Alterations in protein kinase A and different protein kinase C isoforms in the heart during morphine withdrawal

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Abstract

The present study was designed to investigate the possible changes of protein kinase A (PKA) and different isoforms of protein kinase C (PKC): PKC α, PKC δ and PKC ζ after naloxone induced morphine withdrawal in the heart. Male rats were implanted with placebo (naïve) or morphine (tolerant/dependent) pellets for 7 days. On day 8 rats received saline s.c. or naloxone (5 mg/kg s.c.). The protein levels of PKA, PKC δ and PKC ζ were significantly up-regulated in the heart from morphine withdrawal rats. By contrast, morphine withdrawal induced down-regulation of PKC α. These results suggest that both PKA and PKC may be involved in the cardiac adaptive changes observed during morphine withdrawal.

Introduction

Protein kinase A (PKA) and protein kinase C (PKC) play a central role in transducing signal transduction and potentiating intracellular cross talk by phosphorylating diverse substrates, including cell-surface receptors, enzymes and transcription factors. Both protein kinases are expressed in cardiac muscle (for review see Keef et al., 2001). The PKC family is comprised of at least 12 different forms (Hayashi et al., 1999). Based on their structures and ability to bind with the cofactors, these isozymes have been grouped into three subclasses: i) the conventional PKC comprises α, βI, βII and γ isozymes that are activated by Ca2+, diacylglycerol (DAG) and phosphatidylserina (PS); ii) the novel PKC isozymes consisting of δ, ɛ, η and ϴ isozymes that do not respond to Ca2+, but are activated by PS and DAG; iii) the atypical isozymes consisting of ζ and τ/λ that are unresponsive to Ca2+, DAG and phorbol esters, but are also activated by PS. Mammalian hearts have been found to coexpress a number of PKC isozymes, including α, βI, βII, δ, ɛ, and ζ (Naruse and King, 2000).

Accumulating evidence has shown that the cellular and molecular adaptation following long-term opioid exposure results from the phosphorylation of opioid receptor protein, their coupled G proteins, and several related effector proteins. The enzymes producing these changes include second messenger-dependent protein kinases (PKC), cyclic AMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CAMKII), G protein-coupled receptor kinases and mitogen-activated protein kinases (MAPKs), which play important roles in the regulation of opioid signal transduction (for review see Liu and Anand, 2001). Alterations in both PKA and PKC pathways have been suggested as one of the molecular mechanism of opioid tolerance and dependence (Nestler and Aghajanian, 1997). Although the μ opioid receptor is negatively coupled to the adenylate cyclase/cAMP-dependent PKA pathway upon acute stimulation (Childers, 1991), both the PKA and PKC pathways are up-regulated in several brain areas with chronic morphine treatment (Nestler, 1992, Tokuyama et al., 1995). Previous studies in our laboratory have demonstrated that naloxone administration to morphine-dependent rats leads to an enhancement of cAMP levels in the heart (Milanés et al., 2000). In addition, chronic inhibition of PKA with the selective PKA inhibitor, HA-1004 significantly blocks the enhancement of noradrenaline turnover during morphine withdrawal in both the right and left ventricle (Martínez et al., 2003). Studies have also focused on the role of PKC in tolerance and dependence. Acute administration of PKC inhibitors was not able to reverse morphine dependence (Smith et al., 2002). In addition, the chronic inhibition of PKC with calphostin C did not modify the increase in the noradrenaline turnover observed during morphine withdrawal in the heart (Martínez et al., 2003). In contrast, it has been shown that PKC inhibitors prevent the development of opioid physical dependence (Fundytus and Coderre, 1996).

Although the involvement of PKA and PKC pathways in the morphine dependence have been reported, not enough data are available on the characteristic and functional disturbances of the heart protein kinases after chronic morphine treatment and upon drug withdrawal. Therefore, the purpose of the present study was to determine the possible changes in the expression of PKA and PKC isozymes (α, δ, and ζ) after naloxone precipitated morphine withdrawal in the heart.

Section snippets

Material and methods

Male Sprague–Dawley rats (220–240 g at the start of the experiments) were housed four to five per cage under a 12 h light/dark cycle (L:8:00–20:00 h) in a room with controlled temperature (22 ± 2 °C) and humidity (50 ± 10%) and food and water available ad libitum. Animals were pre-handled for several days preceding the experiment to minimize stress, as previously described (Laorden et al., 2000). All surgical and experimental procedures were performed in accordance with the European Communities

Results

Rats treated with morphine showed a significantly lower (P < 0.01; t-test) body weight gain (22.37 ± 2.21 g, n = 68) than animals receiving placebo pellets (52.08 ± 3.14 g, n = 72). Administration of naloxone to control rats resulted in no significant changes in body weight when measured 30 (5.03 ± 0.93 g, n = 16), 60 (4.36 ± 0.60, n = 16) or 90 (2.38 ± 0.55 g, n = 18) min after drug injection, as compared to control rats receiving saline (1.66 ± 0.36 g, n = 13; 4.32 ± 0.61 g, n = 17; 3.69 ± 1.60, respectively; t-test).

Discussion

In the present investigation, we have quantified the immunoreactivity of PKA-Cα subunit, the major catalytic isoform of PKA in most mammalian tissues (Uhler et al., 1986) and different isoforms of PKC (the conventional PKCα, the novel PKCδ and the atypical PKCζ) during morphine withdrawal.

It is known that chronic morphine alters the levels and/or activity of various mu-opioid receptor signalling elements. These chronic adaptive molecular mechanisms involve some protein kinases which are

Acknowledgements

This work was supported by Ministerio de Ciencia y Tecnología (SAF/FEDER 2002-00763, 2003-00756) and Ministerio del Interior, Madrid Spain.

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