Effects of unsaturated fatty acids on calcium-activated potassium current in gastric myocytes of guinea pigs

Abstract

AIM: To investigate the effects of exogenous unsaturated fatty acids on calcium-activated potassium current [I_K(Ca)] in gastric antral circular myocytes of guinea pigs.

METHODS: Gastric myocytes were isolated by collagenase from the antral circular layer of guinea pig stomach. The whole-cell patch clamp technique was used to record I_K(Ca) in the isolated single smooth muscle cells with or without different concentrations of arachidonic acid (AA), linoleic acid (LA), and oleic acid (OA).

RESULTS: AA at concentrations of 2,5 and 10 μmol/L markedly increased I_K(Ca) in a dose-dependent manner. LA at concentrations of 5, 10 and 20 μmol/L also enhanced I_K(Ca) in a dose-dependent manner. The increasing potency of AA, LA, and oleic acid (OA) on I_K(Ca) at the same concentration (10 μmol/L) was in the order of AA>LA>OA. AA (10 μmol/L)-induced increase of I_K(Ca) was not blocked by H-7 (10 μmol/L), an inhibitor of protein kinase C (PKC), or indomethacin (10 μmol/L), an inhibitor of the cyclooxygenase pathway, and 17-octadecynoic acid (10 μmol/L), an inhibitor of the cytochrome P450 pathway, but weakened by nordihydroguaiaretic acid (10 μmol/L), an inhibitor of the lipoxygenase pathway.

CONCLUSION: Unsaturated fatty acids markedly increase I_K(Ca), and the enhancing potencies are related to the number of double bonds in the fatty acid chain. The lipoxygenase pathway of unsaturated fatty acid metabolism is involved in the unsaturated fatty acid-induced increase of I_K(Ca) in gastric antral circular myocytes of guinea pigs.

Key Words: Gastric myocytes; Calcium-activated potassium channel; Unsaturated fatty acids

INTRODUCTION

Unsaturated fatty acids are the major components of membrane lipids and they are mainly released by stimulation of phospholipase A2. Arachidonic acid (AA) and other unsaturated fatty acids modulate the activities of various ion channels[1-3] through direct or indirect pathways. The direct effects are mediated by the interaction between fatty acids and ion channel proteins or through the interference with plasma membranes. The indirect actions on ion channels result from cyclo-oxygenase, lipoxygenase, and epoxygenase metabolites or cellular signal transduction pathways[4]. For example, AA directly affects the activities of cloned human potassium channels mainly existing in heart and brain[5] and Ca²⁺-activated K⁺ channels in rabbit coronary smooth muscle cells[6]. In addition, AA has been shown to modulate ion transient receptor potential channels as a second messenger[7] and to enhance voltage-dependent calcium channels in vascular smooth muscle cells through cytochrome P450 metabolites[8].

The Ca²⁺-activated potassium channel [I_K(ca)] has been considered to play an important role in excitability and functional regulation in excitable cells[9]. Agonists of I_K(ca), such as carbon monoxide and bradykinin, which change the activity of the Ca²⁺-activated potassium channels, can affect the membrane potential and contractility in smooth muscle cells[10,11]. We have shown that NO relaxes gastric antral smooth muscle of the guinea pig through increase of I_K(Ca)[12]. It has been reported that AA affects I_K(Ca) in many cells. It inhibits I_K(Ca) in T84 cells[13], activates I_K(Ca) in vascular smooth muscles[14] and GH(3) cells[15]. In our previous study, we have reported that AA and other unsaturated fatty acids directly inhibit calcium current (I_Ca)[16], chloride current (I_Cl)[17] and muscarinic current (I_CCh)[18] in gastric myocytes of guineapigs. But the effects of AA and other unsaturated fatty acids on I_K(Ca) in gastric myocytes have not yet been reported. In the present study, we investigated the effect of AA and other unsaturated fatty acids on I_K(Ca) in gastric antral circular myocytes of guinea pigs.

MATERIALS AND METHODS

Preparation of cells

Gastric myocytes were isolated enzymatically from the antral circular layer of guinea pig stomachs as described previously[15]. Briefly, EWG/B guinea pigs (obtained from the Experimental Animal Department of Jilin University Clinical College, Certificate No 10-6004) of either sex weighing 300-350 g were euthanized by a lethal dose of IV sodium pentobarbital (50 mg/kg). The antral part of the stomach was dissected from the longitudinal layer using fine scissors and then cut into small segments (2-3 mm). The tissue chunks were then incubated at 36 °C for 25-30 min in a digestion medium consisting of 4 mL Ca²⁺-free physiology solution containing 8 mg bovine serum albumin, 4.5 mg trypsin inhibitor, 4 mg collagenase type II, and 4 mg dithioerythreitol. Single myocytes were kept at 4 °C until use.

Electrophysiological recordings

The isolated cells were transferred to a small chamber (0.1 mL) on the stage of an inverted microscope (IX-70 Olympus, Japan) for 10-15 min to settle down. The cells were superfused continuously with isosmotic solution. An 8-channel perfusion system (L/M-sps-8, List Electronics, Germany) was used to change the solution. Experiments were performed at 20-25 °C and the whole-cell configuration of the patch-clamp technique was applied. Patch-clamp pipettes were manufactured from borosilicate glass capillaries (GC 150T-7.5, Clark Electromedical Instruments, UK) by a two-stage puller (PP-83, Narishige, Japan). The resistance of the patch pipettes was 3-5 MΩ when being filled with pipette solution. Liquid junction potentials were compensated prior to seal formation. The whole-cell holding currents were recorded with an Axopatch 1-D patch-clamp amplifier (Axon Instrument, USA) and an EPC-10 amplifier (HEKA Instrument, Germany).

Drugs and solutions

All drugs were purchased from Sigma Chemical Co, USA. Tyrode’s solution contained NaCl 147, KCl 4, CaCl₂·2H₂O 2, MgCl₂·6H₂O 1.05, NaH₂PO₄·2H₂O 0.42, Na₂HPO₄·2H₂O 1.81 and glucose 5.5 mmol/L, pH was adjusted to 7.35 with NaOH. PSS contained NaCl 134.8, KCl 4.5, MgCl₂·6H₂O 1.0, CaCl₂·2 H₂O 2.0, glucose 5.0 and HEPES 10.0 mmol/L, and pH was adjusted to 7.4 by using Tris. In Ca²⁺-free PSS, 2.0 mmol/L CaCl₂·2H₂O was omitted from PSS. The pH of modified Kraft-Bruhe solution containing 0.5 mmol/L egtazic acid, 10 mmol/L HEPES, MgCl₂·6H₂O 3 mmol/L 3, 50 mmol/L KCl, 10 mmol/L glucose, 50 mmol/L L-glutamata, 20 mmol/L taurine and 20 mmol/L KH₂PO₄, was adjusted to 7.40 with KOH 1 mmol/L. The pipette solution contained 110 mmol/L potassium-aspartic acid, 5 mmol/L Mg-ATP, 5 mmol/L HEPES, 1.0 mmol/L MgCl₂·6H₂O, 20 mmol/L KCl, 0.1 mmol/L egtazic acid, 2.5 mmol/L di-tris-creatine phosphate and 2.5 mmol/L disodium-creatine phosphate, pH was adjusted to 7.30 with Tris. AA, LA and OA were separately prepared at 1 mmol/L. All unsaturated fatty acids were added in external perfusing solution. Indomethacin, 17-octadecynoic acid, nordihydroguaiaretic acid and H-7 were prepared at 1 mmol/L.

Statistical analysis

This experiment was consubstantially compared. The current before perfusion with fatty acids served as controls. All values were expressed as mean±SD. Statistical significance was evaluated by t-test.

RESULTS

Effects of unsaturated fatty acids on I_K(Ca)

Under the whole-cell configuration, the membrane potential was clamped at -60 mV, and I_K(Ca) was elicited by step voltage command pulse from -40 mV to 100 mV for 440 ms with a 20 mV increment at 10 s intervals. AA, an unsaturated fatty acid (with 4 double bonds) significantly increased I_K(Ca) in a dose-dependent manner. AA increased I_K(Ca) by (15.9±3.6)%, (31.9±7.0)% and (46.3±10.4)% at the concentrations of 2, 5 and 10 μmol/L at +60 mV, respectively (n = 8, Figure 1C). Under the whole-cell patch-clamp mode the membrane potential was clamped at -20 mV, the spontaneous transient outward currents (STOCs) due to activation of calcium-activated potassium[19] were then recorded. AA markedly increased STOCs at 10 μmol/L (Figure 1 D). Another unsaturated fatty acid LA (with 2 double bonds) also increased I_K(Ca) by (27.8±4.8)%, (37.9±13.9)% and (70.8±19.9)% at the concentrations of 5, 10 and 20 μmol/L at +60 mV, respectively (n = 8, Figure 1F-G).

Figure 1 Effects of AA and LA on I_K(Ca). A: Raw traces of AA on I_K(Ca) at different concentrations; B: I/V relationship of AA on I_K(Ca). Peak values were normalized to the values obtained at 100mV under control condition (n = 8,^aP>0.05, ^cP<0.05, ^bP<0.01 vs control);C: Dose-dependent increase of AA on I_K(Ca) (n = 8, ^cP<0.05, ^bP<0.01 vs control); D: Increase of AA on STOCs; E: Raw traces of LA on I_K(Ca) at different concentrations; F: I/V relationship of LA on I_K(Ca) (n = 8, ^aP>0.05, ^cP<0.05, ^bP<0.01 vs control); G: Dose-dependent increase of LA on I_K(Ca) (n = 8, ^aP<0.05, ^bP<0.01 vs control).

Comparison of the effects among different unsaturated fatty acids on I_K(Ca)

To determine the enhancing potency of unsaturated fatty acids, the effects of different unsaturated fatty acids on I_K(Ca) were observed. Under the whole-cell configuration, AA, LA, and OA (with one double bond) at the same concentration (10 μmol/L) increased I_K(Ca) by (46.3±10.4)%, (37.9±13.9)% and (13.5±5.1)% at +60 mV, respectively (n = 8, Figure 2). Among them, the increasing potency was in the order of AA (C20: 4, cis-5, 8, 11, 14) > LA (C18: 2, cis-9, 12) > OA (C18: 1, cis-9). The increasing potency of unsaturated fatty acids was in accordance with the number of double bonds in the fatty acid chain.

Figure 2 Comparison of different unsaturated fatty acids on I_K(Ca). A: Raw traces of 10 μmol/L OA, LA and AA on I_K(Ca); B: Increased effect of different unsaturated fatty acids on I_K(Ca) (n = 8, ^aP<0.05, ^bP<0.01 vs control).

Effects of PKC inhibitor and oxygenase inhibitor on AA-induced increase of I_K(Ca)

To determine whether unsaturated fatty acids induced increase of I_K(Ca) directly or indirectly, the effect of AA on I_K(Ca) was observed after pretreatment with indomethacin (indocin, cyclo-oxygenase inhibitor), nordihydroguaiaretic acid (NDGA, lipoxygenase inhibitor), 17-octadecynoic acid (17-ODA, cytochrome P450 inhibitor) and H-7 (protein kinase C inhibitor), which were added in external perfusing solution for about 10-15 min. H-7 (10 μmol/L), indocin (10 μmol/L) and 17-ODA (10 μmol/L) could not block AA-induced increase of I_K(Ca), and AA still increased I_K(Ca) by (41.8±3.7)%, (42.9±10.8)% and (40.8±6.8)% at +60 mV, respectively (Figure 3). There was no significant difference between the two groups before and after pretreatment with H-7 and oxygenase inhibitors (P<0.05, n = 8). But after pretreatment with NDGA 10 μmol/L, AA-induced increase of I_K(Ca) was diminished from 46.3±10.4% of control to (11.3±4.3)% (Figure 3). There was a significant difference between the two groups before and after pretreatment with NDGA (P>0.05, n = 8).

Figure 3 Effects of PKC inhibitor and oxygenase inhibitor on AA-induced increase of I_K(Ca). A, B, C and D: Effects of AA on I_K(Ca) after pretreatment with H-7, indomethacin, 17-octadecynoic acid and nordihydroguaiaretic acid, respectively(n = 8, ^aP>0.05, ^cP<0.05, ^bP<0.01 vs control); E: Comparison of AA on I_K(Ca) before and after pretreatment with H-7, indomethacin, 17-octadecynoic acid and nordihydroguaiaretic acid, respectively(n = 8, ^bP<0.01 vs control, ^aP>0.05 , ^cP<0.05 vs AA).

DISCUSSION

In this study, it was found that unsaturated fatty acids increased I_K(Ca) in a dose-dependent manner and AA increased STOCs also. AA-induced increase of I_K(Ca) was not blocked by H-7, indocin and 17-ODA, but was markedly weakened by NDGA.

Many experiments have shown that AA and other unsaturated fatty acids enhance I_K(Ca). It has been described that AA could directly increase I_K(Ca) in human mesangial cells[20] through lipoxygenase metabolites in rat pituitary tumor cells[21] and cytochrome p-450 epoxygenase products in smooth muscle cells of rat cerebral arteries[22]. The results described here show that unsaturated fatty acids increase I_K(Ca) and the more double bonds they have, the more potent their enhancing effect on I_K(Ca) in gastric antral smooth muscle cells of guinea pigs is. Our previous studies have shown that more double bonds lead to more inhibitory potency on I_Ca[16], and I_CCh[18] in gastric antral smooth muscle cells of guinea pigs; however, saturated fatty acids have no effect on I_Cl[17]. Horimoto et al[23] also reported only fatty acids having more than two double bonds activated the K⁺ channels in freshly dissociated neurons of 10- to 20-day-old rat visual cortex. These data show that double bonds must be satisfied for a given fatty acid to affect ion channels. The double bonds of unsaturated fatty acids might be easily oxidized to form reactive oxygen species or make unsaturated fatty acids to form barrette-like structures, which may optimize the possibility of binding to ion channels to modulate I_K(Ca)[15].

The indirect effects of AA on ion channels require the metabolite transformation of AA[20,21] and activation of PKC[24]. In this study, the lipoxygenase metabolism pathway was involved in AA-induced increase of I_K(Ca), since NDGA markedly diminished AA-induced increase of I_K(Ca), but H-7, indocin and 17-ODA had no effect. Many studies have demonstrated that AA exerts physiological function via lipoxygenase metabolism pathway by modulating ion channels. It has been reported that the lipoxygenase pathway mediates AA-induced vasodilation through a K⁺ channel-dependent mechanism in rat small mesenteric arteries and rat basilar arteries. The effect of AA by lipoxygenase metabolites on I_K(Ca) might play an important role in regulating secretory function of adrenal chromaffin cells in bovine. However, we can not exclude the direct effect of AA on I_K(Ca), since NDGA could not abolish entirely AA-induced increase of I_K(Ca). Unsaturated fatty acids may directly or/and indirectly modulate I_K(Ca).

In summary, I_K(Ca) is increased by unsaturated fatty acids in a dose-dependent manner. There is a correlation between the degree of cis unsaturation and the increasing potency on I_K(Ca). Lipoxygenase metabolism pathway is involved in unsaturated fatty acid-induced increase of I_K(Ca)