Anandamide reduces intracellular Ca2+ concentration through suppression of Na+/Ca2+ exchanger current in rat cardiac myocytes

Abstract

Purpose: Anandamide, one of the endocannabinoids, has been reported to exhibit cardioprotective properties, particularly in its ability to limit the damage produced by ischemia reperfusion injury. However, the mechanisms underlying the effect are not well known. This study is to investigate whether anandamide alter Na(+)/Ca(2+) exchanger and the intracellular free Ca(2+) concentration ([Ca(2+)]i).

Methods: Na(+)/Ca(2+) exchanger current (I(NCX)) was recorded and analysed by using whole-cell patch-clamp technique and [Ca(2+)]i was measured by loading myocytes with the fluorescent Ca(2+) indicator Fura-2/AM.

Results: We found that I(NCX) was enhanced significantly after perfusion with simulated ischemic external solution; [Ca(2+)]i was also significantly increased by simulated ischemic solution. The reversal potential of I(NCX) was shifted to negative potentials in simulated ischemic external solution. Anandamide (1-100 nM) failed to affect I(NCX) and [Ca(2+)]i in normal solution. However, anandamide (1-100 nM) suppressed the increase in INCX in simulated ischemic external solution concentration-dependently and normalized INCX reversal potential. Furthermore, anandamide (100 nM) significantly attenuated the increase in [Ca(2+)]i in simulated ischemic solution. Blocking CB1 receptors with the specific antagonist AM251 (500 nM) failed to affect the effects of anandamide on I(NCX) and [Ca(2+)]i in simulated ischemic solution. CB2 receptor antagonist AM630 (100 nM) eliminated the effects of anandamide on I(NCX) and [Ca(2+)]i in simulated ischemic solution, and CB2 receptor agonist JWH133 (100 nM) simulated the effects of anandamide that suppressed the increase in I(NCX) and [Ca(2+)]i in simulated ischemic solution. In addition, pretreatment with the Gi/o-specific inhibitor pertussis toxin (PTX, 500 ng/ml) eliminated the effects of anandamide and JWH133 on I(NCX) in simulated ischemic solution.

Conclusions: Collectively, these findings suggest that anandamide suppresses calcium overload through inhibition of I(NCX) during perfusion with simulated ischemic solution; the effects may be mediated by CB2 receptor via PTX-sensitive Gi/o proteins. This mechanism is importantly involved in the anti-ischemia injury caused by endocannabinoids.

Figure 1. Recording of Na⁺/Ca²⁺ exchanger current (I_NCX) in rat ventricular myocytes.

(A) Voltage protocol used for measuring I_NCX, the typical membrane currents obtained in a myocyte in the absence (a) or presence 5 mM Ni²⁺ (b). Currents were elicited by ramp pulse from +80 mV to −120 mV at a rate of 90 mV/s. (B) Current–voltage (I-V) relationship before (a) and after (b) application of 5 mM Ni²⁺. (C) I-V relationship for I_NCX (a–b).

Figure 2. Effect of anandamide on I_NCX in rat ventricular myocytes in the normal external solution.

(A) I-V curve of I_NCX in the absence and presence of 100 nM anandamide. (B) Summay data of peak outward (at +80 mV) and inward (at −120 mV) I_NCX from myocytes without or with 1 nM, 10 nM and 100 nM anandamide, n = 10 in each group.

Figure 3. Effect of anandamide on I_NCX in rat ventricular myocytes in the simulated ischemic external solution.

(A) I-V curve of I_NCX in the normal external solution (Control), in the simulated ischemic external solution (IS) and application 100 nM anandamide to the simulated ischemic external solution (Anandamide 100 nM). (B) Anandamide reduced peak outward and inward I_NCX in a concentration-dependent manner in the simulated ischemic external solution. * P<0.05 ** P<0.01 vs Control, ^# P<0.05 ^## P<0.01 vs IS, n = 10 in each group.

Figure 4

Effects of CB1 receptor antagonist AM251 (500 nM) (A) and CB2 receptor antagonist AM630 (100 nM) (B) on anandamide-induced I_NCX change, and CB2 receptor agonist JWH133 (100 nM) on I_NCX (C) in isolated rat ventricular myocytes in the simulated ischemic external solution. * P<0.05 vs Control, ^# P<0.05 vs IS, n = 10 in each group.

Figure 5

Effects of Gi/o protein antagonist pertussis toxin (PTX, 500 ng/mL) on anandamide-induced I_NCX change (A) and JWH133 -induced I_NCX change (B) in isolated rat ventricular myocytes in the simulated ischemic external solution. * P<0.05 vs Control, ^# P<0.05 vs IS, n = 10 in each group.

Figure 6. Effects of anandamide on [Ca²⁺]_i in isolated rat ventricular myocytes in normal Tyrode's solution.

(A) Transient fluorescence ratios (340∶380) were observed in the single Fura-2-loaded ventricular myocyte in normal Tyrode's solution followed by exposure to anandamide 1 nM, 10 nM and 100 nM. (B) Summary data showing the anandamide (1 nM, 10 nM and 100 nM) had no effect on [Ca²⁺]_i in normal Tyrode's solution, n = 10 in each group.

Figure 7. Effects of anandamide 100 nM or NCX inhibitor SEA0400 1 µM on [Ca²⁺]_i in ventricular myocytes in simulated ischemic solution.

(A) Transient fluorescence ratios (340∶380) were observed in the single Fura-2-loaded ventricular myocyte in simulated ischemic solution (IS) followed by exposure to anandamide 100 nM. (B) Summary data showing the anandamide and SEA0400 reduced [Ca²⁺]_i partially in simulated ischemic solution. *P<0.05 **P<0.01 vs Control, ^# P<0.05 vs IS, n = 10 in each group.

Figure 8

Effects of AM251 (500 nM) (A) and AM630 (100 nM) (B) on anandamide-induced [Ca²⁺]_i change, and CB2 receptor agonist JWH133 (100 nM) on [Ca²⁺]_i (C) in isolated rat ventricular myocytes in the simulated ischemic solution. *P<0.05, **P<0.01 vs Control, ^# P<0.05 vs IS, n = 10 in each group.