. Intact NILs were
incubated for 24 h in fL15 alone (left panel) or in fL15 supplemented
with PMA (1026 M; right panel). Then, a single pulse of muscarine
(Musc; 1024 M) was administered for 10 min. Profiles represent the
mean (6SEM) secretion pattern of four independent perifusion experiments. The mean basal level of aMSH release in these experiments
was 98 6 12 pg/minzNIL. See Fig. 1 for other designations.
FIG. 11. Effects of muscarine, isoproterenol, and forskolin on cAMP
content in frog NILs. Intact NILs were incubated in HEPES buffer
alone (C, control) or supplemented with 100 mM muscarine (Musc), 10
mM isoproterenol (Iso), or 50 mM forskolin (FK). Data are the mean
(6SEM) values from 5–14 independent experiments. **, P , 0.01; ***,
P , 0.001 (vs. control).
nor the N-type Ca21 channel blocker v-CgTx could prevent
the elevation of [Ca21]i provoked by muscarine. Likewise,
the stimulatory effect of muscarine on aMSH release was not
significantly affected by addition of nifedipine or v-CgTx in
the perifusion medium. Administration of a high concentration of nifedipine (100 mm), which was supposed to block low
voltage activated Ca21 channels (T channels) (41), did not
inhibit the stimulatory effect of muscarine on aMSH release
either. These data suggest that the Ca21 influx involved in the
secretory response to muscarine may be accounted for by
activation of P- or Q-type channels. Consistent with this
hypothesis, the occurrence of P- and Q-type Ca21 channels
has been recently described in rat melanotrope cells (42).
Alternatively, muscarinic receptors might activate receptoroperated Ca21 channels (43). Electrophysiological studies are
clearly required to investigate the type of Ca21 channels
implicated in the muscarine-induced Ca21 entry.
The present data have shown that muscarine induces a
significant increase in inositol phosphate formation in frog
NILs. In addition, neomycin, a drug known to block PI turnover by directly binding to PIP2 and PIP (44), inhibited the
3531
FIG. 12. Effect of PTX pretreatment on muscarine-induced aMSH
release from perifused frog NILs. Intact NILs were incubated for 18 h
in fL15 alone (left panel) or in fL15 supplemented with PTX (1 mg/ml;
right panel). Then, a single pulse of muscarine (Musc; 1024 M) was
administered for 10 min. Profiles represent the mean (6SEM) secretion pattern of four independent perifusion experiments. The mean
basal level of aMSH release in these experiments was 90 6 6
pg/minzNIL. See Fig. 1 for other designations.
muscarine-evoked stimulation of aMSH release. In contrast,
pretreatment of NILs with PTX did not affect the secretory
response of the tissue to muscarine. These data indicate that
the stimulatory effect of muscarine on frog melanotrope cells
can be ascribed to activation of a phospholipase C (PLC)
through a PTX-insensitive G protein. In agreement with these
findings, it has been shown that M2 and M4 muscarinic receptors are coupled to PTX-sensitive G proteins, whereas M1,
M3, and M5 muscarinic receptors are generally coupled to
PTX-insensitive G proteins (36). Although the implication of
a PLA2 in the mechanism of action of ACh has been described
in various models (45), including frog adrenocortical cells
(46), our data have shown that PLA2 is not involved in the
muscarine-evoked stimulation of aMSH release in the frog
pars intermedia. Finally, the fact that suppression of extracellular calcium totally abolished the stimulatory effect of
muscarine on inositol lipid turnover indicated that the calcium influx provoked by muscarine was necessary for activation of PLC. In agreement with this idea, it has been reported that PLC activity is regulated by physiologically
relevant Ca21 concentrations (47– 49).
Administration of a short pulse of PMA to perifused frog
NILs induced a significant stimulation of aMSH release (data
not shown), indicating the importance of PKC in the regulation of aMSH secretion. In contrast, prolonged exposure of
NILs to PMA, which is known to induce down-regulation of
PKC (50), markedly reduced the effect of muscarine on
aMSH release. These data confirmed that the stimulatory
action of ACh on frog melanotrope cells is mediated through
the PLC/PKC transduction pathway.
It has been previously shown that M2 and M4 muscarinic
receptors are negatively coupled to adenylate cyclase,
whereas activation of odd-numbered receptors is associated
3532
MUSCARINIC CONTROL OF aMSH SECRETION IN FROG
Endo • 1998
Vol 139 • No 8
FIG. 14. Schematic representation summarizing the intracellular
events associated with cholinergic stimulation of frog melanotrope
cells. Binding of ACh to a muscarinic receptor pharmacologically
related to the mammalian M3 receptor induces Ca21 entry via Ni21sensitive calcium channels. ACh also causes activation of PLC
through a PTX-insensitive G protein, generating diacylglycerol (DAG)
and IP3. Concurrently, ACh provokes a modest stimulation of adenylyl cyclase (AC). The increase in [Ca21]i resulting from Ca21 influx is
required for the ACh-induced stimulation of PLC and might be responsible for the activation of adenylyl cyclase (dotted line). The
increase in [Ca21]i and the activation of PKC are both involved in the
ACh-induced stimulation of aMSH release.
FIG. 13. Effect of tyrosine kinase inhibitors on muscarine-induced
aMSH release from perifused frog NILs. A single pulse of muscarine
(Musc; 1024 M; 10 min) was administered under control conditions (A)
or during prolonged infusion of 0.1 mM lavendustin A (B), 10 mM
genistein (C), or 100 mM tyrphostin 25 (C). Profiles represent the mean
(6SEM) secretion pattern of three to six independent perifusion experiments. The mean basal level of aMSH in these experiments was
104 6 14 pg/minzNIL. See Fig. 1 for other designations.
cyclase. In addition to Ca21 mobilization, activation of PKC
is implicated in the secretory response of frog melanotrope
cells to ACh. In contrast, PLA2 and tyrosine kinases do not
appear to play any significant role in the cholinergic stimulation of aMSH release.
Acknowledgment
The authors thank Miss C. Buquet for technical support during cell
culture.
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