Glucose reduces endothelin inhibition of voltage-gated potassium channels in rat arterial smooth muscle cells

Abstract

Prolonged hyperglycaemia impairs vascular reactivity and inhibits voltage-activated K⁺ (Kv) channels. We examined acute effects of altering glucose concentration on the activity and inhibition by endothelin-1 (ET-1) of Kv currents of freshly isolated rat arterial myocytes. Peak Kv currents recorded in glucose-free solution were reversibly reduced within 200 s by increasing extracellular glucose to 4 mm. This inhibitory effect of glucose was abolished by protein kinase C inhibitor peptide (PKC-IP), and Kv currents were further reduced in 10 mm glucose. In current-clamped cells, membrane potentials were more negative in 4 than in 10 mm glucose. In 4 mmd-glucose, 10 nm ET-1 decreased peak Kv current amplitude at +60 mV from 23.5 ± 3.3 to 12.1 ± 3.1 pA pF⁻¹ (n = 6, P < 0.001) and increased the rate of inactivation, decreasing the time constant around fourfold. Inhibition by ET-1 was prevented by PKC-IP. When d-glucose was increased to 10 mm, ET-1 no longer inhibited Kv current (n = 6). Glucose metabolism was required for prevention of ET-1 inhibition of Kv currents, since fructose mimicked the effects of d-glucose, while l-glucose, sucrose or mannitol were without effect. Endothelin receptors were still functional in 10 mmd-glucose, since pinacidil-activated ATP-dependent K⁺ (K_ATP) currents were reduced by 10 nm ET-1. This inhibition was nearly abolished by PKC-IP, indicating that endothelin receptors could still activate PKC in 10 mmd-glucose. These results indicate that changes in extracellular glucose concentration within the physiological range can reduce Kv current amplitude and can have major effects on Kv channel modulation by vasoconstrictors.

Potassium channels contribute to the regulation of contractile tone in vascular smooth muscle, and so to blood vessel diameter, blood flow and blood pressure. Tone is controlled by the intracellular concentration of Ca²⁺ by way of Ca²⁺–calmodulin regulation of myosin light-chain kinase, and the membrane potential of smooth muscle controls intracellular Ca²⁺ by regulating Ca²⁺ entry through voltage-sensitive Ca²⁺ channels and by affecting Ca²⁺ release from intracellular stores (Somlyo, 1985; Nelson et al. 1990). Potassium channels affect tone by their effects on membrane potential. Open K⁺ channels maintain the negative resting potential, while further K⁺ channel activation leads to hyperpolarization, decreased [Ca²⁺]_i and vasodilatation. Conversely, K⁺ channel inhibition causes membrane depolarization and vasoconstriction (Nelson et al. 1990; Nelson & Quayle, 1995).

Recent studies have shown that K⁺ channel activity can be affected by hyperglycaemia. Incubation of rat small coronary arteries for 24 h in a high (23 mm) concentration of glucose reduced voltage-activated K⁺ (Kv) currents of vascular smooth muscle cells (Liu et al. 2001), and high glucose also reduced K_ATP currents of human omental artery (Kinoshita et al. 2004). Both of these effects appear to involve production of superoxide (·O₂⁻) in response to hyperglycaemia. Interaction of superoxide with nitric oxide (NO) may lead to the formation of peroxynitrite (ONOO⁻), which can inhibit both Kv channels and high-conductance Ca²⁺-activated (BK_Ca) channels (Brzezinska et al. 2000; Liu et al. 2002, 2004). Hyperglycaemia is thought to underlie many of the vascular complications of diabetes (Chukwuma, 1992; Williams, 1995; Way et al. 2001), and it has been suggested that K⁺ channel inhibition may contribute to diabetic vascular dysfunction (Liu et al. 2001, 2004). Vasodilator responses are impaired in diabetes (Williams, 1995), and many vasodilators exert part of their effects by activation of K⁺ channels (Quayle et al. 1994; Aiello et al. 1995, 1998; Wellman et al. 1998). Li et al. (2003) have shown recently that impaired cAMP-mediated vasodilator responses in coronary arteries incubated for 24 h in high concentrations of glucose could be attributed mainly to a reduction in Kv channel activity. Vascular Kv and K_ATP channels are also regulated by vasoconstrictors, which inhibit channel activity. For example, angiotensin II inhibits Kv and K_ATP channels both through activation of protein kinase C (PKC) and inhibition of protein kinase A (Clement-Chomienne et al. 1996; Kubo et al. 1997; Hayabuchi et al. 2001b,c) while ET-1 inhibits both channels via PKC (Shimoda et al. 1998; Park et al. 2005). However, the effects of glucose on K⁺ channel inhibition by vasoconstrictors have not been investigated.

In normal individuals, resting blood glucose is 4–6 mm, rising to around 9 mm after a meal. Glucose levels can rise much higher in diabetes, and above 10 mm glucose appears in the urine. The effects of high glucose on K⁺ channels described above have used diabetic levels of glucose, and have generally also involved several hours of incubation in hyperglycaemic solutions. In the present study, we have investigated the acute effects of changes in glucose within the physiological range, both on Kv currents and on their responses to the vasoconstrictor endothelin-1 (ET-1). We find that increases in glucose within the range 0–10 mm cause a modest but immediate reduction in Kv current, and have a profound effect on Kv channel inhibition by ET-1, so that 10 mm glucose abolishes inhibition by 10 nm ET-1. These effects require the metabolism of glucose. In contrast, ET-1 inhibition of K_ATP currents was unaffected by raising glucose to 10 mm.

Methods

Preparation of vascular smooth muscle cells

Adult male Wistar rats were killed by cervical dislocation. The care and killing of the animals conformed to the requirements of the UK Animals (Scientific Procedures) Act 1986. Smooth muscle cells were dissociated from small branches of mesenteric arteries as follows. Arteries were removed, cleaned and dissected in ice-cold solution containing (mm): 137 NaCl, 5.4 KCl, 0.42 Na₂HPO₄, 0.44 NaH₂PO₄, 1 MgCl₂, 10 Hepes and 10 glucose, adjusted with NaOH to pH 7.4. Arteries were then transferred to the same solution except that CaCl₂ was added at 0.1 mm (low-calcium solution) for 10 min, warmed to 35°C in a water-bath, and incubated for 30–35 min in low-calcium solution containing (mg ml⁻¹): 0.9 albumin, 1.4 papain and 0.9 dithioerythritol. This was followed by further digestion for 12.5 min in low-calcium solution containing (mg ml⁻¹): 0.9 albumin, 1.4 collagenase type F and 0.9 hyaluronidase type I-S. Arteries were then washed in low-calcium solution containing 0.9 mg ml⁻¹ albumin. Single smooth muscle cells were obtained by gentle trituration in low-Ca²⁺ solution, stored at 4°C, and used on the day of preparation.

Solutions and chemicals

For conventional whole-cell recording, the intracellular solution contained (mm): 110 KCl, 30 KOH, 1 MgCl₂, 1 CaCl₂, 10 Hepes, 10 EGTA, 1 Na₂ATP and 0.5 GTP; adjusted to pH 7.2. The free [Ca²⁺], calculated using Maxchelator (http://www.stanford.edu/%7Ecpatton/maxc.html), was 20 nm. For perforated patch recording, amphotericin B was made up as stock solution (30 mg ml⁻¹) in DMSO, and 7 μl of stock solution was diluted into 1 ml of pipette solution immediately before recording. Six millimolar K⁺ extracellular solution contained (mm): 134 NaCl, 6 KCl, 1 MgCl₂, 0.1 CaCl₂ and 10 Hepes, adjusted to pH 7.4. For intact artery recordings, CaCl₂ was 1.8 mM and MgCl₂ 1.2 mM. Glucose, or other mono- or disaccharides were added to this solution as indicated in the Results, and mannitol was added where necessary to give a total concentration of 10 mm to maintain osmolarity. In order to minimize the activity of BK_Ca channels, external Ca²⁺ was lowered to 0.1 mm to reduce Ca²⁺ influx, and intracellular Ca²⁺ was buffered to a low level with EGTA. As we have reported previously (Hayabuchi et al. 2001c), under these conditions voltage-activated currents contained very little contribution from BK_Ca channels; the BK_Ca channel blocker penitrem A (100 nm) had no significant effect on the current in response to a 400 ms depolarization from −65 to +60 mV (control, 22.4 ± 0.8 pA pF⁻¹; penitrem A, 22.2 ± 0.3 pA pF⁻¹; n = 6 in each case). The external solution was changed by continuous perfusion of the experimental chamber (volume, 0.4 ml), and complete exchange took about 30 s. All chemicals were purchased from Sigma, except myristoylated protein kinase C inhibitor peptide 19–27 (PKC-IP) which was obtained from Calbiochem (UK).

Data recording and analysis

Whole-cell currents were recorded from single smooth muscle cells using the patch clamp technique. Currents were recorded using an Axopatch 200A amplifier (Axon Instruments) and, for voltage pulses, a P/6 leak subtraction protocol was used. Patch pipettes were made from thick-walled borosilicate glass. Whole-cell currents were filtered at 5 kHz and sampled at 10 kHz. Electrode resistances before sealing were 4–6 MΩ and after sealing were > 1 GΩ. For recordings from intact arteries, segments of third-order mesenteric artery were pinned out under tension across a gap in a Sylgard-based chamber and penetrated with electrodes with resistances of 80–120 MΩ when filled with 2 m KCl. A calibrated voltage source between the bath electrode and earth was used to measure membrane potential. Measurements were accepted if the membrane potential was stable for > 1 min and showed an abrupt return to baseline on withdrawal of the electrode. All experiments were done at 30–32°C. Current–voltage (I–V) relations were measured either at the peak of the current or, for steady-state current, at the end of 400 ms pulses to voltages between −60 and +60 mV. Inactivation time constants were fitted using custom software. Data are expressed as means ± s.e.m. Intergroup differences were analysed by analysis of variance with Bonferroni's test or by Student's unpaired t test as appropriate. A value of P < 0.05 was considered statistically significant.

Results

Extracellular glucose reduces Kv current

Whole-cell patch clamp recordings from rat mesenteric artery smooth muscle cells in a normal extracellular glucose (d-glucose) concentration of 4 mm revealed the presence of voltage-gated K⁺ (Kv) currents induced by 400 ms depolarizing steps from a holding potential of −65 mV to potentials of −30 mV and above (Fig. 1A). We examined the effect of alterations in extracellular glucose concentration by recording from cells in the presence of either 0 or 10 mm glucose. Cells were bathed in the solution for 10–15 min before recording. Mannitol was added to solutions where needed so that [glucose + mannitol] was 10 mm. Kv current densities were higher than control values in 0 mm glucose, but lower in 10 mm glucose, as can be seen from the example records of Fig. 1B and C and the mean current–voltage relations (normalized to cell capacitance) of Fig. 1D. Mean Kv current densities at +60 mV were 29.7 ± 0.9, 24.5 ± 1.5 and 22.7 ± 0.9 pA pF⁻¹ in 0, 4 and 10 mm glucose, respectively (n = 6 in each case). Figure 1E shows mean activation curves measured from tail currents on repolarization to −30 mV. It can be seen that glucose reduced current amplitude without significantly affecting the voltage dependence of activation.

Figure 1. Increasing external glucose reduces Kv current amplitude.

A, Kv currents recorded in response to 400 ms voltage steps in a cell bathed in external solution containing 4 mmd-glucose (6 mm mannitol). In this and subsequent figures, the holding potential was −65 mV and the steps were to voltages between −40 and +60 mV in 20 mV increments. Currents are expressed relative to cell capacitance as pA pF⁻¹. B, Kv currents from a cell bathed in 0 mm glucose solution (10 mm mannitol). C, Kv currents from a cell bathed in 10 mmd-glucose solution. D, mean current–voltage relations for cells bathed in 0 (○) and 10 mm glucose (•); n = 10 cells in each case. E, mean current (relative to cell capacitance) at +60 mV in cells bathed in 4, 0 and 10 mm glucose; n = 6 cells in each case, *P < 0.05 versus 0 mm glucose. F, mean activation curves, measured from tail currents on repolarization to −30 mV from a 400 ms pulse to the indicated voltage in 0 (○) and 10 mm glucose (•). The lines are drawn to the Boltzmann equation, I_T = I_T,max/(1 + exp[−(V−V_0.5)/k]), with V_0.5, k and I_T,max =−6.9 mV, 7.3 mV and 4.37 pA pF⁻¹, and −6.9 mV, 6.0 mV and 3.48 pA pF⁻¹ in 0 and 10 mm glucose, respectively. I_T and I_T,max are the tail current at voltage V and maximum tail current respectively. V_0.5 the voltage at which half-activation occurs, and K is the slope factor.

Since Kv channels are thought to contribute to the resting membrane potential of arterial smooth muscle (Knot & Nelson, 1995; Nelson & Quayle, 1995; Cox, 2005), we also investigated whether the reduction in Kv current with increased glucose concentration was reflected in a depolarization, both in isolated current-clamped cells and in intact segments of mesenteric artery. Figure 2A shows that the resting potential of current-clamped cells was less negative in 10 than in 4 mm glucose (−38.5 ± 1.3 compared to −44.9 ± 1.8 mV). In intact arteries, we measured membrane potentials using a sharp microelectrode after the artery had been exposed to either 4 or 10 mm glucose saline for at least 20 min (Fig. 2B). As for isolated cells, the mean membrane potential was slightly less negative in 10 than in 4 mm glucose (Fig. 2C).

Figure 2. Membrane potentials in low- and high-glucose conditions.

A, mean membrane potentials of current-clamped isolated smooth muscle cells in 4 and 10 mm glucose. Individual cells were exposed to either solution for > 10 min before membrane potentials were recorded shortly after establishing whole-cell current clamp; n = 12 cells in each case; **P < 0.01. B, example sharp microelectrode recordings from segments of third-order mesenteric arteries bathed in 4 (left-hand recording) and 10 mm glucose (right-hand recording). Individual segments were exposed to either 4 or 10 mm glucose solution. The square step indicated by the arrow in each case is a −50 mV calibration pulse. C, mean membrane potentials recorded as in B above from arteries bathed in 4 and 10 mm glucose; n = 10 in each case; *P < 0.05.

The effect of glucose is rapid and depends on PKC

To determine the time course of the effect of glucose on Kv current, we recorded currents in response to voltage steps to +40 mV, repeated every 5 s. Under these conditions, currents ran down slowly with time (Fig. 3A). Raising glucose from 0 to 4 mm caused a rapid reduction in Kv current that was complete within 200 s, and could be reversed on removal of glucose (Fig. 3B). Glucose has been shown to activate PKC in blood vessels (Way et al. 2001), and PKC has been proposed to play a role in modulating the vasodilator response to hyperglycaemia in human visceral arteries (Kinoshita et al. 2004). To test whether the reduction in Kv currents by d-glucose seen here involves PKC, we pre-incubated the cells for 15 min with the membrane-permeant myristoylated PKC inhibitor peptide 19–27 (PKC-IP, 50 μm), which we have previously shown to inhibit PKC activity in these cells (Hayabuchi et al. 2001b,c). Following the period of pre-incubation with PKC-IP, the inhibitory effect of increasing d-glucose concentration from 0 to 4 mm on peak Kv current was abolished, indicating that this process is dependent on activation of PKC by d-glucose (Fig. 3C and D).

Figure 3. PKC-dependent inhibition of Kv current by extracellular glucose.

A, B and C show plots of peak Kv current amplitude activated by 400 ms steps from −65 to +40 mV applied every 5 s to cells under the following conditions: external solution with 0 mmd-glucose (10 mm mannitol) throughout (A); external solution with 0 or 4 mmd-glucose as indicated (B); and external solution with 0 or 4 mmd-glucose following pretreatment with 50 μm PKC inhibitor peptide (C and B). In each case, example current traces recorded at the times indicated (i, ii and iii) are shown above. D, mean Kv current density in experiments like those of B and C; n = 4 cells in each case; *P < 0.05 versus 0 mm glucose.

Endothelin reduces peak Kv current and increases inactivation

The vasoconstrictors angiotensin II and ET-1 have been shown to cause PKC-dependent inhibition of Kv currents in arterial smooth muscle (Clement-Chomienne et al. 1996; Shimoda et al. 1998; Hayabuchi et al. 2001c). Since changes in glucose concentration alter the amplitude of Kv currents in a PKC-dependent manner, we postulated that the modulation of K⁺ currents by vasoconstrictors that activate PKC might be affected by glucose concentration. To test this possibility, we examined the effect of ET-1 on Kv currents in normal and high-glucose solutions.

Figure 4A shows examples of Kv currents recorded in response to 400 ms voltage steps from a cell bathed in 4 mm glucose before and after the application of 10 nm ET-1. It can be seen that ET-1 both caused substantial inhibition of Kv currents and induced a marked inactivation during the 400 ms pulse, similar effects to those of angiotensin II in the same preparation (Hayabuchi et al. 2001c). Mean current–voltage relations for six cells are shown in Fig. 4B; ET-1 reduced the current at +60 mV from 23.5 ± 3.3 to 12.1 ± 3.1 pA pF⁻¹ (n = 6, P < 0.001). The time constants of inactivation, τ, measured by fitting a single exponential to the current decay, are shown in Fig. 4C. At +60 mV, 10 nm ET-1 decreased τ from 1496 ± 90 to 380 ± 80 ms (P < 0.001). These actions of ET-1 were PKC dependent, since pre-incubation of the cells for 15 min with 50 μm PKC-IP abolished the effects of ET-1 in all six cells tested (Fig. 4D, E and F).

Figure 4. Kv current inhibition by endothelin-1.

A, Kv currents activated by 400 ms voltage steps in a cell bathed in 4 mmd-glucose external solution before (control, top traces) and 9 min after addition of 10 nm ET-1 (bottom traces). B, mean current–voltage relations from experiments like that of A, under control conditions (•) and in the presence of 10 nm ET-1 (○). Steady-state currents were measured between 350 and 370 ms and are expressed relative to cell capacitance to allow for variation in cell size; n = 6 cells. C, mean time constants for Kv current inactivation in the absence (•) and presence of 10 nm ET-1 (○). D, E and F, Kv currents, current–voltage relations and inactivation time constants as in A–C above, but from cells that were pretreated with 50 μm PKC inhibitor peptide; •, control; ○,10 nm ET-1; n = 6 cells.

High glucose eliminates the effect of endothelin on Kv currents

When smooth muscle cells were bathed in solution containing 10 mmd-glucose, we found that ET-1 neither reduced peak Kv current nor induced the marked inactivation that was observed in 4 mmd-glucose. A family of Kv currents recorded in 10 mmd-glucose in the absence and presence of 10 nm ET-1 is shown in Fig. 5A and B, and it can be seen that there was little difference in the currents with and without ET-1, as indicated by the mean current–voltage curves at the end of 400 ms pulses (Fig. 5C). Mean currents at +60 mV before and after the addition of ET-1 in 10 mmd-glucose were 22.5 ± 2.5 and 20.6 ± 2.3 pA pF⁻¹ (Fig. 5C; n = 6). To assess whether the effect of glucose showed specificity for the d-isomer, we examined the effect of ET-1 after replacing d-glucose with l-glucose, which is metabolically inert. Figure 5D–F shows that 10 mml-glucose did not block the effect of ET-1 in inhibiting Kv current and increasing its rate of inactivation. Mean currents at +60 mV before and after the addition of ET-1 in 10 mml-glucose were 27.4 ± 3.1 and 13.7 ± 1.3 pA pF⁻¹ (Fig. 5F; n = 6, P < 0.001). These findings suggest that d-glucose affects ET-1 inhibition of Kv by an action that requires metabolism of glucose. It is possible that such metabolic effects might be modified by dialysis of ATP into the cell from the pipette solution, though we would not expect this to provide tight control of intracellular ATP. To investigate this possibility, we compared Kv inhibition by ET-1 in 4 and 10 mm glucose in cells recorded using amphotericin-perforated patches to avoid intracellular dialysis. Figure 5G and H shows that, as for conventional recording, ET-1 inhibited Kv currents in 4 mm glucose (Fig. 5G), but was ineffective in 10 mm glucose (Fig. 5H), suggesting that the effects of glucose are not substantially modified by conventional whole-cell recording.

Figure 5. Endothelin-1 inhibition of Kv currents is abolished in 10 mmd-glucose but not 10 mml-glucose.

A and B, Kv currents recorded from a cell bathed in 10 mmd-glucose solution before (A) and after the addition of 10 nm ET-1 (B). C, mean steady-state current–voltage relations from experiments like that of A and B, under control conditions (•) and in the presence of 10 nm ET-1 (○); n = 6 cells. D, E and F, Kv currents and mean current–voltage relations as in A, B and C except that cells were bathed in solution containing 10 mml-glucose; n = 6 cells. G and H, mean current–voltage relations obtained using amphotericin B perforated patch recording from cells bathed in 4 mmd-glucose (G) or 10 mmd-glucose (H) under control conditions (•) and in the presence of 10 nm ET-1 (○); n = 6 cells in each case.

The effects of high glucose depend on metabolism

To confirm that the effects of high glucose require its metabolism, we investigated the ability of other mono- or disaccharides to substitute for glucose in preventing Kv channel inhibition by ET-1. In each case, the carbohydrate was present at 10 mm, and Kv currents were measured before and 9 min after the addition of 10 nm ET-1. Figure 6 shows that fructose, which can enter glycolysis via the fructose-1-phosphate pathway, prevented Kv current inhibition (Fig. 6A) and inactivation (Fig. 6B) by ET-1 in a similar way to d-glucose. However, sucrose and mannitol (and l-glucose), which are not metabolized, did not affect the inhibitory action of ET-1 (Fig. 6). It can also be seen that the Kv current density is somewhat smaller in d-glucose and fructose than in the non-metabolizable sugars, suggesting that the effect of glucose in reducing Kv current amplitude also requires its metabolism. To test for possible involvement of oxygen free radicals in the effect of glucose on Kv modulation by ET-1, we examined the effect of ET-1 in cells that were exposed to 10 mmd-glucose in the presence of the free radical scavenger N-(2-mercaptopropionyl)-glycine (300 μm). The free radical scavenger did not affect the block of ET-1 inhibition in high-glucose conditions (Fig. 6).

Figure 6. Kv current inhibition by ET-1 in metabolizable and non-metabolizable sugars.

A, mean steady-state Kv current amplitude at the end of a 400 ms pulse to +60 mV before (filled bars) and 9 min after the addition of 10 nm ET-1 (open bars) in cells exposed to external solutions containing the following at 10 mm in each case: d-glucose; fructose; l-glucose, sucrose; mannitol; d-glucose in the presence of the free radical scavenger N-(2-mercaptopropionyl)-glycine (MPG; 300 μm); n = 5–6 cells in each case; **P < 0.01 versus control conditions. B, mean time constants for Kv current inactivation at +60 mV before (filled bars) and 9 min after the addition of 10 nm ET-1 (open bars) in the same experiments as shown in A.

If d-glucose must be metabolized to prevent the inhibitory action of ET-1, metabolic inhibitors should be able to restore the action of ET-1 in the presence of 10 mmd-glucose. We therefore examined Kv channel inhibition by ET-1 in 10 mmd-glucose in the presence of 1 mm iodoacetic acid (IAA) to block glycolysis. Figure 7A shows that the inhibitory actions of ET-1 were restored under these conditions. However, when sodium pyruvate (10 mm) was added to bypass glycolysis and act as a substrate for downstream metabolism, Kv current inhibition by ET-1 was lost (Fig. 7B). We also found that the mitochondrial uncoupler 2,4-dinitrophenol (DNP; 50 μm) restored Kv channel inhibition by ET-1 in 10 mmd-glucose, confirming the need for glucose metabolism to exert this effect (Fig. 7B).

Figure 7. Block of the effects of 10 mmd-glucose by metabolic inhibition.

A, mean steady-state Kv current amplitude at the end of a 400 ms pulse to +60 mV before (filled bars) and 9 min after (open bars) the addition of 10 nm ET-1 in cells exposed to external solutions containing 10 mmd-glucose together with iodoacetic acid (IAA,1 mm), IAA and sodium pyruvate (5 mm), or IAA and 2,4-dinitrophenol (50 μm), as indicated; n = 6 cells in each case; *P < 0.05, **P < 0.01 versus control conditions. B, mean time constants for Kv current inactivation at +60 mV before (filled bars) and 9 min after the addition of 10 nm ET-1 (open bars) in the same experiments as those in A.

High glucose does not affect endothelin inhibition of K_ATP currents

To address the possibility that high glucose caused a non-specific inhibition of the response to endothelin, possibly through a decrease in endothelin receptor function, we examined the effect of ET-1 on a different channel, the K_ATP channel, under control and high-glucose conditions. K_ATP currents, activated by 10 μm pinacidil, were readily observed in arterial smooth muscle cells and were measured as inward currents in symmetrical 140 mm K⁺ at −60 mV to minimize contamination from Kv currents as we have previously described (Hayabuchi et al. 2001a,b). As for Kv currents, 10 nm ET-1 inhibited the pinacidil-induced K_ATP current under control conditions, and the effect of ET-1 was blocked by PKC-IP (Fig. 8A, B and D). When external d-glucose was raised to 10 mm, however, ET-1 remained as effective at inhibiting K_ATP channels as under control conditions, and the inhibition was still PKC dependent (Fig. 8C and D). These results indicate that endothelin receptors were still able to activate PKC in 10 mmd-glucose, and that the effect of high glucose appears specific for Kv channels.

Figure 8. Inhibition of K_ATP current by ET-1.

A, recording of whole-cell current from a cell held at −60 mV in extracellular solution containing 4 mmd-glucose. The extracellular K⁺ concentration was raised from 6 to 140 mm as indicated by the arrow, and inward K_ATP current was activated by pinacidil. Pinacidil (10 μm), ET-1 (10 nm) and glibenclamide (10 μm) were applied as indicated. B, whole-cell recording as in A, but from a cell that had been pretreated with PKC inhibitor peptide (50 μm). C, whole-cell recording as in A, but from a cell bathed in 10 mm extracellular d-glucose. D, mean inhibition of K_ATP current by 10 nm ET-1 in control cells (open bars) and in cells pretreated with 50 μm PKC-IP with extracellular d-glucose of either 4 or 10 mm as indicated; n = 6 cells in each case; **P < 0.01 versus ET-1 alone.

Discussion

In this paper, we show that increasing extracellular glucose concentration within the physiological range has two acute effects on the Kv currents of rat mesenteric artery smooth muscle cells (Fig. 9). It causes a modest decrease in current amplitude, and has a potent effect on inhibition of Kv current by ET-1 such that the inhibitory effect of 10 nm ET-1 is abolished in 10 mmd-glucose.

Figure 9. Diagram summarizing the effects of glucose and endothelin-1 on Kv and K_ATP channels of rat mesenteric arterial smooth muscle.

Endothelin-1 inhibits both Kv and K_ATP channels by a PKC-dependent mechanism. High concentrations of glucose also inhibit Kv channels through a PKC-dependent mechanism (thin arrow), but not to the same extent as ET-1. In addition, 10 mm glucose removes the ability of ET-1 to inhibit Kv channels without affecting its ability to inhibit K_ATP channels. The isoforms of PKC involved in these interactions remain to be determined.

The acute effect of glucose on Kv current

The acute reduction in Kv current in response to increased extracellular d-glucose concentration is complete within a few minutes (Fig. 3), and the effect is relatively small, so that mean Kv current density is about 24% lower in 10 mm glucose than in glucose-free solution. The acute effect of increasing glucose even from 0 to 4 mm is clear within an individual cell (e.g. Fig. 3B). Previous studies in small coronary arteries have shown that more extreme hyperglycaemia (23 mm glucose) applied for 24 h can cause a slightly larger reduction in Kv currents than those we observed (Liu et al. 2001). This longer term effect has been suggested to result from the reaction of superoxide (·O₂⁻), produced in response to hyperglycaemia, with nitric oxide formed by inducible NO synthase to form peroxynitrite (ONOO⁻; Li et al. 2004). Peroxynitrite has been shown previously to inhibit BK_Ca channels (Brzezinska et al. 2000; Liu et al. 2002), and Li et al. (2004) have recently shown that incubation in hyperglycaemic solution leads to increased nitration of tyrosine residues in α-subunits of the channel K_v1.2. Furthermore, ONOO⁻ reduced whole-cell Kv currents in coronary smooth muscle cells and reduced opening of single Kv channels in patches excised from the same cells (Li et al. 2004). While it is possible that such a mechanism is involved in the acute effects of glucose we observe, it seems less likely because our effects occur in isolated arterial smooth muscle cells in the absence of an endothelial source of NO, whereas in the previous studies, intact arteries were incubated in hyperglycaemic solution before isolation of cells. The acute reduction in Kv channel activity by increased glucose does, however, require activation of PKC, since it was abolished in the presence of PKC inhibitor peptide. Hyperglycaemia has been shown to increase levels of diacylglycerol, leading to increased activation of PKC in both cultured aortic smooth muscle cells and in fresh aortic tissue (Inoguchi et al. 1992, 1994; Kunisaki et al. 1994, 1996; Way et al. 2001), and PKC has been implicated in the reduction of K_ATP channel-mediated hyperpolarization and relaxation in human omental arteries exposed to high glucose concentrations (Kinoshita et al. 2004). The involvement of PKC has not, however, been reported in previous studies of Kv inhibition by hyperglycaemia (Liu et al. 2001, 2004).

Prevention of endothelin inhibition by raised glucose concentration

The present study provides the first report, to our knowledge, of a profound effect of extracellular glucose on vasoconstrictor inhibition of Kv channels. In common with several other vasoconstrictors, ET-1 inhibits K⁺ channels by activation of PKC (Fig. 4; Shimoda et al. 1998; Park et al. 2005). Endothelin-1 is a potent inhibitor of Kv currents in mesenteric arterial smooth muscle; 10 nm ET-1 reduced currents by about 50% in 4 mm glucose. Increasing d-glucose to 10 mm abolished the inhibitory action of ET-1. This effect cannot be explained simply in terms of increased glucose having already caused maximal inhibition so that ET-1 can have no further effect, since the inhibitory effect of ET-1 is considerably greater than that of 10 mm glucose. In addition, ET-1 substantially increased the rate of Kv current inactivation, reducing the time constant for inactivation about fourfold, whereas high glucose did not have this effect.

It is clear that block of ET-1 inhibition by glucose requires metabolism of the sugar, since fructose, which is metabolized in essentially the same way as glucose, also abolished the action of ET-1, while the non-metabolized sugars l-glucose, sucrose and mannitol were without effect. Furthermore, the effect of d-glucose can be prevented by blocking glycolysis with iodoacetic acid, and addition of pyruvate as a substrate downstream of glycolysis restores inhibition of the effect of endothelin. The mitochondrial uncoupler 2,4-dintrophenol, which dissipates the mitochondrial membrane potential and so the energy gradient for ATP synthesis, was also able to restore Kv current inhibition by ET-1 in the presence of 10 mmd-glucose, suggesting that glucose must be metabolized as far as ATP synthesis to exert its effect on vasoconstrictor inhibition. Our results do not, however, support the idea that the effect of raised glucose occurs through production of oxygen free radicals such as ·O₂⁻, since 10 mmd-glucose still prevented Kv current inhibition by ET-1 in the presence of the free radical scavenger N-(2-mercaptopropionyl)-glycine. The effect of increased glucose also seems unlikely to occur at the level of the ET-1 receptor or receptor coupling to PKC activation, since ET-1 inhibition of K_ATP channels, which also occurs through activation of PKC (Park et al. 2005), was not affected by increasing d-glucose from 4 to 10 mm (Fig. 8). It is possible that the effect of high glucose concentration on the action of ET-1, like its effect on Kv current density, occurs through activation of PKC. Hyperglycaemia has been reported to activate primarily, but not exclusively, the β-isoform of PKC (Way et al. 2001). Inhibition of K_ATP channels by ET-1 and of K_ATP and Kv channels by angiotensin II in arterial smooth muscle occurs through PKC-ɛ (Hayabuchi et al. 2001b,c; Park et al. 2005). Thus, it is possible that PKC-β activation by glucose might modulate PKC-ɛ-dependent channel inhibition by ET-1. The exact mechanism by which glucose regulates ET-1 signalling to Kv channels and the possible roles of different PKC isoforms remain to be established, and will form the subject of future investigation.

Possible functional implications

Voltage-activated K⁺ channels make an important contribution to the membrane potential of vascular smooth muscle (Leblanc et al. 1994; Knot & Nelson, 1995; Standen & Quayle, 1998; Cheong et al. 2002; Liu & Gutterman, 2002; Cox, 2005) and may play a role in limiting depolarization (Nelson & Quayle, 1995). Our finding that increased glucose, which inhibits Kv, depolarizes isolated smooth muscle cells is consistent with a role for Kv in the resting membrane potential. Our results also suggest a small depolarization in intact arterial segments. Such an effect might be greater in arteries depolarized in response to physiological intra-arterial pressures, where the activation of Kv channels would be expected to be greater.

Voltage-activated K⁺ channels are also activated by β-adrenoceptor stimulation and by activation of PKA, and so are likely to be involved in vasodilator reponses to agents that activate the cAMP–PKA pathway (Aiello et al. 1995, 1998), suggesting that such responses might be impaired under hyperglycaemic conditions. Consistent with this, Li et al. (2003) have reported that dilatations in response to isoprenaline or forskolin are attenuated in coronary arteries that have been incubated in high-glucose solution. Our results show that changes in glucose concentration can also have profound effects on Kv channel inhibition by a vasoconstrictor. These effects on vasoconstrictor action occur rapidly and with relatively small changes in glucose concentration, which are within the physiological range. Such effects might serve to reduce some of the effects of impaired vasodilator function in response to hyperglycaemia by ensuring that, while Kv channel activity is reduced, its further reduction by vasoconstrictors is also limited. Since the effects of increased glucose on ET-1 action are rapid, it is also conceivable that they could play a role during short-term physiological changes in glucose concentration. In future studies it will be important to establish whether glucose can affect channel modulation by other vasoconstrictors in addition to ET-1.