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Endocrinology Vol. 142, No. 5 2022-2031
Copyright © 2001 by The Endocrine Society

ARTICLES

Opioids Suppress Basal and Nicotine-Induced Catecholamine Secretion Via a Stabilizing Effect on Actin Filaments1

E. Dermitzaki, A. Gravanis, M. Venihaki2, C. Stournaras and A. N. Margioris

Department of Clinical Chemistry (E.D., M.V., A.N.M.), Pharmacology (A.G.), and Biochemistry (C.S.), University of Crete School of Medicine, Heraklion GR-711 10, Crete, Greece

Address all correspondence and requests for reprints to: Dr. Andrew N. Margioris, Department of Clinical Chemistry-Biochemistry, University of Crete School of Medicine, Heraklion GR-711 10, Crete, Greece. E-mail: andym{at}med.uoc.gr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Catecholamine secretion and actin filament disassembly are closely coupled in chromaffin cells. Opioid suppression of catecholamine secretion is fast and transient, both characteristics of actin filament involvement. The aim of the present work was to test the hypothesis that opioids suppress catecholamine secretion via an inhibitory effect on actin filament disassembly. For this purpose we used the PC12 rat pheochromocytoma cell line. Norepinephrine and dopamine were measured by enzyme-linked immunosorbent assay or RIA. Polymerized actin was measured by rhodamine-phalloidin and visualized by confocal laser scanning microscopy. Opioids suppressed basal catecholamine secretion. The onset of this effect was fast and transient, peaking at 2 min, and was reversible by opioid antagonists. Synchronously, opioids suppressed actin filament disassembly; this was also reversible by opioid antagonists. Cytochalasin B prevented the inhibitory effect of opioids on catecholamine secretion. In addition, opioids suppressed the stimulatory effect of nicotine on catecholamine secretion and actin depolymerization. Changes in actin cytoskeleton in neuron-like PC12 cells make them resistant to both effects of opioids, i.e. on catecholamine secretion and actin disassembly. In conclusion, our data suggest that the suppressive effect of opioids on basal and nicotine-induced catecholamine secretion may result from an opioid-provoked stabilization of cortical actin. It also appears that basal catecholamine secretion is associated with opioid-sensitive machinery regulating the continuous formation of short-lived areas of cortical actin filament disassembly.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SUBPLASMALEMMAL (cortical) ring consists of actin filaments and binding proteins forming a dense viscoelastic gel underneath cell membranes (1, 2). It now appears that cortical actin constitutes a fast response system regulating the trafficking of secretory granules in several types of cells, including neurons. Indeed, synaptic vesicles are distributed in two areas: a group near active secretion zones docked at plasma membranes and ready to fuse in response to calcium entry, and a second group located inside the actin ring (3, 4). Secretion of the content of the second group of synaptic vesicles involves detachment from actin filaments and translocation toward exocytosis sites. These steps require activation of severing proteins, causing transient filament disassembly (5). In chromaffin cells, the subplasmalemmal ring of actin filaments constitutes a fast response system regulating catecholamine secretion. Nicotine induces catecholamine secretion by provoking actin filament disassembly (6, 7). Furthermore, cytochalasin, an actin filament disrupter, promotes basal and Ca2+-stimulated catecholamine secretion, whereas stabilizers such as phalloidin inhibit it (1, 8, 9). A variety of second messengers, including G proteins, cytosolic calcium, protein kinase C, and phosphatidylinositol 3-kinase, regulate cortical actin disassembly in chromaffin cells (10, 11, 12, 13, 14).

Opioids suppress catecholamine secretion in bovine, human, and rodent adrenals (15, 16, 17, 18, 19). This inhibitory effect of opioids is retained in two pheochromocytoma cell lines, rat PC12 (20) and human KAT45 (21). In chromaffin cells, opioids suppress both basal and nicotine-induced catecholamine secretion. These effects are characterized by acute onset and short duration, both characteristics of actin filament involvement (20). Based on these data, we hypothesized that opioids may inhibit catecholamine secretion via stabilization of actin filaments. To test this hypothesis we used the PC12 rat pheochromocytoma cell line, an in vitro model for the study of chromaffin cell physiology and the regulatory role of endogenous opioids. We examined the effects of several opioid agonists and antagonists on both actin polymerization and norepinephrine and dopamine secretion under basal and nicotine-induced conditions. Subsequently, we also examined the effect of cytochalasin B, an actin filament disrupter, on opioid-mediated suppression of basal and nicotine-induced catecholamine secretion. Finally, we compared the effect of opioids on short- and long-term cultured PC12 cells, because the latter exhibit major changes in their cytoskeletal structure similar or identical to those observed after exposure to nerve growth factor (NGF). Specifically, the following questions have been addressed. 1) Is the suppressing effect of opioids on basal norepinephrine secretion associated with a parallel and synchronous inhibition of actin filament disassembly? 2) Can the inhibitory effect of opioids on basal catecholamine secretion be prevented by prior actin filament disassembly? 3) Is opioid-mediated inhibition of nicotine-induced norepinephrine secretion associated with an opioid-induced stabilization of actin filaments? 4) Do the cytoskeletal changes that follow long-term PC12 culture affect their response to opioids? For this purpose we examined the effects of several opioid agonists, antagonists, and/or cytochalasin B on basal and nicotine-induced norepinephrine and dopamine secretion and actin polymerization. Our data suggest that opioids suppress basal catecholamine secretion via an inhibitory effect on spontaneous actin filament depolymerization. The suppressive effect of opioids on nicotine-induced catecholamine secretion appears to be associated with an opioid-mediated stabilization of actin filaments. Finally, the close association between actin filaments, catecholamine secretion, and opioid suppression can also be shown in older PC12 cells, where changes in actin cytoskeleton confer resistance to both effects of opioids, i.e. catecholamine secretion and actin disassembly.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
PC12 cell culture
PC12 cells were obtained from Dr. M. Greenberg (Children’s Hospital, Boston, MA), the late G. Guroff (Section on Growth Factors, NICHHD, NIH, Bethesda, MD), and American Type Culture Collection (Manassas, VA). The PC12 cells were cultured in flat-bottom wells (6-well plates of 9.5-cm2 surface area/well, or 24-well plates of 1.9-cm2 surface area/well; Costar Europe Ltd., Eindhoven, The Netherlands) at an initial concentration of 1.5 x 105 cells/cm2. The cells were grown in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) containing 10 mM L-glutamine, 15 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% horse serum, and 5% FCS in 5% CO2 at 37 C. Newly plated cells were cultured for 2–3 days in the growth medium just described and subsequently were left in serum-free medium supplemented with 0.1% BSA (fraction V).

Materials
Human pheochromocytomas (22) and PC12 cells (including our own passages) possess predominantly the K1 type of opioid-binding sites and, to a lesser degree, {delta} and a few µ sites. Based on this profile the following synthetic opioid agonists and antagonists have been used: the pure {kappa}-opioid receptor agonist U-69593 [5{alpha},7{alpha},8ß-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-(4,5)-dec-7–8-yl]benzeneacetamide; Upjohn Co., Kalamazoo, MI] and the {kappa}1/{kappa}2-opioid receptor agonist {alpha}S1-casomorphin, which also exhibits some affinity toward {delta}- and µ-opioid receptors (Tyr-Val-Pro-Phe-Pro; a gift from Dr. E. Castanas, University of Crete School of Medicine, Crete, Greece) (23), the µ- and {delta}-opioid agonist DADLE ([D-Ala2,D-Leu5]enkephalin; Sigma, St. Louis, MO), the µ-opioid agonist DAGO ([D-Ala2,N-Me-Phe4,Gly-ol]enkephalin; Peninsula Laboratories, Inc., Belmont, CA), the {delta}2-opioid agonist DSLET ([D-Ser2]-Leu-enkephalin-Thr; Sigma), the selective {kappa}-opioid receptor antagonist nor-binaltorphimine [nor-BNI; 17,17'-(dicyclopropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-binorphinan-3,4',14,14'-tetrol; RBI, Natick, MA],and the general opioid antagonist naloxone [5{alpha}-4,5-epoxy-3,14-dihydro-17-(2-propenyl)morphinan-6-one; Sigma]. BSA (fraction V) and PBS were obtained from Life Technologies, Inc., cytochalasin B and poly-L-lysine were obtained from Sigma, Coomassie Brilliant Blue was purchased from Serva (Heidelberg, Germany), and rhodamine-phalloidin was obtained from Molecular Probes, Inc. (Eugene, OR).

Measurement of catecholamines
Cells were grown in six-well plates, coated with poly-L-lysine, at a concentration of 106 cells/well. Cells were incubated with opioid agonists and/or antagonists for several time intervals; 1 ml supernatant was transferred to tubes containing 0.1 M HCl for measurement of catecholamines. It should be noted that catecholamines are stable in an acid environment.

Norepinephrine was measured by a competitive enzyme immunoassay (ICN Pharmaceuticals, Inc., Costa Mesa, CA). Briefly, norepinephrine was extracted from samples and acylated to N-acylnorepinephrine using catechol-O-methyltransferase (EC 2.1.1.6) as enzyme and S-adenosyl-L-methionine as coenzyme, followed by rapid addition of the acylation reagent provided. Optical density was read on a Dynatech Corp. Microelisa reader (Chantilly, VA) at 405 nm. The concentration of noradrenaline was inversely proportional to optical density. The sensitivity of the assay was 3 ng/ml.

Dopamine was measured by RIA (IBL, Hamburg, Germany) using 125I as tracer. It was extracted by a cis-diole-specific substrate, converted to 3-methoxytyramine using catechol-O-methyltransferase as enzyme and S-adenosyl-L-methionine as coenzyme, and simultaneously acylated to N-acyl-3-methoxytyramine. Bounded 125I-labeled antigen was precipitated with a second antibody and counted in a {gamma}-counter (1275 Minigamma, LKB Wallac, Inc., Turku, Finland). The sensitivity of the method was 30 pg/ml, its intraassay coefficient of variation was 7%, and its interassay coefficient of variation was 11.5%.

Measurement of filamentous actin
Cells were grown in 24-well plates at an initial concentration of 3 x 105 cells/well. At the end of each experiment, the cells were harvested and transferred to tubes, followed by washing with PBS, and centrifuged at 1500 rpm. The cells were fixed and permeabilized by exposure to 3.7% formaldehyde for 15 min, followed by exposure to 0.2% Triton for 5 min. The cells were incubated with rhodamine-phalloidin at a final concentration of 1.5 µM in PBS for 30 min in the dark. Phalloidin, a toxin, binds specifically to polymerized actin. Rhodamine, conjugated to phalloidin, plays the role of tracer measured by fluorescence. Subsequently, the cells were washed twice with PBS and reconstituted in 0.1 M NaOH, and the fluorescent signals were measured on a fluorescence spectrometer (LS-3B, Perkin-Elmer Corp., Norwalk, CT). Normalization was achieved by expressing all row data per mg total cellular protein, i.e. at the end of each experiment the cells were lysed by sonication on ice (model 72434, Bioblock Scientific, Cedex, France), and the protein concentration of each well was measured using a modification of the Bradford Coomassie Brilliant Blue G250 method with BSA (fraction V) as standard (24).

Nicotine experiments
Cells were grown in 6-well plates coated with poly-L-lysine at a concentration of 106 cells/well. At the time of the experiment, cells were exposed to nicotine alone or its vehicles for several time intervals. The supernatants were collected, acidified with 0.1 N HCl, and stored for catecholamine measurement. For actin measurement, cells were grown in 24-well plates, exposed to nicotine or its vehicles, and harvested as described above. The content of filamentous actin was conjugated to rhodamine-phalloidin and measured in a fluorometer. The effect of opioids on nicotine-induced catecholamine secretion was examined at the maximal response to the latter, i.e. at the peak catecholamine response to nicotine. At this time point, opioids or their vehicles were added for 2 min, and subsequently, cells and supernatants were collected as described above for actin and catecholamine measurements.

Confocal laser scanning microscopy
Cells were grown on 22 x 22-mm coverslips. At the end of each incubation period, cells were fixed by exposure to 3.7% formaldehyde and permeabilized by 0.2% Triton for 10 min (25) or by immersion in -20 C acetone (26). Cells were then incubated with 0.1% NaBH4 for 10 min and washed, and 0.5% BSA was added for 15 min. Subsequently, cells were incubated with rhodamine conjugated to phalloidin for 40 min at room temperature. The coverslips were analyzed using a confocal laser scanning module (Leica Corp., Lasertechnik, Heidelberg, Germany) attached to an inverted microscope (IM35, Carl Zeiss, Oberkochen, Germany) equipped with an argon-krypton ion laser. Confocal images were acquired using a 63/1.25 oil immersion objective and CLSM software (Leica Corp., Lasertechnik).

Statistical analysis
Results are presented as either normalized row data (concentration of norepinephrine or fluorometric units measured in our fluorescence spectrometer) or the percent change compared with parallel control values. To be able to compare results from independent experiments, the concentrations of norepinephrine, dopamine, and polymerized actin were normalized per total cellular protein measured by the Bradford Coomassie Brilliant Blue G250 method (Serva, Heidelberg, Germany) using BSA as the standard (27) as previously modified (24). For statistical evaluation of our data we used ANOVA, post-hoc comparison of means followed by two multiple comparison tests: Fisher’s least significance difference test and the Newman-Keuls test. For the data expressed as the percent change over parallel control values we used the nonparametric Kruskal-Wallis test for several independent samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Opioids suppress acutely basal norepinephrine secretion
Opioids exerted a fast, transient, and reproducible suppressive effect on basal norepinephrine secretion from PC12 cells, which peaked between 2–5 min and ended at approximately 15 min. Figure 1AGo depicts graphically the timedependent effect of the {kappa}-opioid receptor agonist U-69593 at 10-6 M, on norepinephrine secretion from PC12 cells, whereas Fig. 1BGo depicts the dose-response curve at 2 min. U-69593 at 10-6 M suppressed norepinephrine secretion at 2 min to 4 ± 0.8 ng norepinephrine/mg total cellular protein (mean ± SE; n = 11 from five independent experiments) compared with 8.3 ± 2.1 ng in parallel controls, i.e. cells exposed only to the substance’s vehicle (P < 0.001). This effect amounts to an approximately 50% suppression of basal norepinephrine secretion. The degree and timing of peak suppression were remarkably constant and reproducible, as they can be deduced from the low SD and SE of pooled data from five independent experiments. The µ-opioid agonist DAGO suppressed norepinephrine secretion to 5.8 ± 1.2 ng compared with 9.4 ± 1.8 ng in parallel controls (n = 6, three independent experiments; P < 0.001). The µ- and {delta}-opioid agonist DADLE suppressed norepinephrine secretion to 5 ± 0.9 ng compared with 5.8 ± 1 ng in parallel controls (n = 9, three experiments; P < 0.01), whereas the {delta}2-opioid agonist DSLET did not have any statistically significant effect. The synthetic opioid agonist {alpha}S1-casomorphin (a potent agonist for the {kappa}1/{kappa}2-opioid receptors and a weak µ and {delta} agonist) suppressed norepinephrine secretion to 4.9 ± 0.2 ng compared with 6.1 ± 0.9 ng in parallel controls (n = 4, two independent experiments; P < 0.05).



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  		Figure 1. Suppressive effect of {kappa}-opioids on basal norepinephrine secretion. A, The time response of basal norepinephrine secretion from PC12 cells in culture to the synthetic {kappa}-opioid receptor agonist U-69593 at 10-6 M. B, The dose-response curve to U-69593 at 2 min of exposure. Values represent the mean ± SE (n = 11 obtained from five independent experiments). Data are expressed as percentages of parallel controls exposed only to vehicles. *, P < 0.05; ***, P < 0.001 (significant difference compared with controls).

 
Cytochalasin B prevents the suppressive effect of opioids on basal norepinephrine secretion
The suppressive effect of opioid agonists on basal norepinephrine secretion was prevented by opioid antagonists or cytochalasin, an actin filament disrupter. These data suggest that the effect of opioids involved activation of opioid receptors, which resulted in alterations of actin filaments. Figure 2AGo depicts the antagonizing effect of the selective {kappa}opioid receptor antagonist nor-BNI and the general opioid antagonist naloxone on the suppressive effect of U-69593, DAGO, and DADLE on basal norepinephrine secretion at 2 min. Nor-BNI prevented the inhibitory effect of the {kappa}-opioid agonist U-69593 on norepinephrine secretion. Specifically, although U-69593 suppressed norepinephrine secretion to 48.3 ± 10% (mean ± SE; n = 11) of the parallel control value, the simultaneous presence of nor-BNI prevented the suppressive effect of U-69593 to levels comparable to those in unexposed controls, i.e. to 135.4 ± 25% (P < 0.01). Similarly, the simultaneous presence of naloxone prevented the suppressive effect of DAGO from 62 ± 6% to 125 ± 28% (P < 0.05) and that of DADLE from 78.8 ± 6.4% to 100 ± 10%. Figure 2BGo shows that the simultaneous presence of cytochalasin B at 10-5 M prevented the suppressive effect of U-69593, DAGO, and DADLE on norepinephrine secretion. Indeed, cytochalasin B prevented the suppressive effect of U-69593 (from 48.3 ± 10% achieved by U-69593 to 96 ± 13%; n = 11; P < 0.05), of DAGO (from 62 ± 6% to 114 ± 42%; n = 11; P < 0.05), and of DADLE (from 78.8 ± 6.4% to 173 ± 101).



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  		Figure 2. Inhibition of the suppressive effect of opioids by either opioid antagonists or cytochalasin B. A, The suppressive effect on norepinephrine secretion at 2 min of exposure to the opioid agonists U-69593, DAGO, and DADLE (each at 10-6 M) was blocked by the general opioid antagonist naloxone or the specific {kappa}-opioid antagonist nor-BNI (each at 10-5 M). B, The suppressive effect on norepinephrine secretion at 2 min of the opioid agonists U-69593, DAGO, and DADLE (each at 10-6 M) was also blocked by the actin filament disrupter cytochalasin B, which did not have any effect of its own. Data are expressed as a percentage of the control value in three independent experiments. **, P < 0.01; ***, P < 0.001 (significant difference between cells exposed to opioid agonists and cells exposed to opioid agonists plus antagonists or cytochalasin B).

 
Opioids acutely increase the total cellular content of actin filaments
Under basal conditions, i.e. in unstimulated PC12 cells, opioids increased the total cellular content of polymerized actin in parallel to their effect on catecholamine secretion. Figure 3Go depicts the effect of opioid agonists with or without antagonists on the total cellular content of polymerized actin at 2 min. Thus, 10-6 M U-69593 at 2 min brought about a 2-fold increase in the content of polymerized actin to 25 ± 8 [mean ± SE; fluorometric units (U) per mg total cellular protein] compared with 13 ± 4 U in parallel controls (n = 6, two independent experiments; P < 0.01). Similarly, 10-6 M of the µ- and {delta}-opioid agonist DADLE at 2 min increased the concentration of polymerized actin to 27 ± 1 U compared with 13 ± 4 U in parallel controls (n = 4, two independent experiments; P < 0.01), whereas the µ-opioid agonist DAGO increased actin to 25 ± 5 U compared with 13 ± 4 U in parallel controls (n = 6, two independent experiments; P < 0.5). Figure 4BGo depicts the confocal data of cells exposed to the {kappa}-opioid agonist U-69593 at 10-6 M compared with those of cells exposed to its vehicles (Fig. 4AGo). The confocal microscopy data supplement the results obtained by rhodamine-phalloidin. Indeed, {kappa}-opioids increased the intracellular content of filamentous actin at precisely the same time point at which catecholamine secretion was suppressed, i.e. between 2–5 min. Figure 4BGo depicts an opioid-mediated increase in rhodamine-phalloidin staining confined mainly to the subplasmalemmal area beneath the cell membrane. Furthermore, intense staining for microfilament was seen at the lower part of cell bodies in the area attached to the well surface (Fig. 4BGo). Moreover, the cellular content of filamentous actin returned to normal and was completely indistinguishable from parallel control values at 30 min of exposure to opioid agonists (Fig. 4Go, C and D).



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  		Figure 3. Effects of opioids on polymerized actin. This graph depicts the acute effects of various opioid agonists with or without opioid antagonists on actin polymerization measured by the rhodamine-phalloidin method. The opioid agonists U-69593, DAGO, and DADLE caused a 2-fold increase in polymerized actin at 2 min of exposure ({square}). These effects were completely blocked by the general opioid antagonist naloxone and the specific {kappa}-opioid antagonist nor-BNI ({blacksquare}). Neither nor-BNI nor naloxone alone had any effect on polymerized actin (left column). Data are expressed as a percentage of the parallel control value in three independent experiments. *, P < 0.05; **, P < 0.01 (significant difference between cells exposed to opioid agonists and cells exposed to opioid agonists plus antagonist).

 


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  		Figure 4. Confocal images of PC12 cells with rhodamine-phalloidin staining. A, Parallel controls; B, cells treated with 10-6 M U-69593 for 5 min. The {kappa}-opioid agonist increased the content of polymerized actin at the subplasmalemmal area. D, The changes at 30 min of exposure to 10-6 M U-69593. The filamentous actin content in the treated cells is indistinguishable from that in the parallel controls (C). The order of photographs represents succeeding sections starting from the upper cytoplasmic region and ending at the attachment site. Section thickness was adjusted to 0.5 µm. Scale bar, 10 µm.

 
The stimulatory effect of opioids was prevented by the specific {kappa}-opioid antagonist nor-BNI and the general opioid antagonist naloxone. Thus, although exposure of PC12 cells to U-69593 at 2 min increased the content of polymerized actin by 187 ± 38% (n = 6; P < 0.01), the addition of nor-BNI kept its level to 94 ± 14% of that in parallel controls (Fig. 3Go). Naloxone antagonized the stimulatory effects of DADLE and DAGO on actin polymerization. Thus, exposure to DADLE increased the concentration of polymerized actin by 194 ± 29% (n = 6; P < 0.01), whereas the presence of naloxone kept its level to 127 ± 39%. Similarly, exposure to DAGO increased the concentration of polymerized actin by 195 ± 17% (n = 7; P < 0.5), whereas the presence of naloxone kept its level to 129 ± 24%. It should be noted that neither nor-BNI nor naloxone had any effect of its own. Figure 3Go shows that 10-6 M nor-BNI at 2 min showed a polymerized actin content of 24 ± 8 U compared with 23 ± 7 U in parallel controls (n = 9, three independent experiments).

The suppressive effect of opioids on norepinephrine secretion appears to be synchronous to their stimulatory effect on actin filament content. Figure 5Go illustrates this synchronicity at 2 min of exposure to the {kappa}-opioid receptor agonist U-69593. Figure 5AGo depicts the suppressive effect of U-69593 at 10-6 M on norepinephrine secretion, whereas Fig. 5BGo depicts the effect of the same concentration of U-69593 on the total cellular content of actin filaments. Both effects were undetectable by 15 min of exposure.



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  		Figure 5. Synchronicity of the two effects of opioids. A, The peak (left column) and the end (right column) of the effect of U-69593 on norepinephrine secretion. B, The peak (left column) and the end (right column) of the effect of U-69593 on actin polymerization. Data are expressed as a percentage of the parallel control value in three independent experiments. Statistical significance is depicted as explained in Fig. 2Go.

 
Change in actin cytoskeleton in older PC12 cells confers resistance to both effects of opioids, i.e. on catecholamine secretion and actin disassembly
Figure 6CGo ({square}) depicts newly cultured PC12 cells, and Fig. 6CGo () depicts PC12 changes in morphology after a 3-week culture. The latter cells appear identical to those observed after exposure to NGF. These changes were accompanied by the development of resistance to both effects of opioids, i.e. on catecholamine secretion and actin polymerization. Figure 6AGo depicts the catecholamine response of differentiated and nondifferentiated PC12 cells to opioids. Both types of PC12 cells secrete dopamine as well as norepinephrine. Although nondifferentiated PC12 cells responded to opioids by suppressing their secretion of catecholamines (either norepinephrine or dopamine), the differentiated PC12 cells did not. For the shake of simplicity, Fig. 6AGo depicts norepinephrine data of nondifferentiated PC12 cells and dopamine data of differentiated cells. Figure 6AGo ({blacksquare}) depicts the lack of effect of opioid agonists on catecholamine secretion of older PC12 cells at 2 min. Figure 6BGo ({blacksquare}) depicts the effect of opioid agonists on actin polymerization at 2 min, i.e. U-69593 did not increase the concentration of actin filaments in long-cultured PC12 cells. It should be noted here that a significant decrease in actin filament content was observed instead, reaching 1489 ± 440 U/mg total cellular protein content (n = 6, two independent experiments; P < 0.001) compared with 3356 ± 1041 U in parallel controls. DADLE had a similar, but less pronounced, effect, i.e. a decrease to 1850 ± 784 U (n = 5, two experiments; P < 0.01) compared with 3356 ± 1041 U in the parallel controls. DAGO decreased the amount of polymerized actin to 653 ± 163 U (n = 3, one experiment) compared with 1474 ± 237 U in parallel controls. The different response of older PC12 cells to opioids vis-à-vis actin polymerization and catecholamine secretion should not be attributed to the loss of opioid receptors, as in a different set of experiments in our laboratory we found that long-term cultured PC12 cells exhibit an increase in the number of {kappa}-opioid-binding sites, whereas µ and {delta} remain the same (data not shown). It is thus possible that the cytoskeletal changes in the older cells make them resistant to the opioid effects at a postreceptor level.



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  		Figure 6. Morphological changes in long-term cultured cells. PC12 cells cultured for more than 3 weeks undergo profound changes in appearance, similar to those after exposure to NGF. C: {square}, Newly cultured PC12 cells forming dense floating cell accumulations; {blacksquare}, long-term cultured PC12 cells attaching individually to the bottom of the flask assuming a neuron-like phenotype. Changes in actin cytoskeleton in older PC12 cells confer resistance to both effects of opioids, i.e. on catecholamine secretion and on actin depolymerization A: {blacksquare}, Lack of effect of opioid agonists at 2 min on catecholamine secretion from PC12 cells cultured for more than 3 weeks. B: {blacksquare}, Lack of stimulatory effect at 2 min of opioids on actin polymerization. Data are expressed as a percentage of the parallel control value. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (significant difference compared with controls).

 
Opioids antagonize the effects of nicotine on norepinephrine secretion and on actin filaments
As expected, nicotine-stimulated norepinephrine secretion disassembled actin filaments. Opioids not only antagonized the stimulatory effect of nicotine on norepinephrine secretion, but also prevented its disassembling effect on actin filaments. Figure 7AGo depicts the time-response curve of norepinephrine secretion to nicotine at 10-6 M. The peak response was observed at approximately 20 min, at which point nicotine increased norepinephrine secretion to 52 ± 16 ng/mg total cellular protein (mean ± SE) compared with 30 ± 16 ng in parallel controls (n = 8, of three independent experiments; P < 0.05). Figure 7BGo shows that the presence of the {kappa}-opioid agonist U-69593 suppressed nicotine-induced norepinephrine secretion to 35 ± 13 ng/mg total cellular protein (right column) compared with 52 ± 16 ng nicotine alone (left column; n = 8, of three independent experiments; statistical comparison between nicotine and nicotine plus U-69593, P < 0.05). Figure 7CGo shows that at the same time nicotine reduced the concentration of actin filaments to 39 ± 5% of the parallel control value (left column; n = 8, three independent experiments; P < 0.001). Indeed, nicotine decreased polymerized actin to 30 ± 4 U/mg total cellular protein (mean ± SE) compared with 80 ± 22 U in parallel controls (n = 8, three independent experiments; P < 0.001). The addition of U-69593 prevented most of this suppressive effect of nicotine to only 82.8 ± 25.9% of the parallel control value (right column; n = 8, three independent experiments; statistical comparison between nicotine and nicotine plus U-69593, P < 0.05), i.e. the concentration of actin filaments after exposure to nicotine plus U-69593 was 64 ± 15 U compared with 30 ± 4 U after exposure to nicotine alone (statistical comparison between nicotine and nicotine plus U-69593, P < 0.05).



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  		Figure 7. Opioids antagonized both effects of nicotine: on norepinephrine secretion and on actin filament status. A, The time-response curve of norepinephrine to nicotine. B, The {kappa}-opioid agonist U-69593 suppressed (right column) nicotine-induced norepinephrine secretion (left column) at 20 min. C, Nicotine-induced norepinephrine secretion was associated with a simultaneous decrease in polymerized actin (left column), whereas the U-69593 block of nicotine-induced secretion was also associated with a simultaneous block of nicotine-mediated decrease in polymerized actin (right column). Statistical significance is depicted as explained in Fig. 1Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The association between actin filament disassembly and secretion is now well established. Indeed, transient depolymerization of actin filaments in the subplasmalemmal ring is followed by an increase in the secretory activity of a wide variety of cells, including norepinephrine-secreting SH-SY5Y neuroblastoma cells (28), guinea pig parotid acinar cells (29), renin-secreting kidney juxtaglomerular granular cells (30), insulin-secreting HIT-T15 pancreatic B cells (31), and several others. Chromaffin and PC12 rat pheochromocytoma cells also exhibit this association. Activation of nicotinic cholinergic receptors in chromaffin cells results in a transient disassembly of actin filaments in parallel to the amplified catecholamine secretion (32), whereas pertussis toxin, a highly selective inhibitor of G protein signaling, also reduces their content in polymerized actin (33). On the other hand, stimulation of the severing protein 14–3-3 or sciderin results in actin depolymerization and a parallel increase in catecholamine secretion from chromaffin cells (6, 34). These data, by showing a close association between catecholamine secretion and actin depolymerization, suggest that the secretory activity of chromaffin cells is coupled to actin filament disassembly. This hypothesis is strengthened by immunocytochemical data showing that the exocytosis sites of catecholamines in chromaffin cells are preferentially localized in areas of filamentous actin disassembly (7). Furthermore, in experiments using electron microscopy, it has been shown that nicotine stimulation of chromaffin cells is followed closely by the development of multiple polygonal areas in the subplasmalemmal actin ring devoid of filaments and containing catecholamine secretory granules (35). In short, the subplasmalemmal ring appears to be composed of filamentous actin and actin-binding proteins; it forms a dense viscoelastic gel that entraps catecholamine-containing secretory granules and prevents them from reaching their exocytosis sites. Upon stimulation, severing proteins are activated curving tunnels through which secretory granules pass. Our data, by showing a close association between filament depolymerization and basal or stimulated catecholamine secretion, are in agreement with this model and its application in PC12 cells (36, 37).

Opioids exert an almost universal suppressive effect on the secretion of classical neurotransmitters, neuropeptides, and hormones. They exert these effects on neurons, adrenal chromaffin, and other cells. This effect is opioid receptor mediated and usually paracrine in manner, i.e. the effector is produced in the vicinity of target cells. Opioid production by adrenal chromaffin cells is one of the oldest and best documented models of extracranial opioid production. Each type of chromaffin cell appears specialized to a single family of endogenous opioids. Thus, chromaffin cells located in the outer medullary zone, which synthesize the phenylethanolamine N-methyltransferase enzyme that converts noradrenaline to adrenaline and secrete mainly adrenaline, specialize in the production of proenkephalin-derived opioids, the principal endogenous {delta}-opioid receptor agonists. In addition, these cells have been shown to posses mainly {delta}-opioid receptors on their surface, suggesting an autocrine/paracrine effect of their opioids. It should be noted that these cells represent the most abundant type of chromaffin cell in the adrenal medulla. On the other hand, chromaffin cells located at the center of the medulla (inner zone chromaffin cells), which lack phenylethanolamine N-methyltransferase and thus secrete mainly noradrenaline, produce opioid products of the prodynorphin gene and posses {kappa}-opioid receptors on their surface. For reasons not yet understood, PC12 rat pheochromocytoma cells and perhaps most human pheochromocytomas exhibit inner zone chromaffin cell characteristics vis-à-vis in the type of catecholamine produced and secreted and in the opioid and opioid receptor profile (20, 22, 38). We have previously shown that opioids suppress chromaffin and PC12 cell basal and nicotine-induced catecholamine secretion in a fast, transient, and opioid antagonist-reversible manner (20). All of these characteristics are suggestive of actin filament involvement. It should be noted here that the opioid receptors are coupled to G proteins and adenylate cyclase. It is interesting that G proteins and their effectors have all been shown to affect the subplasmalemmal actin filament ring of chromaffin cells (12). Because of the above-mentioned characteristics of the opioid effect on adrenal chromaffin cells, we hypothesized that opioids may affect cortical actin filaments, which by their nature represent a fast response system regulating trafficking of synaptic and other secretory granules. Our data appear to confirm this hypothesis. More specifically, we found that opioids suppressed basal catecholamine secretion in a rapid, but transient, manner, and their effect peaked at 2–5 min and ended a few minutes later. The physiological significance of this effect is not clear. It is possible that locally produced opioids participate in paracrine regulatory mechanisms, providing momentary adaptations of basal catecholamine secretion that may be local as well as the classical systemic effects. Simultaneously, opioids stabilized actin filaments, suggesting that their inhibitory effect on catecholamine secretion may be due to this stabilizing effect, probably the result of an inhibitory effect on actin filament severing proteins. Indeed, these two events are so perfectly synchronous that it implies an almost certain cause and effect relationship. {kappa}-Opioid receptors appear to be the dominant mediators of both effects of opioids on PC12 cells. However, in normal adrenal chromaffin cells {delta}-opioids may be the principal regulator of actin filaments for reasons described above. The simultaneous presence of cytochalasin B prevents the suppressive effect of opioids on basal catecholamine secretion. It should be noted that cytochalasin, by disrupting actin filaments, potentiates the effects of several inducers of catecholamine secretion from chromaffin cells (13). This is understandable if we accept that the induction of catecholamine secretion involves disruption of the integrity of the subplasmalemmal actin ring. Indeed, this is not limited only to catecholamine secretion. For example, cytochalasin abolishes the augmented secretion of surfactant from bronchial epithelium cells after ß-adrenergic stimulation (39). In our experiments cytochalasin blocked the inhibitory effect of opioids on basal norepinephrine release from PC12 cells. This highly reproducible effect of cytochalasin suggests that the effect of opioids involves polymerized actin. Indeed, this is the first time that the effect of opioids on secretion has been associated with alterations in actin filament dynamics. Opioid-induced suppression of actin filament disassembly may be viewed as an inhibitory effect on a spontaneous and constant low level disruption of actin filaments responsible for basal catecholamine secretion. To our knowledge, this is the first report documenting an association between basal catecholamine secretion and actin filament dynamics.

The effects of opioids on actin polymerization and norepinephrine secretion under basal conditions were synchronous and fast, both peaking at 2 min. Similarly, opioids suppressed equally quickly (within 2 min of exposure) the nicotine-induced noradrenaline secretion and actin depolymerization, which, however, peaked at 20 min. It thus appears that the effect of opioids on the subplasmalemal actin ring is a fast response system, controlling both basal and stimulated trafficking of secretory granules. Long-term culture of PC12 cells results in changes in their morphology similar to those observed after NGF treatment (40). {kappa}-Opioids did not have any effect on catecholamine secretion while decreasing instead of increasing the concentration of actin filaments. These results may be explained by either the profound changes in cytoskeleton in the neuron-like PC12 cells or by changes in low threshold calcium currents, which sustain a higher level of severing protein activation (41). It has been proposed that the opioid-mediated inhibition of nicotine-induced catecholamine secretion in cultured chromaffin cells may not involve opioid receptors, as naloxone could not prevent it (42, 43). It should be noted, however, that these data were obtained using high concentrations of opioids. In our experiments opioids were effective in suppressing catecholamine release at physiological doses, i.e. between 109–106 M, whereas the general opioid antagonist naloxone and the specific {kappa}-opioid antagonist nor-BNI were able to completely reverse the effects of opioids on both basal and nicotine-induced catecholamine secretion and on actin reorganization. Nicotinic stimulation of chromaffin cells results in transient disassembly of cortical actin filaments (32), an effect synchronous to their induction of catecholamine secretion (7). Opioids antagonized the stimulatory effect of nicotine on norepinephrine secretion and simultaneously prevented nicotine-mediated disassembly of actin filaments. The inhibitory effect of U-69593 on nicotine-induced depolymerization of actin probably represents two independent and antagonizing effects of the two drugs on actin polymerization, the net effect of which results in attenuation of the nicotine effect of catecholamine secretion.

In conclusion, our data suggest that the suppressive effect of opioids on basal and nicotine-induced catecholamine secretion involves a simultaneous inhibitory effect of opioids on cortical actin depolymerization, i.e. opioids stabilize actin filaments. We also hypothesize that basal catecholamine secretion is associated with a constant and spontaneous low level severing of subplasmalemmal actin. Opioids suppress basal catecholamine release by inhibiting this spontaneous actin depolymerization, probably via an inhibitory effect on severing proteins. The inhibitory effect of opioids on nicotine-induced catecholamine secretion appears to be due to a parallel inhibitory effect of opioids on nicotine-induced actin filament disruption. Changes in actin cytoskeleton in older PC12 cells confer resistance to both effects of opioids, i.e. on catecholamine secretion and on actin depolymerization.


   Acknowledgments
 
We thank Dr. C. Tsatsanis for his constructive remarks.


   Footnotes
 
1 This work was supported by Medicon Hellas Co. (Gerakas, Athens, Greece) and the Greek Ministry of Health [KESY grants to A.N.M. (335/1997) and to C.S. (119/1998)]. Back

2 Present address: Division of Endocrinology, Department of Pediatrics, Children’s Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115. Back

Received October 30, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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