Phosphodiesterase 4 inhibitors, rolipram and diazepam block the adaptive changes observed during morphine withdrawal in the heart

Abstract

In this study, we investigated whether morphine dependence was inhibited by phosphodiesterase (PDE) 4 inhibitors rolipram and diazepam, since a role for the cyclic AMP systems in the development of morphine dependence was reported. Dependence of morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone. In order to determine the effect of rolipram or diazepam the animals were injected with these drugs for seven days and 30 min before the administration of naloxone. When opioid withdrawal was precipitated, enhancement of noradrenaline (NA) turnover in the heart was observed 30 min after naloxone administration. Moreover, morphine withdrawal induces Fos expression, increase in cyclic AMP and cyclic GMP levels. Co-administration of rolipram or diazepam with morphine during the pre-treatment period significantly reduces the signs of withdrawal symptoms, the enhancement of NA turnover, the increase in cyclic AMP and the Fos expression. However, these inhibitors did not modify the levels of cyclic GMP. These findings demonstrated that co-administration of rolipram or diazepam with morphine abolish the development of morphine dependence and suggest that these compounds prevent the up-regulation of the cyclic AMP pathway and the associated increase in cyclic AMP level after naloxone administration.

Introduction

The clinical use of morphine is limited in practice by its tendency to cause tolerance and dependence with prolonged or repeated administration. The development of dependence and tolerance has been postulated to be the physiological response that compensates for chronic activation of opioid receptor. However, the exact mechanism underlying these physiological changes has not been elucidated.

Accumulating evidence has shown that the cellular and molecular adaptation following long-term opioid exposure results from the phosphorylation of opioid receptor protein, its coupled G proteins, and several related effector proteins. The enzymes producing these changes include second messengers such as cyclic AMP-dependent protein kinase A (PKA) or cyclic GMP-dependent protein kinase G (PKG) which play important roles in the regulation of opioid signal transduction (Burton et al., 1991, Liu and Anand, 2001, Wegener and Kunmer, 1994). Although the μ opioid receptor is negatively coupled to the adenylate cyclase/cAMP-dependent PKA pathway upon acute stimulation (Childers, 1991), the pathway is up-regulated in several brain areas with chronic morphine treatment (Nestler, 1992, Tokuyama et al., 1995). In addition, previous studies in our laboratory have demonstrated that withdrawal from morphine is associated with a marked increase in the ventricular levels of cyclic AMP in parallel with enhancement of noradrenaline (NA) turnover, suggesting that the up-regulation of the cyclic AMP pathway plays an important role in the development of morphine dependence at heart level (Milanés et al., 2000).

Several intracellular messengers, including transcription factors, are modified by chronic treatment with opiates and other drugs of abuse (Blendy and Maldonado, 1998, Benavides et al., 2003). The transcription factor Fos has been shown to be altered in several brain areas following morphine withdrawal (Curran et al., 1996, Laorden et al., 2002a, Laorden et al., 2002b) and has been widely used as an indicator of cellular activity (Morgan and Curran, 1991). In addition, previous studies from our laboratory have shown that naloxone administration to rats treated chronically with morphine enhances Fos expression in different cardiac tissues (González-Cuello et al., 2003, González-Cuello et al., 2004b). It was also proved treatment with phosphodiesterase inhibitors, 3-isobutyl-l-methylxanthine (IBMX), rolipram, nefiracetam and enprofyline in combination with morphine attenuates the development of morphine dependence in the central nervous system (Itoh et al., 1998, Itoh et al., 2000, Wang et al., 1999, Hamdy et al., 2001, Mamiya et al., 2001). However, there are not studies about the role of PDE4 inhibitors rolipram (Beavo, 1995) and diazepam (Collado et al., 1998) to suppress the processes of the development of morphine dependence at heart levels. Diazepam has been described like a selective PDE4 inhibitor in cardiac tissue and its cardiac effects seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors (Marín and Hernández, 2002, Juan-Fita et al., 2003, Juan-Fita et al., 2006).

One of the main PDE isoenzymes that hydrolyses cyclic AMP in the rat heart is PDE4 (Nicholson et al., 1991, Verde et al., 1999). We therefore investigated if rolipram and diazepam, which act on the cyclic AMP system, can inhibit the enhancement of noradrenaline turnover, cyclic AMP, cyclic GMP and Fos expression observed during morphine withdrawal in the heart.