Activities of cAMP-dependent protein kinase and protein kinase C are modulated by desensitized nicotinic receptors in the rat brain

Neuroscience Letters 367 (2004) 19–22

Activities of cAMP-dependent protein kinase and protein kinase C are modulated by desensitized nicotinic receptors in the rat brain Xiulan Sun a,b , Yue Liu a,c , Gang Hu b , Hai Wang a,∗ a

Beijing Institute of Pharmcology and Toxicology, Beijing 100850, PR China b Nanjing Medical University, Nanjing 210029, PR China c Thadweik Academy of Medicine, Beijing 100850, PR China

Received 3 December 2003; received in revised form 19 April 2004; accepted 18 May 2004

Abstract When rats were treated with different dosages of nicotine, nicotinic acetylcholine receptors (nAChRs) were observed in activated, sub-acute desensitized, acute desensitized, and chronic desensitized states. The activities of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) were assayed in the respective rat brains. The results showed that the activities of PKA and PKC could not be modified when brain nAChRs were in an activated state. However, the activities of PKA and PKC decreased when brain nAChRs were in a sub-acute state, an acute state or a chronic desensitized state induced by repeated administration of nicotine. These results suggest that desensitized nAChRs in the rat brain can inhibit the activities of PKA and PKC, which may be responsible for nicotine dependence. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Nicotine; Nicotinic acetylcholine receptor; Desensitization; cAMP-dependent protein kinase; Protein kinase C

Nicotine, as the major addictive component of tobacco, produces effects that are commonly seen with other addictive drugs such as opioids and cocaine. The biological basis of smoking habits is known to be due to nicotine dependence. Nicotine is a highly selective ligand for nicotinic acetylcholine receptors (nAChRs), binds readily to nAChRs and exerts its function by acting on nAChRs. However, repeated or chronic exposure to nicotine induces nAChR desensitization, which results in a decrease or loss of nicotine-like responses to agonists. Desensitization is a general characteristic of ligand-gated ion channels, such as nAChRs, GABA and 5-hydroxytryptamine (5-HT) receptors [8]. Studies from our lab have shown that nAChR desensitization is not a non-functional state. We proposed that desensitized nAChRs could increase the sensitivity of brain muscarinic receptors to agonists, promote the coupling relationship between muscarinic receptors and adenylate cyclase (AC), and stimulate phosphatidylinositol turnover [21]. Brain mitogen-activated protein kinase (MAPK), phosphatidylinositol, and epidermal growth factor receptor (EGFR) signaling pathways were ∗ Corresponding author. Tel.: +86 10 6693 2651; fax: +86 10 6821 1656. E-mail address: [email protected] (H. Wang).

also identified and proposed as possible targets in response to chronic nicotine treatment [9]. Thus, it is reasonable to suggest that desensitized nAChRs can modulate the signal transduction system in the brain. Accumulating evidence shows that cellular and molecular adaptation, following long-term opioid exposure, results from the phosphorylation of opioid receptor proteins, their coupled G protein, and several related effector proteins. The enzymes producing these changes include the second messenger-dependent protein kinases, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and Ca2+ /camodulin-dependent protein kinase (CaMK), which play important roles in the regulation of opioid signal transduction [11,13]. nAChRs are not G protein-coupled receptors, their related effectors are cations since they belong to the ligand-gated ion channel receptor family. Whether nAChR desensitization induced by nicotine modulates the function of second messengers remains unclear. PKA and PKC are the most important signal messengers, and nAChRs are recognized to have phosphorylation sites for at least three protein kinases, PKA, PKC and tyrosine protein kinase [5,6,25]. Most previous studies have focused on the regulatory effects of PKA and PKC on nAChRs. For example, PKC and PKA can phosphorylate several amino

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.05.072

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X. Sun et al. / Neuroscience Letters 367 (2004) 19–22

acid sites of nAChRs, and modulate the onset and recovery of nAChR desensitization [13,16,19]. However, no reports exist on the modulation of PKA and PKC activities by desensitized nAChRs in the brain. Male Sprague–Dawley rats, weighing 180–200 g, obtained from the laboratory animal center of Beijing Institute of Pharmacology and Toxicology, were used in this study. The animals were housed individually in a controlled environment with a 12 h/12 h light/dark cycle and received food and water ad libitum. The study was conducted in accord with the principles and procedures of the NIH Guide for the Care and Use of Laboratory Animals. The rats were randomly divided into four groups: in the first group, nicotine 1.0 mg/kg was intravenously administered and rats were decapitated within 3 min; in the second group, nicotine 1.0 mg/kg was intravenously administered and rats were decapitated after 10 min; in the third group, nicotine at 0.75 and 2.0 mg/kg was intravenously administered at 10-min intervals [24,27] and rats were decapitated after the final 10-min interval; in the fourth group, the rats were treated with nicotine for 14 days at doses of 2.4 mg/kg/day via two daily subcutaneous injections [17], then decapitated. The activities of PKA and PKC were then determined for the brain tissue of each group. Brain tissue of 1 g was homogenized in 5 ml of cold extraction buffer (25 mM Tris–HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM ␤-mercaptoethanol, 1 ␮g/ml leupeptin, and 1 ␮g/ml aprotinin) with a cold homogenizer. The lysate was then centrifuged for 5 min at 14,000 × g and 4 ◦ C in a microcentrifuge. The supernatant was collected for determining PKC and PKA activities. The extracts were assayed on the same day they were prepared to retain maximal activity and obtain optimal results. The most common method for assaying PKC or PKA activity involves measuring the transfer of 32 P-labeled phosphate to a protein or peptide substrate that could be captured on phosphocellulose filters via weak electrostatic interactions. However, in the presence of multiple kinases, the 32 P-labeled peptides/proteins bound to the phosphocellulose filter might reflect kinase activity other than that due to PKC or PKA. To increase the specificity of the PKC or PKA assay, Promega’s Signa TECT® PKC or PKA assay system was used. This systems employs the biotinylated peptides neurogranin or kemptide, which are specific for PKC or PKA, respectively, providing sufficient sensitivity to detect the enzyme at levels typically found within most biological samples. PKA and PKC activities were assayed by transferring phosphorus (32 P) into the two specific substrates. The biotinylated 32 P-labeled substrate was recovered from the reaction mix with the SAM2® Biotin Capture Membrane, which is a novel streptavidin matrix. After washing and drying, the samples on the SAM2® membrane were cut out and placed into individual scintillation vials. Finally, scintillation fluid was added to the vials and samples were analyzed by scintillation counting.

The kinase activities of PKA were carried out in duplicate in a 20 ␮l reaction consisting of 200 mM Tris–HCl, pH 7.4, 100 mM MgCl2 , 0.5 mg/ml BSA, 0.025 mM cAMP, PKA biotinylated peptide substrate and ␥-32 P-ATP mix. Control reactions without substrate were also performed to determine background counts. The kinase activities of PKC were determined in a reaction consisting of 1.25 mM EGTA, 2 mM CaCl2 , 0.5 mg/ml BSA, PKC biotinylated peptide substrate, ␥-32 P-ATP mix. The activities were determined in the presence and absence of 1.6 mg/ml phosphatidylserine and 0.16 mg/ml diacylglycerol. The reactions were initiated by adding the enzyme samples and incubated at 30 ◦ C for 5 min. Reactions were terminated by adding termination buffer (7.5 M guanidine hydrochloride). Enzyme activities were determined as picomole 32 P transferred from ␥-32 P-ATP to the peptide substrate in 1 min at 30 ◦ C. Brain PKA activities were determined after the rats were given nicotine. The basal line of PKA activity was calculated as 0.662 ± 0.207 pmol/min/mg protein. PKA activities were not influenced in the first group, 3 min after nicotine administration, but decreased to 0.50 ± 0.07 pmol/min/mg protein in the second group, 10 min after nicotine was given at the single intravenous dose of 1.0 mg/kg. Interestingly, PKA activities also decreased in the third and fourth groups, when rats were treated with repeated administration of nicotine, including 10 min after intravenous nicotine injections at doses of 0.75 and 2.0 mg/kg at 10-min intervals (0.40 ± 0.06 pmol/min/mg protein), as well as after long-term nicotine treatment at the dose of 2.4 mg/kg/day, s.c., b.i.d., for 14 days (0.521 ± 0.165 pmol/min/mg protein; Fig. 1A–D). Under the same experimental conditions, PKC activities were also determined in the above brain preparations. The basal level of PKC activity was calculated as 0.200 ± 0.083 pmol/min/mg protein, and similar trends in protein kinase activities were observed in the four groups of rats. PKC activities were not influenced 3 min after nicotine administration, but decreased 10 min after nicotine was given at a single intravenous dose of 1.0 mg/kg, and when nicotine was repeatedly given at intravenous doses of 0.75 and 2.0 mg/kg at 10-min intervals, as well as long-term treatment of 2.4 mg/kg/day, s.c., b.i.d., for 14 days. The activities were 0.09 ± 0.01 pmol/min/mg protein, 0.07 ± 0.009 pmol/min/mg protein, and 0.07 ± 0.02 pmol/min/mg protein, respectively (Fig. 1E–H). nAChRs exist in four different states: resting, activated, desensitized, and inactivated. Upon binding an agonist, nAChRs briefly enter the open conformation of an ion channel, which provides a fluid-filled pathway through the membrane inducing an influx of cations such as K+ , Na+ , and Ca2+ . As a member of the ligand-gated ion channel family, nAChRs can be desensitized by repeated or chronic exposure to nicotine. The physiological significance of nAChR desensitization has been investigated since the 1950s. Under physiological conditions, concentrations of acetylcholine (ACh) (1 mM) evoked by nervous pulse cause synchronized activation of nearby nAChRs with little or no desensitiza-

(A) activation

(B) sub-acute desensitization

PKA (pmol/min/mg)

PKA (pmol/min/mg)

X. Sun et al. / Neuroscience Letters 367 (2004) 19–22

1.2 1 0.8 0.6 0.4 0.2 0

**

0.2

0.4 0.2 0

1.2 1 0.8 0.6 0.4 0.2 0

**

(E) activation

(F) sub-acute desensitization PKC (pmol/min/mg)

0

PKA (pmol/min/mg)

0.8 0.4

*

(D) chronic desensitization

1 0.6

0.6

PKC (pmol/min/mg)

PKA (pmol/min/mg)

(C) acute desensitization

1 0.8

0.3 0.2 0.1

0.2 0.15 0.1

(H) chronic desensitization PKC (pmol/min/mg)

0

(G) acute desensitization 0.15 0.12 0.09 0.06 0.03 0

**

*

0.05

PKC (pmol/min/mg)

0

0.25

0.15 0.12 0.09

*

0.06 0.03 0

Fig. 1. Brain PKA (A–D) and PKC (E–H) activities in the absence (䊐) or in the presence (䊏) of nicotine pretreatment. Activation of nAChRs were induced by intravenous administration of nicotine at the dose of 1.0 mg/kg for 3 min; sub-acute desensitization of nAChRs were induced by nicotine 1.0 mg/kg, i.v., for 10 min; acute desensitization were induced by repeated administration of nicotine (0.75 and 2.0 mg/kg, i.v., at 10-min interval); chronic desensitization of nAChRs were induced by repeated administration of nicotine at doses of 2.4 mg/kg/day for 14 days. Values were expressed as mean ± S.D., n = 8, ∗ P < 0.5, ∗∗ P < 0.01, vs. control groups.

tion. When smoking, nicotine obtained from tobacco arrives much more slowly at concentrations of 50–600 nM, which is much lower than physiological concentrations of ACh. Furthermore, nicotine is present for much longer, in part, because nicotine is not degraded by acetylcholinesterase. This longer exposure to a low concentration of nicotine favors nAChR desensitization, after a transient activation [3].

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In fact, slow treatment with a low concentration of nicotine can cause some nAChR desensitization without activation because the desensitized conformation of the nAChRs has a higher affinity for agonists than the resting or open conformation [1,4]. Therefore, desensitized nAChRs in the brain are especially responsible for nicotine addiction [2,3]. The behavior manifestations of brain nAChRs in different functional states were investigated in our lab using nAChR-mediated rat EEG seizure discharges: 1.0 mg/kg nicotine could activate nAChRs lasting 30 s to 5 min [24,27]; nAChRs began to enter sub-acute desensitization 5 min after nicotine treatment, and acute desensitization to nicotine resulted after two intravenous doses of nicotine at 0.75 and 2.0 mg/kg at 10-min intervals [27]. When rats were treated with nicotine at doses of 2.4 mg/kg/day for 14 days via two daily subcutaneous injections, nAChRs entered a chronic desensitized state and the number of receptors was up-regulated [17]. In the present study, diverse effects of nicotine treatment on brain PKC and PKA activities were observed. The results show that the activities of PKA and PKC were not influenced by activated nAChRs. However, when nAChRs were desensitized, PKA and PKC activities were inhibited, indicating that desensitized nAChRs could modulate the functions of PKA and PKC. When nAChRs were in sub-acute or acute desensitized states, the activities of PKA or PKC decreased, similar to acute treatment with morphine or heroin. Acute exposure to morphine or heroin could inhibit AC by inducing Gi proteins to couple with opium receptors, decreasing cyclic AMP (cAMP) levels, which are responsible for lower PKA activity [12,26]. Compared to the resting state, chronic desensitized nAChRs also induce decreases in the activities of PKA and PKC, which is opposite to the effect of chronic morphine administration. Chronic treatment with morphine could increase the activity of AC, and up-regulate the AC/cAMP signal transduction system, promoting a cAMP-dependent phosphorylation process [14]. The results of this study indicate that chronic nicotine dependence is different from morphine dependence. Nicotine addiction is a mentally dependent process, which is more subject to biochemical changes such as neurotransmitter releases and enzyme activities. Nicotine addiction could also be viewed as a form of neuroadaptation to nicotine, or nicotine-related brain disorders. The present study shows that such disturbances also reflect neuroadaptive changes in signal transduction function. However, the mechanism by which desensitized nAChR modulates protein kinase activities remains unclear. The data from our lab, and others, shows that nAChR desensitization only displays a decrease or loss of response to agonists and is not a non-functional state. For example, desensitized nAChRs can increase the activity of muscarinic receptors [20,22,23] and have a neuroprotective modulatory effect on cellular model systems induced by various types of injury [7,10,15,18]. Thereby, it is reasonable to assume that desensitized nAChRs may modulate PKA and PKC activities by affecting the function of other receptors.

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Interestingly, the present study demonstrates that there are similar trends in the changes in activity of PKC and PKA, suggesting a possible interactive relationship between the PKC-dependent signal transduction pathway and the PKA-dependent signal transduction pathway. It has been shown that receptor-mediated activation of both adenylate cyclase and the phosphatidylinositide hydrolysis system occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either PKA or Ca2+ /phospholipid-dependent PKC. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signaling systems could occur [26]. These results indicate that there might be cross-talk between PKA and PKC. In conclusion, the activities of PKA and PKC were not influenced by activated nAChRs, but their activities were inhibited by desensitized nAChRs induced by nicotine administration. The present study suggests that nicotine dependence could also modulate the function of PKA- and PKC-related signal transduction systems. Further study is needed to identify the detailed mechanism by which nicotine causes this effect, to determine whether the modulation by desensitized nAChRs is direct or indirect in vitro, and to demonstrate the subtype of nAChRs involved in this effect.

Acknowledgements This work was supported by a grant from the National Natural Science Foundation of China (No. 30371641).

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