Activation of the ERK signalling pathway contributes to the adaptive changes in rat hearts during naloxone-induced morphine withdrawal

Abstract

Background and purpose: We have previously demonstrated that morphine withdrawal induced hyperactivity of the heart by activation of noradrenergic pathways innervating the left and right ventricle, as evaluated by noradrenaline turnover and c-Fos expression. The extracellular signal-regulated kinase (ERK) has been implicated in drug addiction, but its role in activation of the heart during morphine dependence remains poorly understood. Here, we have looked for activation of ERK during morphine withdrawal and if this activation induced gene expression.

Experimental approach: Dependence on morphine was induced by s.c. implantation of morphine pellets for 7 days. Morphine withdrawal was precipitated on day 8 by injection of naloxone (2 mg kg(-1), s.c.). ERK1/2, their phosphorylated forms and c-Fos were measured by western blotting and immunohistochemistry of cardiac tissue.

Key results: Naloxone-induced morphine withdrawal activated ERK1/2 and increased c-Fos expression in cardiac tissues. c-Fos expression was blocked by SL327, a drug that prevents ERK activation.

Conclusions and implications: These results indicate that signalling through the ERKs is necessary for morphine withdrawal-induced hyperactivity of the heart and suggest that this pathway may also be involved in activation of immediate-early genes in both cytosolic and nuclear effector mechanisms that have the potential to bring about long-term changes in the heart.

Figure 1

Effect of morphine and morphine withdrawal on the body weight of rats. The body weight was recorded on the days of pellet implantation and on the day of killing (day 8), before receiving any injection (a), 60 (b) and 90 min (c) after saline or naloxone administration. Data represent means±s.e.m, n=20–25. ^###P<0.001 versus placebo; ^***P<0.001 versus placebo+naloxone; ⁺⁺⁺P<0.001 versus morphine+saline.

Figure 2

Morphine withdrawal stimulates ERK1/2 phosphorylation in the right ventricle. Representative immunoblots of phospho (p)ERK1 and pERK2 in ventricular tissue isolated from placebo or morphine-dependent rats after subcutaneous administration of saline or naloxone at the time indicated. Actin was used as an internal loading control. For quantification, optical densities of pERK1 and pERK2 immunoreactive bands were measured, normalised to the background values, and expressed as percentages of controls. Data represent means±s.e.m. (n=3–4). ⁺P<0.05; ⁺⁺P<0.01 versus morphine+saline. ^*P<0.05, ^**P<0.01 versus placebo+naloxone. ERK, extracellular signal-regulated kinase; m, morphine; nx, naloxone; P, placebo; s, saline .

Figure 3

Morphine withdrawal stimulates ERK1/2 phosphorylation in the left ventricle. Representative immunoblots of phospho (p)ERK1 and pERK2 in the left ventricle tissue isolated from placebo- or morphine-dependent rats after subcutaneous administration of saline or naloxone at the time indicated. Actin was used as an internal loading control. For quantification, optical densities of pERK1 and pERK2 immunoreactive bands were measured, normalised to the background values, and expressed as percentages of controls. Data represent means±s.e.m. (n=4–6). ⁺P<0.05; ⁺⁺P<0.01, ⁺⁺⁺P<0.001 versus morphine+saline. ^**P<0.01, ^***P<0.001 versus placebo+naloxone. ERK, extracellular signal-regulated kinase; m, morphine; nx, naloxone; P, placebo; s, saline.

Figure 4

Time course of total ERK during morphine withdrawal. Representative immunoblots of total ERK in the right and left ventricle isolated from placebo or morphine-dependent rats after subcutaneous administration of saline or naloxone at the time indicated. Actin was used as an internal loading control. For quantification, optical densities of total ERK immunoreactive bands were measured, normalised to the background values, and expressed as percentages of controls. Data represent means±s.e.m. (n=4). ERK, extracellular signal-regulated kinase; m, morphine; nx, naloxone; P, placebo; s, saline.

Figure 5

Morphine withdrawal activates ERK1/2 in the right and left ventricle. Rats were made dependent on morphine for 7 days and, on day 8, were injected saline or naloxone (subcutaneous). Controls received placebo pellets at the same time schedule and on day 8 were given with saline or naloxone. At 90 min after injections, rats were perfused and the right and left ventricle was processed for phospho (p)ERK1/2 immunohistochemistry. Photographs show the immunohistochemical detection of pERK1/2 in the left ventricular wall (a, b, c). These results are representative of four independent experiments. Normaski interference optics. Scale bar 30 μm (a), 20 μm (b, c). ERK, extracellular signal-regulated kinase.

Figure 6

Morphine withdrawal stimulates c-Fos expression in the right and left ventricle. (a) Representative immunoblots of c-Fos in samples isolated from placebo- or morphine-dependent rats 90 min after subcutaneous administration of saline or naloxone. Actin was used as an internal loading control. For quantification, optical densities of c-Fos immunoreactive bands were measured, normalised to the background values, and expressed as percentages of controls. Data represent means±s.e.m. (n=4–6). ⁺⁺⁺P<0.001 versus morphine+saline.^***P<0.001 versus placebo+naloxone. (b) Represents photomicrographs of c-Fos immunoreactvity in the right and left ventricular wall and in the septum, after naloxone-precipitated withdrawal. Results are representative of four independent experiments. Normaski interference optics. Scale bar 58 μm. m, morphine; nx, naloxone; P, placebo; s, saline; v, vehicle (DMSO).

Figure 7

Immunoblots of ERK1/2 in right ventricles isolated from placebo- or morphine-dependent rats after subcutaneous administration of saline or naloxone in absence or presence of SL327 (100 mg kg⁻¹), 1 h before saline or naloxone injection. Actin was used as an internal loading control. Phospho (p)ERK1 and pERK2 immunoreactive bands were measured, normalised to the background values, and expressed as percentages of controls. Data correspond to mean±s.e.m. (n=4). ^*P<0.05, ^**P<0.01 versus its control group. ERK, extracellular signal-regulated kinase; m, morphine; nx, naloxone; p, placebo; s, saline; ,sl, SL327; v, vehicle (DMSO).

Figure 8

Morphine withdrawal stimulates c-Fos expression in the right and left ventricle. Representative immunoblots of c-Fos in the right and in the left ventricle tissue isolated from placebo- or morphine-dependent rats, 90 min after subcutaneous administration of naloxone in the absence or presence of SL327 (100 mg kg⁻¹) 1 h before naloxone. Actin was used as an internal loading control. c-Fos immunoreactive bands were measured, normalised to the background values and expressed as percentages of controls. Data correspond to mean±s.e.m. (n=4). ⁺⁺P<0.01, ⁺⁺⁺P<0.001 versus morphine+SL+saline; ^***P<0.001 versus placebo+veh +naloxone. m, morphine; nx, naloxone; p, placebo; s, saline; ,sl, SL327; v, vehicle (DMSO).