Methadone but not morphine inhibits lubiprostone-stimulated Cl- currents in T84 intestinal cells and recombinant human ClC-2, but not CFTR Cl- currents

Abstract

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl(-) channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl(-) currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl(-) currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl(-) currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl2-inhibited Cl(-) currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl(-) currents with half-maximal inhibition at 100 and 200-230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl(-) currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl(-) currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl(-) currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl(-) currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.

Fig. 1

Effects of methadone and morphine on lubiprostone-stimulated Isc across T84 cell cultures and effect of naloxone. a T84 cells were mounted in an Ussing chamber under short circuit conditions, and first treated with 300 μM 1-ethyl-2-benzimidazolinone (1-EBIO) and then with either 5 μM morphine, 5 μM methadone, or no addition. 250 nM lubiprostone was then added, and Isc was measured. Mean ± SEM are plotted. Number of filters (n) measured under each condition is indicated. *P < 0.0005 versus lubiprostone alone. b Indicated concentrations of methadone were added before treatment with lubiprostone. Mean ± SEM are plotted; n is indicated. *P < 0.0005 methadone versus lubiprostone; #P < 0.001 methadone versus lubiprostone; **P < 0.05 methadone versus lubiprostone. c Effects of selected concentrations of methadone and morphine on lubiprostone-stimulated Isc are shown. Isc values plotted were taken at 16 min after start of the experiment, and 12 min after addition of lubiprostone. Mean ± SEM are plotted; n is indicated. d T84 cells were mounted in an Ussing chamber under short circuit conditions, and first treated with 300 μM 1-EBIO and then with nothing, followed by 10 μM naloxone, 1 μM methadone, or 10 μM naloxone plus 1 μM methadone. 250 nM lubiprostone was then added to all, and Isc was measured. Data are plotted as mean ± SEM and n is indicated. *P < 0.0005 versus lubi ± naloxone

Fig. 2

Effects of methadone and morphine on A lubiprostone-stimulated hClC-2 Cl⁻ currents; B forskolin/IBMX-stimulated hClC-2 Cl⁻ currents, and C forskolin/IBMX-stimulated CFTR Cl⁻ currents. HEK293 cells expressing hClC-2 or hCFTR were used. Cl⁻ currents were measured at −140 mV, 200 ms, and normalized to capacitance. In A, hClC-2-transfected HEK293 cells were treated with (a) 100 nM lubiprostone, followed by 100 nM morphine and then by 1 μM methadone or with (b) 100 nM lubiprostone, followed by 1 μM methadone and then by 100 nM morphine. Data are plotted as mean ± SEM (n = 3). In (a) *P < 0.001 versus control; #P < 0.02 versus lubi + morph; **P < 0.01 versus lubi; in (b) ##P < 0.005 versus control; ***P < 0.005 versus lubi. In B, hClC-2-transfected HEK293 cells were treated with (a) 5 μM forskolin/20 μM IBMX, followed by 100 nM morphine and then 1 μM methadone or with (b) 5 μM forskolin/20 μM IBMX followed by 1 μM methadone and then 100 nM morphine. Data are presented as mean ± SEM (n = 3). In (a) *P < 0.001 versus control; #P < 0.025 versus F/I & F/I + morph; **P < 0.01 versus control; in (b) ##P < 0.0005 versus control & F/I + meth; ***P < 0.01 versus control. In C, recombinant hCFTR-transfected HEK293 cells were treated with (a) 5 μM forskolin/20 μM IBMX followed by 100 nM morphine and then 1 μM methadone or with (b) 5 μM forskolin/20 μM IBMX followed by 1 μM methadone and then 100 nM morphine. Data are plotted as mean ± SEM (n = 4). In (a) *P < 0.0005 versus control; in (b) *P < 0.0005 versus control, and #P < 0.0025 versus control. Control (c) is the Cl⁻ current without lubiprostone being added

Fig. 3

Cl⁻ currents expressed in hClC-2-transfected HEK293EBNA cells are time dependent and voltage activated: effects of a CdCl₂ and b lubiprostone. Cl⁻ currents were measured in recombinant hClC-2-transfected HEK293EBNA cells by whole cell patch clamp. a Representative current recordings are shown for hClC-2-transfected cells before and after addition of 300 μM CdCl₂ (cell capacitance = 31.6 pF) and corresponding I/V curves (I at 200 ms), plotted as mean ± SEM (n = 3) are also shown. *P < 0.005 and #P < 0.001 versus respective +CdCl₂. At −120 and −100 mV, P < 0.0005 and P < 0.025, respectively, versus +CdCl₂. In b, hClC-2 Cl⁻ current recordings were made at defined lubiprostone concentrations. I at −140 mV, 200 ms, normalized to capacitance (pA/pF) was plotted versus lubiprostone concentration. Data are plotted as mean ± SEM, n = 4 cells. Curve was fitted (using Origin) with a modified Michaelis–Menton equation: EC₅₀ = 28.2 ± 2.2 nM (4) and the V _max = 489.8 ± 22.9 pA/pF (4). Inset shows the Hill plot of the data for the four cells, each a different color, (in black are means ± SEM for comparison, but not used for Hill plot calculations). Hill coefficient = 0.91 ± 0.02 (4), R = 0.96 ± 0.09 (8), and P = 0.000183 or P < 0.0005

Fig. 4

Effect of methadone on a control and b lubiprostone-stimulated Cl⁻ currents in hClC-2- and mock-transfected HEK293EBNA cells. Cl⁻ currents were measured in recombinant hClC-2- and mock-transfected HEK293EBNA cells by whole cell patch clamp. a Typical Cl⁻ current recordings before (control) and after addition of 5 μM methadone are shown in hClC-2-transfected HEK293EBNA cells (cell capacitance = 30.2 pF). Also shown is the I/V curve (I at 200 ms) with 1 μM methadone, plotted as mean ± SEM (n = 3). *P < 0.01 versus meth and at −120, −100, and −60 mV P < 0.01 versus meth; at −80 mV P < 0.005. b Typical Cl⁻ current recordings before (control) and after 20 nM lubiprostone, followed by 1 μM methadone are shown in hClC-2-transfected and mock-transfected HEK293EBNA cells, with cell capacitances = 21.2 and 28.8 pF, respectively. Corresponding I–V curves (I at 200 ms) are also shown plotted as mean ± SEM (n = 3). *P < 0.025 versus mock-transfected; #P < 0.05 control versus lubi and lubi versus meth

Fig. 5

Effects of selected concentrations of methadone and morphine on Cl⁻ currents in hClC-2-expressing HEK293EBNA cells without lubiprostone (−lubi) and with lubiprostone (+lubi). hClC-2 Cl⁻ current recordings were made at selected concentrations of methadone and morphine in the absence (−lubi) or the presence (+lubi) of 100 nM lubiprostone. I at −140 mV and 200 ms is plotted as mean ± SEM (n = 3, except where indicated as n = 4). Experiments were carried out by adding compounds either before or after the cells were patched. #P < 0.025 and ##P < 0.05, both versus meth after patch. **P < 0.025 and ###P < 0.01 both meth after patch versus morph before patch. Concentrations of methadone resulting in half-maximal inhibition were 100 nM for control currents and 230 nM for +lubi currents meth added after patch, and 200 nM for +lubi currents meth added before patch

Fig. 6

Effect of forskolin/IBMX, followed by methadone and then CdCl₂ on Cl⁻ currents in hClC-2-transfected HEK293EBNA cells (a), (b); and (c) the effect of the specific PKA inhibitor, myristoylated PKI, on forskolin/IBMX- and lubiprostone-stimulated Cl⁻ currents in hClC-2-expressing HEK293EBNA cells. a Typical hClC-2 currents are shown before (control) and after addition of 5 μM forskolin/20 μM IBMX, followed by 1 μM methadone and then 300 μM CdCl₂ (cell capacitance = 32.6 pF). Corresponding I/V curves are also shown expressed as I at 200 ms. Data are plotted as mean ± SEM, n = 3. *P < 0.001 versus meth, **P < 0.0005 versus control and CdCl₂, ##P < 0.025 versus CdCl₂, F/I versus meth −140 to −60 mV P < 0.005–0.05; F/I versus CdCl₂ −140 to −60 mV P < 0.0005–0.025. b Cl⁻ currents (at 200 ms and −140 mV) in hClC-2-expressing HEK293EBNA cells before (control, c) and after 5 μM forskolin/20 μM IBMX (F/I) addition, followed by 1 μM methadone (meth), and followed by CdCl₂, are plotted as mean ± SEM, n = 3. *P < 0.001 versus control, #P < 0.005 versus meth, **P < 0.0005 versus CdCl₂, ##P < 0.02 versus meth. c Cl⁻ currents (at 200 ms and −140 mV) in hClC-2-expressing HEK293EBNA cells before (control, c) and after 0.4 μM mPKI addition, followed by sequentially adding 5 μM forskolin/20 μM IBMX, 100 nM lubiprostone, 1 μM methadone, and finally 300 μM CdCl₂ are plotted as mean ± SEM, n = 3 *P < 0.01 versus F/I, mPKI and c, #P < 0.05 versus meth, ##P < 0.005, and **P < 0.025 both versus CdCl₂