The PKs PKA and ERK 1/2 are involved in phosphorylation of TH at Serine 40 and 31 during morphine withdrawal in rat hearts

Abstract

Background and purpose: Our previous studies have shown that morphine withdrawal induced hyperactivity of cardiac noradrenergic pathways. The purpose of the present study was to evaluate the effects of morphine withdrawal on site-specific phosphorylation of TH in the heart.

Experimental approach: Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets in rats. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg kg(-1)). TH phosphorylation was determined by quantitative blot immunolabelling using phosphorylation state-specific antibodies.

Key results: Naloxone-induced morphine withdrawal induced phosphorylation of TH at serine (Ser)40 and Ser31 in the right ventricle, associated with both an increase in total TH levels and an enhancement of TH activity. When HA-1004 (PK A inhibitor) was infused, concomitantly with morphine, it diminished the increase in noradrenaline turnover, total TH levels and TH phosphorylation at Ser40 in morphine-withdrawn rats. In contrast, the infusion of calphostin C (PKC inhibitor), did not modify the morphine withdrawal-induced increase in noradrenaline turnover and total TH levels. In addition, we show that the ability of morphine withdrawal to stimulate phosphorylation at Ser31 was reduced by SL327, an inhibitor of ERK 1/2 activation.

Conclusions and implications: The present findings demonstrate that the enhancement of total TH levels and the increased phosphorylation state of TH during morphine withdrawal were dependent on PKA and ERK activities and suggest that these transduction pathways might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine withdrawal.

Figure 1

Immunoblots of PKA (a) and PKCδ (b) in right ventricle from placebo (pla, p)- or morphine (mor, m)-dependent rats after naloxone (nx, n)-precipitated withdrawal in vehicle (veh, v) infused rats and in animals chronically administered with HA-1004 (HA) or calphostin C (CA). Animals received s.c. implantation of pla or mor (75 mg) pellets for 7 days and concomitantly were infused with veh, HA (40 nmol day⁻¹) or CA (40 pmol day⁻¹). On day 8, rats were injected with saline (s) s.c. or nx; 2 mg kg⁻¹) and were decapitated 90 min later. The immunoreactivity corresponding to PKA or PKCδ is expressed as a percentage of that in the control group (pla+veh+nx; defined as 100%). Data are the mean±s.e.mean (n=4). ^***P<0.001 versus the (pla+veh+nx;); ⁺⁺⁺P<0.001 versus the group treated with (mor+HA+nx) or (mor+CA+nx); ^##P<0.01 versus (pla+veh+nx). Bottom panels: representative bands from autoradiograms at the known apparent molecular weight for PKA and PKCδ. β-actin was used as an internal loading control.

Figure 2

Normetanephrine (NMN)/noradrenaline (NA) ratio in the right ventricle 60 (a) or 90 min (b) after saline or naloxone (nx, n) administration to placebo-(pla, p) or morphine (mor, m)-treated rats receiving vehicle (veh, v), HA1004 or calphostin. Data are the mean±s.e.mean (n=6–7). ^***P<0.001 versus the group receiving saline instead of nx; ⁺⁺⁺P<0.001 versus the group treated with pla instead of mor; ^###P<0.001 versus the group treated with veh+mor+nx.

Figure 3

Western blotting analysis of TH immunoreactivity levels in the right ventricle 60 (a) or 90 min (b) after saline or naloxone (nx, n) administration to placebo--(pla, p) or morphine (mor, m)-treated rats receiving vehicle (veh, v), HA1004 or calphostin. The immunoreactivity corresponding to total TH is expressed as a percentage of that in the control group (pla+veh+saline; defined as 100%). Data are the mean±s.e.mean (n=4–6). ^*P<0.05, ^***P<0.001 versus the group receiving saline instead of nx; ⁺P<0.05, ⁺⁺P<0.01 versus the group pretreated with pla instead of mor; ^##P<0.01 versus the group receiving veh instead of HA. Bottom panels: representative bands from autoradiograms at the known apparent molecular weight for TH. β-actin was used as an internal loading control.

Figure 4

Western blotting analysis of TH phospho (p) Ser40 in the right ventricle 60 (a) or 90 min (b) after saline or naloxone (nx, n) administration to placebo- (pla, p) or morphine (mor, m)-treated rats receiving vehicle (veh, v), or HA1004. The immunoreactivity corresponding to TH phospho-Ser40 is expressed as a percentage of that in the control group (pla+veh+saline; defined as 100%). Data are the mean±s.e.mean (n=4–6). ^*P<0.05 versus the group receiving saline instead of nx; ⁺P<0.05 versus the group pretreated with pla instead of mor; ^#P<0.05 versus the group receiving veh instead of HA. Bottom panels: representative bands from autoradiograms at the known apparent molecular weight for TH. β-actin was used as an internal loading control.

Figure 5

Western blotting analysis of TH phospho (p) Ser31 in the right ventricle 60 (a) or 90 min (b) after saline or naloxone (nx, n) administration to placebo- (pla, p) or morphine (mor, m)-treated rats. The immunoreactivity corresponding to TH phospho-Ser31 is expressed as a percentage of that in the control group (pla+saline; defined as 100%). Data are the mean±s.e.mean (n=4–6). ^*P<0.05 versus the group receiving saline instead of nx; ⁺P<0.05 versus the group pretreated with pla instead of mor. Bottom panels: representative bands from autoradiograms at the known apparent molecular weight for TH. β-actin was used as an internal loading control.

Figure 6

ERK1/2 (a, b) and TH phospho (p) Ser31 (c) immunoblots and TH activity (d) in right ventricle from placebo-(pla, p) or morphine(mor, m)-dependent rats 90 min after s.c. administration of naloxone (nx, n) in the absence or presence of SL327 (SL, 100 mg kg⁻¹, i.p.), 1 h before nx. Phospho(p)ERK1/2 or pSer31 TH immunoreactivity bands were measured, normalized to the background values and expressed as percentage of controls. Data are the mean±s.e.mean (n=4–6). ^***P<0.001 versus the group receiving vehicle (veh, v) instead of SL; ⁺⁺P<0.01 versus the group pretreated with pla instead of mor; ^##P<0.01 versus mor+SL+nx; ^†††P<0.001 versus pla+nx.