K(ATP) channels mediate the antihypertrophic effects afforded by κ-opioid receptor stimulation in neonatal rat ventricular myocytes

Abstract

Recent evidence suggests that κ-opioid receptor (OR) agonists and K(ATP) channel activation exert antihypertrophic effects on cardiac myocytes. We studied the role of K(ATP) channels in the antihypertrophic effects of ORs in primary cultures of neonatal rat ventricular myocytes exposed for 48 h to the α(1) adrenoceptor agonist phenylephrine and the relative contributions of mitochondrial K(ATP) (mitoK(ATP)) and sarcolemmal K(ATP) (sarcK(ATP)). Furthermore, we elucidated the pathway between ORs and K(ATP) channels and their impact on intracellular Ca(2+) ([Ca(2+)](i)) transients. Hypertrophy of cardiomyocytes was characterized by increases in i) total protein content; ii) cell size and iii) [(3)H]leucine incorporation. Phenylephrine (10 μM) increased the three parameters. Trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective κ-opioid receptor agonist, prevented phenylephrine-induced hypertrophy and [Ca(2+)](i) transients. The effect of U50,488H was abolished by nor-binaltorphimine, a selective κ-OR antagonist, indicating that the effect was κ-OR-mediated. The protein kinase C inhibitor chelerythrine and the K(ATP) channel inhibitors glibenclamide (50 μM), a nonselective K(ATP) antagonist, and 5-hydroxydecanoic acid (100 μM), a mitochondrial selective K(ATP) antagonist, reversed the antihypertrophic effect of U50,488H, and there was no significant difference between the two K(ATP) channel blockers. Moreover, we also determined the expression of the Kir6.2 subunits of the K(ATP) channel, which increased in response to U50,488H in the presence of phenylephrine, but was suppressed by chelerythrine, glibenclamide and 5-hydroxydecanoic acid. U50,488H also attenuated the elevation of [Ca(2+)](i). This study suggests that K(ATP), and particularly the mitochondrial K(ATP,) mediates the antihypertrophic effects of κ-opioid receptor stimulation via the PKC signaling pathway.

Figure 1.

Effects of U50,488H, nor-binaltorphimine (NBI), glibenclamide (Gli), 5-hydroxydecanoic acid (5-HD) or chelerythrine (CHE) on the (B) peak amplitude and (C) frequency of the spontaneous [Ca²⁺]_i transient and (D) resting Ca²⁺ in cultured ventricular myocytes from the neonatal rats treated with phenylephrine (PE). (A) Representative tracings. The myocytes were cultured in wells with a coverslip. After the cells were cultured for 2 days, the coverslip with myocytes was incubated with Fura-2/AM at a concentration of 4 μM in a medium for 25 min. The unincorporated dye was removed by washing twice with fresh medium. Then the cytosolic calcium transient of multiple cells were measured by the TILL imaging system with a spec-trofluorometric method. The various treatments were added respectively at certain time points. Values are presented as mean ± SEM; n=4 in each group. **P<0.01 vs. control; ^##P<0.01 vs. PE group, ⁺⁺P<0.01 vs. PE+U50 group. PE, 10 μM phenylephrine; U50, 1 μM U50,488H; NBI, 1 μM nor-binaltorphi-mine; Gli, 50 μM glibenclamide; 5-HD, 100 μM 5-hydroxydecanoic acid; CHE, 1 μM chelerythrine.

Figure 2.

Effects of U50,488H, nor-binaltorphimine (NBI), glibenclamide (Gli), 5-hydroxydecanoic acid (5-HD) or chelerythrine (CHE) on (A) protein content, (B) cell size and (C) [³H]leucine uptake in cultured ventricular myocytes from the neonatal rats treated with phenylephrine (PE). Methods and times of cell culture are as described in Materials and methods. After the cells were cultured for 2-3 days, the medium was changed to DMEM supplemented with 0.4% calf serum. The various treatments were added to the medium as described in Materials and methods and cultured for 48 h. Values are presented as mean ± SEM; n=6. ^**P<0.01 vs. control; ^##P<0.01 vs. PE group; ⁺P<0.05, ⁺⁺P<0.01 vs. PE+U50 group. PE, 10 μM phenylephrine; U50, 1 μM U50,488H; NBI, 1 μM nor-binaltorphimine; Gli, 50 μM glibenclamide; 5-HD, 100 μM 5-hydroxydecanoic acid; CHE, 1 μM chelerythrine.

Figure 3.

Effects of U50,488H, nor-binaltorphimine (NBI), glibenclamide (Gli), 5-hydroxydecanoic acid (5-HD) and chelerythrine (CHE) on Kir6.2 expression in cultured myocardial cells from neonatal rats treated with phenylephrine (PE). Apart from the cells (1×10⁶ cells per flask), the method and time of cell culture were the same as in Fig. 2. The different treatments were added to the medium at the same time and cultured for 48 h. (A) Representative autoradiograms of Kir6.2 and β-actin. Lane 1, normal control; lane 2, PE 10 μM; lane 3, PE 10 μM + U50 1 μM; lane 4, PE 10 μM + NBI 1 μM + U50 1 μM; lane 5, PE 10 μM + 5-HD 100 μM + U50 1 μM; lane 6, PE 10 μM + Gli 50 μM + U50 1 μM; lane 7, PE 10 μM + CHE 1 μM + U50 1 μM. (B) Relative levels of Kir6.2 expressed as the absorbance ratio of each group:control (%). Values are presented as the mean ± SEM., n=4 in each group. ^##P<0.01 vs. PE group. ⁺⁺P<0.01 vs. PE + U50 group. PE, 10 μM phenylephrine; U50, 1 μM U50,488H; NBI, 1 μM nor-binaltorphimine; Gli, 50 μM glibenclamide; 5-HD, 100 μM 5-hydroxydecanoic acid; CHE, 1 μM chelerythrine.