Atria1 natriuretic polypeptide secretion via selective activa .tion of K-opioid receptor: role of dynorphin KENICHI
YAMADA,
SHO YOSHIDA,
AND
YUTAKA
SHIMADA
Division of Clinical Research and Internal Medicine, Sakura National Hospital, Sakura, Chiba 285; and Second Department of Internal Medicine, Department of Anatomy, School of Medic line, Chiba University, Chiba 280, Japan
YAMADA,
KENICHI,
SHO
YOSHIDA,
AND
YUTAKA
SHIMADA.
Atria1 natriuretic polypeptide secretion via selective activation of K-opioid receptor: role of dynorphin. Am. J. Physiol. 261 (Endocrinol. Metab. 24): E293-E297, 1991.-The present study was designed to investigate the direct effect of dynorphin on atria1 natriuretic polypeptide (ANP) secretion in cultured rat atria1 cardiocytes via a K-opioid receptor activation as well as the involvement of adenosine 3’,5’-cyclic monophosphate (CAMP) system in the secretion of ANP from cardiocytes. Dynorphin stimulated ANP secretion dose and time dependently from 2-day cultured atria1 cardiocytes. The dynorphininduced ANP secretion was partially antagonized by MR2266, a selective K-opioid receptor antagonist. U-62066E, a selective K-opioid receptor agonist, stimulated ANP secretion. This stimulation was also antagonized by MR2266. However, no stimulation of ANP secretion was seen with [D-Ala*,D-Leu5]enkephalin, methionine (Met)-enkephalin, or [ D-Ala*,N-MePhe4,Gly”-ollenkephalin. Dynorphin at 10m6 M significantly decreased the production of CAMP in the cultured cardiocytes. However, lo-’ M Met-enkephalin had no effect on CAMP at all. The decrease in CAMP production by the addition of dynorphin was partially antagonized with a simultaneous addition of MR2266. The dynorphin-induced ANP secretion, as well as the basal secretion, were significantly decreased by the addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, as compared with the respective controls. Dibutyryl CAMP at lo-” M significantly decreased the basal secretion of ANP as compared with the control. Therefore, the present studies show that dynorphin selectively stimulates ANP secretion, at least in part, via the activation of a specific K-opioid receptor. selectivity;
adenosine
3’,5’-cyclic
monophosphate
KNOWN that opiates, in general, modulate the secretion of neurotransmitters or neuropeptides (5). Morphine or p-opioid agonists are known to block cholinergic transmission by pre- and postsynaptic mechanisms in many preparations, including autonomic ganglia (3). K-Opioid receptor agonists cause diuresis mainly through the decrease in plasma arginine vasopressin (AVP) levels (30), and K-opioid receptor activation actually reduces the increase in AVP secretion evoked by stimuli on the final common pathway in the process of AVP secretion (31). Thus it could be proposed that Kopioid receptors might be involved in the modulation of the secretion of neurohomones such as AVP. Mammalian atria1 cardiocytes secrete a potent natriIT IS WELL
0193-1849/91
$1.50 Copyright
uretic and vasoactive polypeptide hormone termed atria1 natriuretic peptide (ANP) (6, 7, 15). It has been shown that the secretion of ANP into the circulation is stimulated by cardiodynamic changes such as atria1 distension (ZO), volume expansion (19), and the administration of pressor doses of AVP, angiotensin II, and phenylephrine (21). It has recently been reported that ANP is not only a systemic circulating hormone but possesses the function of a neuropeptide hormone as well (27). The in vitro secretion of ANP after the stimulation by neurotransmitters, including a-adrenergic and cholinergic agonists, has been demonstrated (22). Dynorphin, which is known as an endogenous K-opioid peptide (4), has been reported to exist in the rat heart as a postulated neurotransmitter or neuromodulator at a relatively high concentration (9, 26). In fact, opioid administration in vivo produces a large increase in plasma ANP levels (28). Therefore, dynorphin might act directly on K-opioid receptors in the heart for the local regulation of ANP secretion. Peptide receptor-associated effects on neurotransmission, in general, have been thought to be modulated by second messengers such as cyclic nucleotides (8). In the brain, opiates have been proposed to act by triggering receptor-mediated inhibition of adenylate cyclase activity, thereby decreasing the formation of adenosine 3’,5’cyclic monophosphate (CAMP) (29). Pharmacological studies have shown that the spinal antinociceptive effect of opiate in the tail-flick test is prevented by the simultaneous injection of aminophylline, a CAMP phosphodiesterase inhibitor (14). Furthermore, K-opioid receptors were reported to be selectively and negatively coupled to adenylate cyclase in the rat spinal cord (1). Therefore, the present study was carried out to determine the direct effect of dynorphin on ANP secretion in cultured rat atria1 cardiocytes via K-opioid receptor activation and to determine the involvement of the CAMP system in the secretion of ANP from cardiocytes. METHODS
Cell culture. Neonatal atria1 cardiocytes were prepared by a modification of the technique originally described by Harary and Farley (12). Atria removed from neonatal rats (2-4 days old) were rinsed and minced well in the preparation medium [(in g/l) 6.8 NaCl, 0.14 NaH2P04. 2Hz0, 0.2 CaClzo 2H20, 0.2 MgS04* 7H20, 1.0 glucose,
0 1991 the American
Physiological
Society
E293
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E294
K-OPIOID
RECEPTOR
SELECTIVITY
and 2.2 NaHC03] and then were first incubated for 30 min at 37°C with gentle stirring in the medium [(in g/l) 8.0 NaCl, 0.4 KCl, 1.0 glucose, and 0.35 NaHCOz] (10 ml) containing 0.1% trypsin. After the incubation, the trypsin digest was discarded, and remaining tissue was retained. Fresh medium (5 ml) containing trypsin was added to the flask, and the incubation was continued for another 30 min. The resulting cell suspension was decanted into a centrifuge tube in which the medium solution containing 10% inactivated horse serum as a trypsin inhibitor was present. Fresh trypsin medium was again added to the remaining tissue in the flask, and the same procedure was repeated again. The combined cells were centrifuged, resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal calf serum (FCS), passed through 100 pm of mesh, and centrifuged. The dispersed cells were seeded into a gelatinized plastic dish (60 x 15 mm; Falcon) and incubated at 37°C for 30 min. During the incubation, most nonmyocardial cells became attached to the floor of the dish, while myocytes remained suspended. The nonattached cells were then seeded into multiwell plates (12 wells; Falcon) in 1 ml DMEM supplemented with 20% FCS. The cells were incubated at 37°C under a humidified atmosphere of 95% air-5% COZ. Cultured myocytes began beating after 24 h and continued for at least 7 days. The cells after a 2-day incubation were replaced with fresh DMEM without FCS, and various agents were added to investigate their effects on ANP secretion. To measure the productin of CAMP, after preincubation with 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX) for 10 min, an incubation was carried out for 5 min at 37°C with or without agents and was stopped with the addition of cold trichloroacetic acid (TCA) (final concentration 5%). After the extraction of TCA with cold saturated ether, samples were lyophilized, dissolved in radioimmunoassay (RIA) buffer, and used for the CAMP assay. Assay. RIA of rat ANP (rANP) was carried out according to the previous method (32) by the use of rabbit antihuman ANP-( l-28) antiserum. This antibody showed identical cross-reactivity with cu-rANP-( l28) and y-rANP-(l-126) on an equimolar basis. Synthetic rANP-( l-28) (Peptide Institute, Osaka, Japan) was used as standard. Dilution curves of the medium in which atria1 myocytes had been incubated ran parallel with that of standard cu-rANP, suggesting that cu-rANPlike immunoreactivity was secreted by the cells. Analysis of rANP-like immunoreactivity in the culture medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a large molecular weight form (14,OOO), probably y-rANP, was predominant (>99%), together with a negligible amount of a small molecular weight form (3,000), which was coeluted with cu-rANP(l-28), findings consistent with those reported previously (2). The assay of CAMP was performed with a CAMP assay kit. Materials. ANP (rat and human), AVP, dynorphin A-( l-17), [o-Ala2,N-Me-Phe4,Gly5-ollenkephalin (DAGO), [D-Ala2,D-Le$]enkephalin (DADLE), leucine (Leu)-enkephalin, and methionine (Met)-enkephalin were purchased from Peptide Institute, Osaka, Japan.
IN
ANP
SECRETION
(-)-2-(3-Furylmethyl)-noretazocine (MR2266) was kindly supplied by Boehringer-Ingelheim, FRG, and (5a~, 7ar,8P)-(f)-3,4-dichloro-N-methyl-N-[7-(l-pyrrolidinyl)-1-oxaspiro(4,5)dec-&yl]benzeneacetamide methane sulphonate (U-62066E) was supplied by Upjohn, Kalamazoo, MI. Dibutyryl CAMP (DBcAMP), trypsin, and IBMX were purchased from Sigma, St. Louis, MO. Naloxone hydrochloride was from Endo-Laboratories, Garden City, NY. Sep-Pak Cls cartridges were obtained from waters Associates, Millipore, Milford, MA. Other reagents used were of analytical grade. A CAMP assay kit was obtained from Yamasa, Tokyo, Japan. Analysis. Data are expressed as means t SE. Statistical analyses were accomplished by an unpaired Student’s t test. RESULTS
Atria1 cardiocytes after a &day culture secreted immunoreactive rANP (irANP) time dependently into serum-free medium; the secretion of irANP due to 10D6 M dynorphin as well as basal secretion was linear over at least 4 h (Fig. 1). Therefore, in the subsequent experiments, unless otherwise indicated, 2-h incubation periods were used. As shown in Fig. 2, dynorphin stimulated the secretion of irANP dose dependently. Then, to evaluate the specificity of K-opioid receptor activation due to dynorphin, the effect of K-opioid receptor antagonist was examined. Dynorphin-induced irANP secretion was partially inhibited by the addition of MR2266, a relatively selective Kopioid receptor antagonist (13) (Table 1). Naloxone, a nonspecific opioid antagonist, had a lesser effect. Furth ermore, the effect of various opiate agonists was examined to evaluate the selectivity of the opiate-induced irANP secretion. As shown in Table 1, U-62066E, a selective K-opioid receptor agonist (18, 30)) significantly stimulated the secretion of irANP. This increase was
U
1
2
incubation
3
4
5
time h)
FIG. 1. Secretion of rat atria1 natriuretic peptide (rANP) from 2day cultured rat atria1 myocytes as function of time. Rat atria1 myocytes (lo5 cells) were incubated at 37°C in 1 ml serum-free medium for indicated times in absence (vehicle control; 0) or presence (0) of 10s6 M dynorphin. Each point is represented by 6 experiments. iANP, immunoreactive ANP.
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K-OPIOID
* 1
-
(-4
RECEPTOR
1o-7
1o-8
Dynorphin
T
SELECTIVITY
**
1o-6
1. Effect of opioid agonists and antagonists on irANP secretion from cultured atria1 myocytes
TABLE
Addition
+ MR2266
(10v6 M) (lo-” M) (10s4 M) (lo-”
E295
SECRETION
(M)
2. Effect of dynorphin on secretion of immunoreactive rANP (irANP) from cultured rat atria1 myocytes. Each bar represented by 6 * P < 0.05 and ** P < 0.01 vs. control (unpaired Student’s experiments. t test).
+ MR2266 + MR2266 + naloxone
ANP
Secondly, the effect of the produced CAMP was examined. The dynorphin-induced irANP secretion and the basal secretion were significantly decreased with the addition of IBMX, a phosphodiesterase inhibitor, as compared with the respective controls (Fig. 3). When 10B5 M MR2266 was coincubated with low6 M dynorphin in the presence of IBMX, dynorphin-induced irANP secretion was further suppressed, down to the level of the control in the presence of IBMX. In addition, lOa M DBcAMP significantly decreased the basal secretion of irANP in comparison with the control [basal irANP secretion (n = 5): from 0.82 t 0.05 ng/105 cells to 0.64 t 0.09 ng/105 cells when 10D3 M DBcAMP was added (P < 0.05)].
FIG.
Control (vehicle) Dynorphin (lo-” M) Dynorphin (lo-” M) Dynorphin ( 10s6 M) Dynorphin ( 10s6 M) U-62066E (lo+ M) U-62066E (1O-6 M) DADLE (lo-” M) Met-enkephalin (lo-” DAGO (1O-6 M)
IN
n
ng irANP/ 10” Cells
8 10 6 5
0.90t0.04 1.60*0.12* 1.35kO.05 1.12+0.08t 1.41kO.04 1.21+0.06$ 0.89+0.07§ 0.88&0.04
4 4 4 4 4 4
M)
M)
DISCUSSION
The present studies first showed that dynorphin stimulated the in vitro secretion of ANP with K-opioid receptor specificity and selectivity. The dose-dependent increase in ANP secretion due to dynorphin was partially suppressed with a selective K-opioid receptor antagonist. The dynorphin-induced ANP secretion takes place, at least in part, via a selective activation of K-opioid receptor in atria1 myocytes. It has been reported that opiate administration in vivo produced a large increase in plasma ANP (11, 28). The presence of cardiac innervation of dynorphin as well as enkephalin has been suggested (9). In fact, immunoreactive dynorphin was detected in rat heart at a relatively high concentration (26). The effect of Met-enkephalin,
0.89t0.04 0.90t0.04
Values are means t SE; n, no. of experiments. irANP, immunoreactive rat atria1 natriuretic polypeptide. See text for other definitions. * P < 0.01 vs. control; t P < 0.05 vs. 10v6 M dynorphin; $ P < 0.05 control; Q P < 0.05 vs. U62066E (unpaired Student’s t test).
c-1
Dynorphin
1OD6M + MR 2266 1O+M
vs.
2. Effect of opiates on CAMP production in cultured atria1 myocytes
TABLE
Control (vehicle) Dynorphin (lo-” Dynorphin (lo-” Met-enkephalin
Addition
n
M) M) + MR2266 ( 10m6 M)
5 5 5 5
(lo-”
M)
pmol/105
Cells
0.26t0.03 0.16t0.03* 0.23t0.04 0.27t0.02
Values are means & SE; n, no. of experiments. Phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine; 0.5 mM) was present in medium during lo-min preincubation, and then 5-min incubation was (unpaired Student’s t carried out with agents. * P < 0.05 vs. control test).
then antagonized by the addition of MR2266. On the contrary, DADLE (&opioid), Met-enkephalin (p/8opioid), and DAGO (p-opioid) did not stimulate the secretion of irANP (Table 1). To examine the involvement of CAMP in the dynorphin-induced irANP secretion, CAMP production during the incubation with dynorphin was first estimated. Dynorphin at 10e6 M significantly decreased the production of CAMP (Table 2). However, 10m6 M Met-enkephalin did not affect CAMP at all. The decrease in CAMP production by the addition of dynorphin was partially antagonized with a simultaneous addition of MR2266.
0
IBMX (0.5mM)
* h-+ -
-
FIG. 3. Effect of phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) on basal and dynorphin-induced ANP secretion from cultured rat atria1 myocytes. IBMX (0.5 mM; +) or vehicle (-) was added in incubation medium. In addition, when 10D5 M MR2266 was coincubated with low6 M dynorphin in presence of IBMX, dynorphin-induced iANP secretion was suppressed almost to level of basal secretion in presence of IBMX. * P C 0.05 vs. respective controls in absence of IBMX. ** P < 0.01 vs. 10B6 M dynorphin-induced iANP secretion in absence of IBMX. $ P < 0.05 vs. 10s6 M dynorphin-induced iANP secretion in presence of IBMX. Statistical analyses are carried out by unpaired Student’s t test. Each bar is represented by 5-6 experiments. See text for definitions.
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E296
K-OPIOID
RECEPTOR
SELECTIVITY
which is the active enkephalin on p-receptors, on the secretion of ANP in vitro was not observed in our study, as has also been previously reported (17). The present study also failed to detect any effect due to a-opiate agonists. This evidence suggests that the increase in plasma ANP levels evoked by p-opiate agonists such as morphine might not be a direct effect but rather might occur secondarily to the hemodynamic changes they may induce. On the other hand, dynorphin stimulates the secretion of ANP directly via an activation of cardiac Kopioid receptors. Therefore, endogenous dynorphin may serve, at least in part, as a cotransmitter with norepinephrine or acetylcholine released from cardiac innervating nerves in selectively modulating ANP secretion. Although a slight difference between the potency of the secretions caused by dynorphin and U-62066E, a K-receptor agonist, was observed, this might be due to the difference of affinity to the receptor or to the difference of the function of tissue handling (i.e., susceptibility to proteases). In any event, this still remains to be determined. A role for cyclic neucleotides in the modulation of ANP secretion has been suggested. It has recently been reported that, in the spinal cord, K-opioid agonists selectively inhibit adenylate cyclase and that its inhibition requires the presence of GTP (1, 16). In our study with the use of myocytes, CAMP production was decreased with dynorphin but not with Met-enkephalin. Furthermore, the dynorphin-induced decrease in CAMP production was partially antagonized by MR2266. This evidence suggests that K-opioid receptor activation is coupled with adenylate cyclase and selectively inhibits its activity in myocytes. Presynaptic modulation of opiates at choroid terminals appeared to involve a G protein (10). The same mechanism may be involved in the action of dynorphin on the myocytes. Furthermore, the addition of the phosphodiesterase inhibitor IBMX, while increasing the CAMP content in myocytes, decreased the dynorphin-induced ANP secretion as well as the basal secretion. Moreover, an incubation with DBcAMP, just as with IBMX, also decreased the basal secretion of ANP, raising the possibility that the increased CAMP itself inhibits dynorphin-induced ANP secretion as well as basal secretion. Thus opiate receptor selectivity might be suggestive of distinct highly specific roles for receptor types in the processing of information, even in myocytes, as has been reported in the spinal cord (25). It has been reported that ANP works negatively against the secretion of AVP from the central nervous system and against the peripheral action of AVP to maintain the homeostasis of water-electrolyte balance (23, 24). The evidence that dynorphin as a K-opioid receptor agonist has an inhibitory effect on the secretion of AVP (30,31) and a stimulatory effect on the secretion of ANP shows that dynorphin, as a neuromodulator or a neuromediator, might play an important role in controlling the water-electrolyte balance. In conclusion, dynorphin selectively stimulates ANP secretion, at least in part, via a specific K-opioid receptor activation. 1
l
1
l
IN
ANP
SECRETION
We thank J. Mabuchi for technical assistance and Dr. T. Suda, Tokyo Women’s Medical College, for donating 12”1-labeled dynorphin. We are grateful to the following companies for giving us the drugs used in this study: Upjohn for U-62066E, Boehringer-Ingelheim for MR2266, and Mitsubishi Yuka for ANP antiserum. This work was partially supported by a research grant for cardiovascular diseases (2A-1) from the Ministry of Health and Welfare, Tokyo, Japan. Address for reprint requests: K. Yamada, Div. of Clinical Research and Internal Medicine, Sakura National Hospital, 2-36-2 Ebaradai, Sakura, Chiba 285, Japan. Received
26 November
1990; accepted
in final
form
7 May
1991.
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YAMADA,
SHO YOSHIDA,
AND
YUTAKA
SHIMADA
Division of Clinical Research and Internal Medicine, Sakura National Hospital, Sakura, Chiba 285; and Second Department of Internal Medicine, Department of Anatomy, School of Medic line, Chiba University, Chiba 280, Japan
YAMADA,
KENICHI,
SHO
YOSHIDA,
AND
YUTAKA
SHIMADA.
Atria1 natriuretic polypeptide secretion via selective activation of K-opioid receptor: role of dynorphin. Am. J. Physiol. 261 (Endocrinol. Metab. 24): E293-E297, 1991.-The present study was designed to investigate the direct effect of dynorphin on atria1 natriuretic polypeptide (ANP) secretion in cultured rat atria1 cardiocytes via a K-opioid receptor activation as well as the involvement of adenosine 3’,5’-cyclic monophosphate (CAMP) system in the secretion of ANP from cardiocytes. Dynorphin stimulated ANP secretion dose and time dependently from 2-day cultured atria1 cardiocytes. The dynorphininduced ANP secretion was partially antagonized by MR2266, a selective K-opioid receptor antagonist. U-62066E, a selective K-opioid receptor agonist, stimulated ANP secretion. This stimulation was also antagonized by MR2266. However, no stimulation of ANP secretion was seen with [D-Ala*,D-Leu5]enkephalin, methionine (Met)-enkephalin, or [ D-Ala*,N-MePhe4,Gly”-ollenkephalin. Dynorphin at 10m6 M significantly decreased the production of CAMP in the cultured cardiocytes. However, lo-’ M Met-enkephalin had no effect on CAMP at all. The decrease in CAMP production by the addition of dynorphin was partially antagonized with a simultaneous addition of MR2266. The dynorphin-induced ANP secretion, as well as the basal secretion, were significantly decreased by the addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, as compared with the respective controls. Dibutyryl CAMP at lo-” M significantly decreased the basal secretion of ANP as compared with the control. Therefore, the present studies show that dynorphin selectively stimulates ANP secretion, at least in part, via the activation of a specific K-opioid receptor. selectivity;
adenosine
3’,5’-cyclic
monophosphate
KNOWN that opiates, in general, modulate the secretion of neurotransmitters or neuropeptides (5). Morphine or p-opioid agonists are known to block cholinergic transmission by pre- and postsynaptic mechanisms in many preparations, including autonomic ganglia (3). K-Opioid receptor agonists cause diuresis mainly through the decrease in plasma arginine vasopressin (AVP) levels (30), and K-opioid receptor activation actually reduces the increase in AVP secretion evoked by stimuli on the final common pathway in the process of AVP secretion (31). Thus it could be proposed that Kopioid receptors might be involved in the modulation of the secretion of neurohomones such as AVP. Mammalian atria1 cardiocytes secrete a potent natriIT IS WELL
0193-1849/91
$1.50 Copyright
uretic and vasoactive polypeptide hormone termed atria1 natriuretic peptide (ANP) (6, 7, 15). It has been shown that the secretion of ANP into the circulation is stimulated by cardiodynamic changes such as atria1 distension (ZO), volume expansion (19), and the administration of pressor doses of AVP, angiotensin II, and phenylephrine (21). It has recently been reported that ANP is not only a systemic circulating hormone but possesses the function of a neuropeptide hormone as well (27). The in vitro secretion of ANP after the stimulation by neurotransmitters, including a-adrenergic and cholinergic agonists, has been demonstrated (22). Dynorphin, which is known as an endogenous K-opioid peptide (4), has been reported to exist in the rat heart as a postulated neurotransmitter or neuromodulator at a relatively high concentration (9, 26). In fact, opioid administration in vivo produces a large increase in plasma ANP levels (28). Therefore, dynorphin might act directly on K-opioid receptors in the heart for the local regulation of ANP secretion. Peptide receptor-associated effects on neurotransmission, in general, have been thought to be modulated by second messengers such as cyclic nucleotides (8). In the brain, opiates have been proposed to act by triggering receptor-mediated inhibition of adenylate cyclase activity, thereby decreasing the formation of adenosine 3’,5’cyclic monophosphate (CAMP) (29). Pharmacological studies have shown that the spinal antinociceptive effect of opiate in the tail-flick test is prevented by the simultaneous injection of aminophylline, a CAMP phosphodiesterase inhibitor (14). Furthermore, K-opioid receptors were reported to be selectively and negatively coupled to adenylate cyclase in the rat spinal cord (1). Therefore, the present study was carried out to determine the direct effect of dynorphin on ANP secretion in cultured rat atria1 cardiocytes via K-opioid receptor activation and to determine the involvement of the CAMP system in the secretion of ANP from cardiocytes. METHODS
Cell culture. Neonatal atria1 cardiocytes were prepared by a modification of the technique originally described by Harary and Farley (12). Atria removed from neonatal rats (2-4 days old) were rinsed and minced well in the preparation medium [(in g/l) 6.8 NaCl, 0.14 NaH2P04. 2Hz0, 0.2 CaClzo 2H20, 0.2 MgS04* 7H20, 1.0 glucose,
0 1991 the American
Physiological
Society
E293
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E294
K-OPIOID
RECEPTOR
SELECTIVITY
and 2.2 NaHC03] and then were first incubated for 30 min at 37°C with gentle stirring in the medium [(in g/l) 8.0 NaCl, 0.4 KCl, 1.0 glucose, and 0.35 NaHCOz] (10 ml) containing 0.1% trypsin. After the incubation, the trypsin digest was discarded, and remaining tissue was retained. Fresh medium (5 ml) containing trypsin was added to the flask, and the incubation was continued for another 30 min. The resulting cell suspension was decanted into a centrifuge tube in which the medium solution containing 10% inactivated horse serum as a trypsin inhibitor was present. Fresh trypsin medium was again added to the remaining tissue in the flask, and the same procedure was repeated again. The combined cells were centrifuged, resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal calf serum (FCS), passed through 100 pm of mesh, and centrifuged. The dispersed cells were seeded into a gelatinized plastic dish (60 x 15 mm; Falcon) and incubated at 37°C for 30 min. During the incubation, most nonmyocardial cells became attached to the floor of the dish, while myocytes remained suspended. The nonattached cells were then seeded into multiwell plates (12 wells; Falcon) in 1 ml DMEM supplemented with 20% FCS. The cells were incubated at 37°C under a humidified atmosphere of 95% air-5% COZ. Cultured myocytes began beating after 24 h and continued for at least 7 days. The cells after a 2-day incubation were replaced with fresh DMEM without FCS, and various agents were added to investigate their effects on ANP secretion. To measure the productin of CAMP, after preincubation with 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX) for 10 min, an incubation was carried out for 5 min at 37°C with or without agents and was stopped with the addition of cold trichloroacetic acid (TCA) (final concentration 5%). After the extraction of TCA with cold saturated ether, samples were lyophilized, dissolved in radioimmunoassay (RIA) buffer, and used for the CAMP assay. Assay. RIA of rat ANP (rANP) was carried out according to the previous method (32) by the use of rabbit antihuman ANP-( l-28) antiserum. This antibody showed identical cross-reactivity with cu-rANP-( l28) and y-rANP-(l-126) on an equimolar basis. Synthetic rANP-( l-28) (Peptide Institute, Osaka, Japan) was used as standard. Dilution curves of the medium in which atria1 myocytes had been incubated ran parallel with that of standard cu-rANP, suggesting that cu-rANPlike immunoreactivity was secreted by the cells. Analysis of rANP-like immunoreactivity in the culture medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a large molecular weight form (14,OOO), probably y-rANP, was predominant (>99%), together with a negligible amount of a small molecular weight form (3,000), which was coeluted with cu-rANP(l-28), findings consistent with those reported previously (2). The assay of CAMP was performed with a CAMP assay kit. Materials. ANP (rat and human), AVP, dynorphin A-( l-17), [o-Ala2,N-Me-Phe4,Gly5-ollenkephalin (DAGO), [D-Ala2,D-Le$]enkephalin (DADLE), leucine (Leu)-enkephalin, and methionine (Met)-enkephalin were purchased from Peptide Institute, Osaka, Japan.
IN
ANP
SECRETION
(-)-2-(3-Furylmethyl)-noretazocine (MR2266) was kindly supplied by Boehringer-Ingelheim, FRG, and (5a~, 7ar,8P)-(f)-3,4-dichloro-N-methyl-N-[7-(l-pyrrolidinyl)-1-oxaspiro(4,5)dec-&yl]benzeneacetamide methane sulphonate (U-62066E) was supplied by Upjohn, Kalamazoo, MI. Dibutyryl CAMP (DBcAMP), trypsin, and IBMX were purchased from Sigma, St. Louis, MO. Naloxone hydrochloride was from Endo-Laboratories, Garden City, NY. Sep-Pak Cls cartridges were obtained from waters Associates, Millipore, Milford, MA. Other reagents used were of analytical grade. A CAMP assay kit was obtained from Yamasa, Tokyo, Japan. Analysis. Data are expressed as means t SE. Statistical analyses were accomplished by an unpaired Student’s t test. RESULTS
Atria1 cardiocytes after a &day culture secreted immunoreactive rANP (irANP) time dependently into serum-free medium; the secretion of irANP due to 10D6 M dynorphin as well as basal secretion was linear over at least 4 h (Fig. 1). Therefore, in the subsequent experiments, unless otherwise indicated, 2-h incubation periods were used. As shown in Fig. 2, dynorphin stimulated the secretion of irANP dose dependently. Then, to evaluate the specificity of K-opioid receptor activation due to dynorphin, the effect of K-opioid receptor antagonist was examined. Dynorphin-induced irANP secretion was partially inhibited by the addition of MR2266, a relatively selective Kopioid receptor antagonist (13) (Table 1). Naloxone, a nonspecific opioid antagonist, had a lesser effect. Furth ermore, the effect of various opiate agonists was examined to evaluate the selectivity of the opiate-induced irANP secretion. As shown in Table 1, U-62066E, a selective K-opioid receptor agonist (18, 30)) significantly stimulated the secretion of irANP. This increase was
U
1
2
incubation
3
4
5
time h)
FIG. 1. Secretion of rat atria1 natriuretic peptide (rANP) from 2day cultured rat atria1 myocytes as function of time. Rat atria1 myocytes (lo5 cells) were incubated at 37°C in 1 ml serum-free medium for indicated times in absence (vehicle control; 0) or presence (0) of 10s6 M dynorphin. Each point is represented by 6 experiments. iANP, immunoreactive ANP.
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K-OPIOID
* 1
-
(-4
RECEPTOR
1o-7
1o-8
Dynorphin
T
SELECTIVITY
**
1o-6
1. Effect of opioid agonists and antagonists on irANP secretion from cultured atria1 myocytes
TABLE
Addition
+ MR2266
(10v6 M) (lo-” M) (10s4 M) (lo-”
E295
SECRETION
(M)
2. Effect of dynorphin on secretion of immunoreactive rANP (irANP) from cultured rat atria1 myocytes. Each bar represented by 6 * P < 0.05 and ** P < 0.01 vs. control (unpaired Student’s experiments. t test).
+ MR2266 + MR2266 + naloxone
ANP
Secondly, the effect of the produced CAMP was examined. The dynorphin-induced irANP secretion and the basal secretion were significantly decreased with the addition of IBMX, a phosphodiesterase inhibitor, as compared with the respective controls (Fig. 3). When 10B5 M MR2266 was coincubated with low6 M dynorphin in the presence of IBMX, dynorphin-induced irANP secretion was further suppressed, down to the level of the control in the presence of IBMX. In addition, lOa M DBcAMP significantly decreased the basal secretion of irANP in comparison with the control [basal irANP secretion (n = 5): from 0.82 t 0.05 ng/105 cells to 0.64 t 0.09 ng/105 cells when 10D3 M DBcAMP was added (P < 0.05)].
FIG.
Control (vehicle) Dynorphin (lo-” M) Dynorphin (lo-” M) Dynorphin ( 10s6 M) Dynorphin ( 10s6 M) U-62066E (lo+ M) U-62066E (1O-6 M) DADLE (lo-” M) Met-enkephalin (lo-” DAGO (1O-6 M)
IN
n
ng irANP/ 10” Cells
8 10 6 5
0.90t0.04 1.60*0.12* 1.35kO.05 1.12+0.08t 1.41kO.04 1.21+0.06$ 0.89+0.07§ 0.88&0.04
4 4 4 4 4 4
M)
M)
DISCUSSION
The present studies first showed that dynorphin stimulated the in vitro secretion of ANP with K-opioid receptor specificity and selectivity. The dose-dependent increase in ANP secretion due to dynorphin was partially suppressed with a selective K-opioid receptor antagonist. The dynorphin-induced ANP secretion takes place, at least in part, via a selective activation of K-opioid receptor in atria1 myocytes. It has been reported that opiate administration in vivo produced a large increase in plasma ANP (11, 28). The presence of cardiac innervation of dynorphin as well as enkephalin has been suggested (9). In fact, immunoreactive dynorphin was detected in rat heart at a relatively high concentration (26). The effect of Met-enkephalin,
0.89t0.04 0.90t0.04
Values are means t SE; n, no. of experiments. irANP, immunoreactive rat atria1 natriuretic polypeptide. See text for other definitions. * P < 0.01 vs. control; t P < 0.05 vs. 10v6 M dynorphin; $ P < 0.05 control; Q P < 0.05 vs. U62066E (unpaired Student’s t test).
c-1
Dynorphin
1OD6M + MR 2266 1O+M
vs.
2. Effect of opiates on CAMP production in cultured atria1 myocytes
TABLE
Control (vehicle) Dynorphin (lo-” Dynorphin (lo-” Met-enkephalin
Addition
n
M) M) + MR2266 ( 10m6 M)
5 5 5 5
(lo-”
M)
pmol/105
Cells
0.26t0.03 0.16t0.03* 0.23t0.04 0.27t0.02
Values are means & SE; n, no. of experiments. Phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine; 0.5 mM) was present in medium during lo-min preincubation, and then 5-min incubation was (unpaired Student’s t carried out with agents. * P < 0.05 vs. control test).
then antagonized by the addition of MR2266. On the contrary, DADLE (&opioid), Met-enkephalin (p/8opioid), and DAGO (p-opioid) did not stimulate the secretion of irANP (Table 1). To examine the involvement of CAMP in the dynorphin-induced irANP secretion, CAMP production during the incubation with dynorphin was first estimated. Dynorphin at 10e6 M significantly decreased the production of CAMP (Table 2). However, 10m6 M Met-enkephalin did not affect CAMP at all. The decrease in CAMP production by the addition of dynorphin was partially antagonized with a simultaneous addition of MR2266.
0
IBMX (0.5mM)
* h-+ -
-
FIG. 3. Effect of phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) on basal and dynorphin-induced ANP secretion from cultured rat atria1 myocytes. IBMX (0.5 mM; +) or vehicle (-) was added in incubation medium. In addition, when 10D5 M MR2266 was coincubated with low6 M dynorphin in presence of IBMX, dynorphin-induced iANP secretion was suppressed almost to level of basal secretion in presence of IBMX. * P C 0.05 vs. respective controls in absence of IBMX. ** P < 0.01 vs. 10B6 M dynorphin-induced iANP secretion in absence of IBMX. $ P < 0.05 vs. 10s6 M dynorphin-induced iANP secretion in presence of IBMX. Statistical analyses are carried out by unpaired Student’s t test. Each bar is represented by 5-6 experiments. See text for definitions.
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E296
K-OPIOID
RECEPTOR
SELECTIVITY
which is the active enkephalin on p-receptors, on the secretion of ANP in vitro was not observed in our study, as has also been previously reported (17). The present study also failed to detect any effect due to a-opiate agonists. This evidence suggests that the increase in plasma ANP levels evoked by p-opiate agonists such as morphine might not be a direct effect but rather might occur secondarily to the hemodynamic changes they may induce. On the other hand, dynorphin stimulates the secretion of ANP directly via an activation of cardiac Kopioid receptors. Therefore, endogenous dynorphin may serve, at least in part, as a cotransmitter with norepinephrine or acetylcholine released from cardiac innervating nerves in selectively modulating ANP secretion. Although a slight difference between the potency of the secretions caused by dynorphin and U-62066E, a K-receptor agonist, was observed, this might be due to the difference of affinity to the receptor or to the difference of the function of tissue handling (i.e., susceptibility to proteases). In any event, this still remains to be determined. A role for cyclic neucleotides in the modulation of ANP secretion has been suggested. It has recently been reported that, in the spinal cord, K-opioid agonists selectively inhibit adenylate cyclase and that its inhibition requires the presence of GTP (1, 16). In our study with the use of myocytes, CAMP production was decreased with dynorphin but not with Met-enkephalin. Furthermore, the dynorphin-induced decrease in CAMP production was partially antagonized by MR2266. This evidence suggests that K-opioid receptor activation is coupled with adenylate cyclase and selectively inhibits its activity in myocytes. Presynaptic modulation of opiates at choroid terminals appeared to involve a G protein (10). The same mechanism may be involved in the action of dynorphin on the myocytes. Furthermore, the addition of the phosphodiesterase inhibitor IBMX, while increasing the CAMP content in myocytes, decreased the dynorphin-induced ANP secretion as well as the basal secretion. Moreover, an incubation with DBcAMP, just as with IBMX, also decreased the basal secretion of ANP, raising the possibility that the increased CAMP itself inhibits dynorphin-induced ANP secretion as well as basal secretion. Thus opiate receptor selectivity might be suggestive of distinct highly specific roles for receptor types in the processing of information, even in myocytes, as has been reported in the spinal cord (25). It has been reported that ANP works negatively against the secretion of AVP from the central nervous system and against the peripheral action of AVP to maintain the homeostasis of water-electrolyte balance (23, 24). The evidence that dynorphin as a K-opioid receptor agonist has an inhibitory effect on the secretion of AVP (30,31) and a stimulatory effect on the secretion of ANP shows that dynorphin, as a neuromodulator or a neuromediator, might play an important role in controlling the water-electrolyte balance. In conclusion, dynorphin selectively stimulates ANP secretion, at least in part, via a specific K-opioid receptor activation. 1
l
1
l
IN
ANP
SECRETION
We thank J. Mabuchi for technical assistance and Dr. T. Suda, Tokyo Women’s Medical College, for donating 12”1-labeled dynorphin. We are grateful to the following companies for giving us the drugs used in this study: Upjohn for U-62066E, Boehringer-Ingelheim for MR2266, and Mitsubishi Yuka for ANP antiserum. This work was partially supported by a research grant for cardiovascular diseases (2A-1) from the Ministry of Health and Welfare, Tokyo, Japan. Address for reprint requests: K. Yamada, Div. of Clinical Research and Internal Medicine, Sakura National Hospital, 2-36-2 Ebaradai, Sakura, Chiba 285, Japan. Received
26 November
1990; accepted
in final
form
7 May
1991.
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