KMUP-1 activates BK_Ca channels in basilar artery myocytes via cyclic nucleotide-dependent protein kinases

Abstract

1. This study investigated whether KMUP-1, a synthetic xanthine-based derivative, augments the delayed-rectifier potassium (K_DR)- or large-conductance Ca²⁺-activated potassium (BK_Ca) channel activity in rat basilar arteries through protein kinase-dependent and -independent mechanisms.

2. Cerebral smooth muscle cells were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K⁺- and Ca²⁺ channel activities.

3. KMUP-1 (1 μM) had no effect on the K_DR current but dramatically enhanced BK_Ca channel activity. This increased BK_Ca current activity was abolished by charybdotoxin (100 nM) and iberiotoxin (100 nM). Like KMUP-1, the membrane-permeable analogs of cGMP (8-Br-cGMP) and cAMP (8-Br-cAMP) enhanced the BK_Ca current.

4. BK_Ca current activation by KMUP-1 was markedly inhibited by a soluble guanylate cyclase inhibitor (ODQ 10 μM), an adenylate cyclase inhibitor (SQ 22536 10 μM), competitive antagonists of cGMP and cAMP (Rp-cGMP, 100 μM and Rp-cAMP, 100 μM), and cGMP- and cAMP-dependent protein kinase inhibitors (KT5823, 300 nM and KT5720, 300 nM).

5. Voltage-dependent L-type Ca²⁺ current was significantly suppressed by KMUP-1 (1 μM), and nearly abolished by a calcium channel blocker (nifedipine, 1 μM).

6. In conclusion, KMUP-1 stimulates BK_Ca currents by enhancing the activity of cGMP-dependent protein kinase, and in part this is due to increasing cAMP-dependent protein kinase. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and the relaxation of cerebral arteries.

Introduction

Relaxation of blood vessels can be mediated by several mechanisms. One major mechanism of vasodilatation is activation of guanylate cyclase and increased production of cGMP (Rembold, 1992). A second major mechanism that mediates vasodilatation is activation of adenylate cyclase (AC) and production of cAMP. Previous evidence (Faraci & Heistad, 1998) suggests that several types of K⁺ channels are present in cerebral blood vessels, and that activation of these channels may constitute a key mechanism of relaxation in cerebral blood vessels. Activation of K⁺ channels in arterial smooth muscle hyperpolarizes the cell membrane, and subsequently closes voltage-dependent calcium channels, resulting in a decrease in intracellular calcium, and vascular relaxation (Kitazono et al., 1995). The membrane potential of cerebral arterial muscle measured in vitro has ranged widely from approximately −40 to −70 mV, and changes in this potential of only a few millivolts are associated with significant changes in vascular tone (Nelson & Quayle, 1995).

Large-conductance calcium-activated potassium (BK_Ca) channels were first described in skeletal muscle (Latorre et al., 1982), chromaffin cells (Marty, 1981), and vascular smooth muscle, including cerebral vessels (Brayden & Nelson, 1992). Pharmacological agents commonly used to inhibit BK_Ca channels include tetraethylammonium (TEA; ⩽1 mmol l⁻¹), charybdotoxin (ChTX), and iberiotoxin (IbTX) (Giangiacomo et al., 1995). ChTX blocks BK_Ca channels in arterial smooth muscle, although it may inhibit some other types of K⁺ channels in other tissues (Galvez et al., 1990). IbTX is a highly selective blocker of BK_Ca channels. BK_Ca channels are activated by increases in intracellular Ca²⁺ and membrane depolarization (Nelson & Quayle, 1995). High levels of Ca²⁺, of the order of 3–10 μmol l⁻¹, are required for K⁺ channel activity in the physiological range of membrane potentials (−60 to −30 mV) in relaxed cells (Jackson & Blair, 1998). On the other hand, the functional role of BK_Ca channels is enhanced in arterial smooth muscle during chronic hypertension. This phenomenon occurs similarly throughout the vasculature, including the aorta (Rusch et al., 1992) and carotid artery, and the mesenteric, femoral (Asano et al., 1993), and cerebral vascular beds (Paterno et al., 1997). Therefore, increased BK_Ca channel function in arterial smooth muscle cells may provide a protective mechanism against progressive increases in blood pressure. This negative-feedback mechanism would modulate increased pressure and vascular tone, and subsequently limit pressure-induced vasoconstriction and preserve local blood flow.

In arterial smooth muscle patch-clamp experiments, cGMP and cAMP activate protein kinase G (PKG) and protein kinase A (PKA), respectively, and this leads to BK_Ca activation (Robertson et al., 1993; Schubert et al., 1996; White et al., 2000). BK_Ca channels play an important role in regulating the smooth muscle contractility and in controlling the diameter of small myogenic cerebral arteries (Brayden & Nelson, 1992; Nelson et al., 1995). Thus any agent (e.g., NO) that activates BK_Ca channels would not only tend to hyperpolarize and relax arteries but it would also alter how arteries respond to changes in pressure.

Delayed rectifier potassium (K_DR) channels have been described in nearly all excitable membranes including vascular muscle. These channels are activated by membrane depolarization with threshold potentials for substantial activation of ∼−30 mV. When cells are depolarized, these potassium channels are activated, resulting in an outward current that returns the membrane potential toward the resting level (Nelson & Quayle, 1995). Thus, the K_DR channel appears to be a negative feedback system to regulate vascular tone. K_DR channels are inhibited by 4-aminopyridine, cesium, and high concentrations of TEA (Hirst & Edwards, 1989). Relatively little is known about the physiological importance of these potassium channels in the cerebral circulation.

KMUP-1, a chemically synthetic xanthine-based derivative, has been demonstrated to raise cyclic nucleotides, inhibit phosphodiesterases (PDEs), and activate K⁺ channels resulting in relaxations in aortic (Wu et al., 2001), corporeal carvenosa (Lin et al., 2002), and tracheal smooth muscles (Wu et al., 2004). Recently, we proposed that tracheal relaxations of KMUP-1 could be mediated via two major pathways, either through (1) activation of K⁺ channels that are independent of cellular cyclic nucleotides; or (2) increases in both cAMP and cGMP, followed by stimulation of PKA and PKG cascades. Increased PKA and PKG appear to activate K⁺ channels, thus resulting in the lowering of cellular Ca²⁺ levels (Wu et al., 2004). The main objective of this study was to investigate further the mechanism by which KMUP-1 could modulate BK_Ca channels in rat basilar artery myocytes. Both conventional and perforated patch-clamp techniques were used to determine whether KMUP-1 enhanced the BK_Ca channel activity through cGMP/cAMP-dependent and -independent signaling pathways.

Methods

Animal procedures and tissue preparations

All procedures and protocols were approved by the Animal Care and Use Committee at Kaohsiung Medical University. Briefly, female Sprague–Dawley rats (10–12 weeks of age) were killed by carbon dioxide asphyxiation. The brain was carefully removed and placed in cold phosphate-buffered saline containing (in mM) 138 NaCl, 3 KCl, 10 Na₂HPO₄, 2 NaH₂PO₄, 5 glucose, 0.1 CaCl₂, and 0.1 MgSO₄ (pH 7.4). Basilar arteries were dissected free of the surrounding tissue and cut into 2 mm segments.

Preparation of isolated arterial smooth muscle cells

Smooth muscle cells from rat basilar arteries were enzymatically isolated as previously described (Welsh et al., 2000). In brief, arterial segments were placed in warm (37°C) cell isolation medium containing (in mM) 60 NaCl, 80 Na-glutamate, 5 KCl, 2 MgCl₂, 10 glucose, and 10 HEPES with 1 mg ml⁻¹ albumin (pH 7.2) for 10 min. After this equilibration step, arterial segments were initially incubated (37°C) in 1 mg ml⁻¹ papain and 3 mg ml⁻¹ dithioerythritol for 20 min. This was followed by a second incubation (37°C) in isolation medium containing 100 μM Ca²⁺, 0.7 mg ml⁻¹ type F collagenase, and 0.4 mg ml⁻¹ type H collagenase for 10 min. After enzyme treatment, the tissue was washed three times in ice-cold isolation medium and triturated with a fire polished pipette to release the myocytes. Cells were stored in ice-cold isolation medium for use on the same day.

Patch-clamp electrophysiology

Conventional whole cell patch-clamp electrophysiology was used to measure the K_DR currents in basilar artery myocytes. In brief, basilar artery myoctyes were placed in a recording dish and perfused with a solution containing (in mM) 120 NaCl, 3 NaHCO₃, 4.2 KCl, 1.2 KH₂PO₄, 2 MgCl₂, 0.1 CaCl₂, 10 glucose, and 10 HEPES. A recording electrode was pulled from borosilicate glass (resistance 4–7 MΩ), the tip was covered with sticky wax and backfilled with pipette solution containing (in mM): 110 K-gluconate, 30 KCl, 0.5 MgCl₂, 5 HEPES, 5 EGTA, 5 Na₂ATP, and 1 GTP (pH 7.2, KOH) and was gently lowered onto a smooth muscle cell. Negative pressure was briefly applied to rupture the membrane and a gigaohm seal was obtained. Cells were subsequently voltage clamped (−60 mV). Membrane currents were recorded on an Axopatch 700A amplifier (Axon Instruments, Union City, CA, U.S.A.), filtered at 1 kHz using a low-pass Bessel filter, digitized at 5 kHz, and stored on a computer for subsequent analysis with Clampfit 9.0. A 1 M NaCl–agar salt bridge between the bath and the Ag-AgCl reference electrode was used to minimize offset potentials. All electrical recordings were performed at room temperature and cell capacitance averaged 16.5±0.6 pF.

Whole cell BK_Ca currents were measured using the conventional or perforated patch-clamp configuration. Under both recording conditions, the bathing solution contained (in mM): 140 NaCl, 5 KCl, 1.8 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4, NaOH). In comparison, the pipette solution contained (in mM) 110 K-gluconate, 30 KCl, 0.5 MgCl₂, 5 HEPES, 5 EGTA, 5 Na₂ATP, and 1 GTP (pH 7.2, KOH). Amphotericin B (200 μg ml⁻¹) was included in the pipette solution for the perforated patch-clamp recordings. With respect to employing the inside-out patch-clamp configuration to monitor single-channel BK_Ca activity, recording pipettes were backfilled with a solution containing (in mM) 140 NaCl, 6 KCl, 0.5 MgCl₂, and 10 HEPES. The bath solution contained (in mM): 140 KCl, 1 MgCl₂, 10 HEPES, 1.8 CaCl₂, 5 EGTA, and 10 glucose. Note, the free Ca²⁺ concentration of these solutions was first calculated using Max Chelator Sliders software (C. Patton, Standford University) and secondarily measured with a Ca²⁺-selective electrode (Corning, Acton, MA, U.S.A.). Single-channel activity in excised patches was recorded at 0 mV, filtered at 2.5 kHz, and digitized at 10 kHz. To measure voltage-dependent Ca²⁺ currents, KCl inside the pipette solution was replaced with equimolar CsCl, and the pH was adjusted to 7.2 with CsOH, whereas the bathing solution contained 1 μM tetrodotoxin and 10 mM tetraethylammonium chloride.

Experimental procedures

Voltage-clamped cells were equilibrated for 15 min prior to experimentation. Following equilibration, whole cell K_DR and BK_Ca currents were monitored in the presence and absence of KMUP-1 (1 μM), ChTX (100 nM), or IbTX (100 nM). To ascertain whether PKG or PKA signaling was involved in the KMUP-1-induced increases in BK_Ca, cerebral smooth muscle cells were preincubated for 15 min with ODQ (10 μM), SQ 22536 (10 μM), KT5823 (300 nM), KT5720 (300 nM), Rp-cGMP (100 μM), or Rp-cAMP (100 μM) prior to the addition of KMUP-1. ODQ, SQ 22536, KT5823 and KT5720 were continuously superfused in the bath whereas Rp-cGMP and Rp-cAMP were added to the pipette solution. In general, the net current–voltage (I-V) relationship was determined at 5 min intervals by measuring the peak current at the end of a 300 ms pulse to voltages between −70 and +40 mV for K_DR currents, and 0 and +40 mV for BK_Ca currents. To evoke whole-cell Ca²⁺ currents, cells were clamped at −40 mV with step depolarizations (200 ms) from −40 to 10 mV and the currents were recorded in the presence and absence of KMUP-1 or nifedipine.

Chemicals

Buffer reagents, 4-aminopyridine (4-AP), 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-br-cAMP), 8-bromo-guanosine 3′,5′-cyclic monophosphate (8-br-cGMP), ChTX, collagenase (type F and H), dithioerythritol, IbTX, isoproterenol, KT 5720, KT 5823, ODQ, papain, and SQ 22536 were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO, U.S.A.). All drugs and reagents were dissolved in distilled water unless otherwise stated. ChTX and ODQ were dissolved in DMSO at 10 mM; KMUP-1 was dissolved in 10% absolute alcohol, 10% propylene glycol, and 2% 1 N HCl at 10 mM. Serial dilutions were made in phosphate buffer solution, with the final solvent concentration ⩽0.01%.

Data analysis and statistics

For single-channel analysis, BK_Ca channel activity (NP₀) was determined from continuous gap-free data by using Clampfit 9.0. The NP₀ was calculated from the following equation:

where i is the number of channels open, t_i is the open time for each level i and T is the total time of analysis.

For BK_Ca currents analysis, I−I_min/I_max–I_min was used to normalize the currents. I_max and I_min indicate the values of control BK_Ca current at 40 and 0 mV, respectively, where I represents the values of BK_Ca current for test agent measured between 0 and 40 mV.

Data are expressed as means±s.e. and n indicates the number of cells. Repeated measure ANOVAs compared values at a given voltage. When appropriate, a Tukey-Kramer pairwise comparison was used for post hoc analysis. P-values ⩽0.05 were considered statistically significant.

Results

Lack of modulation of K_DR currents by KMUP-1

Using conventional whole-cell patch-clamp and pipette solutions that minimize BK_Ca channel activity, the K_DR current was isolated in rat basilar artery smooth muscle cells. In general, brief voltage steps positive to −30 mV activated K_DR (Figure 1b). In the basilar artery myocytes, the K_DR current was recognized by the addition of 4-AP (5 mM) as previously described (Luykenaar et al., 2004). Superfused KMUP-1 (1 μM) had no significant effect on the K_DR current. In contrast, perfusion with 4-AP did significantly inhibit this current (38.6±4.1% at +40 mV) (Figure 1c).

Figure 1.

Effects of KMUP-1 on delayed rectifier K⁺ (K_DR) current in myocytes isolated from rat basilar arteries. (a) Voltage protocol was designed to measure steady-state activation of the K_DR current. (b) Representative recordings of K_DR current before and after the addition of KMUP-1 (1 μM) or 4-AP (5 mM). (c) Average current–voltage (I–V) relationships under control conditions and in the presence of 1 μM KMUP-1 (n=6).

Activation of BK_Ca currents by KMUP-1

Conventional and perforated patch-clamp was used to assess the effect of KMUP-1 on the regulation of outward BK_Ca conductance. Openings of BK_Ca channels were identified based on the characteristic single-channel conductance and blocked by ChTX or IbTX as previously described (Jaggar et al., 2002; Xi et al., 2004). The BK_Ca channels were also recognized by their conductance over the voltage range of −40 to +40 mV (249.5±3.2 pS, n=6) in excised inside-out patches bathed in symmetrical 140 mM KCl (unpublished data). In brief, rat basilar artery myocytes were voltage clamped at 0 mV to inactivate voltage-dependent K⁺ currents (Wu et al., 1999), and continuously superfused with an isotonic physiological solution containing 1.8 mM Ca²⁺ ±1 μM KMUP-1 (Figure 2b). When KMUP-1 was <1 μM in the perfusate, there was little or no effect on BK_Ca channels. As noted in Figure 2c, the KMUP-1-induced increase in BK_Ca current (+30 mV: 1.1±0.1 to 2.4±0.3 pA pF⁻¹; +40 mV: 2.0±0.2 to 4.1±0.7 pA pF⁻¹, n=7, P<0.05) was inhibited by ChTX (100 nM) or IbTX (100 nM; +30 mV: 1.1±0.1 to 0.8±0.1 pA pF⁻¹; +40 mV: 2.0±0.2 to 1.5±0.1 pA pF⁻¹, n=7, P<0.05). The increases of BK_Ca current were consistently observed 5–10 min after the addition of KMUP-1, with the peak steady-state level occurring by 30 min (Figure 2d). Similarly, the membrane-permeable analogs of cGMP (8-Br-cGMP, 1 μM; +30 mV: 1.1±0.1 to 1.8±0.2 pA pF⁻¹; +40 mV: 2.1±0.2 to 3.2±0.5 pA pF⁻¹, n=6, P<0.05) and cAMP (8-Br-cAMP, 1 μM; +30 mV: 1.4±0.2 to 2.0±0.2 pA pF⁻¹; +40 mV: 2.0±0.2 to 3.0±0.3 pA pF⁻¹, n=6, P<0.05) increased BK_Ca activity, a response that was completely abolished in the presence of ChTX (100 nM) and IbTX (100 nM; +30 mV: 1.2±0.1 to 0.8±0.1 pA pF⁻¹; +40 mV: 2.1±0.2 to 1.5±0.2 pA pF⁻¹, n=6, P<0.05; Figure 3).

Figure 2.

Effects of KMUP-1 (1 μM) on large-conductance Ca²⁺-activated K⁺ (BK_Ca) currents. Cells were bathed in high Ca²⁺ solution containing 1.8 mM CaCl₂. (a) Voltage protocol. (b) Representative recordings of BK_Ca currents under control conditions and in the presence of KMUP-1, KMUP-1+ChTX (100 nM) or KMUP-1+IbTX (100 nM). (c) Average I–V relationships under control conditions and in the presence of KMUP-1, KMUP-1+ChTX, or KMUP-1+IbTX. (d) Time course of KMUP-1 on BK_Ca currents. The horizontal bars of the diagram indicate the periods of drug perfusion (n=7). ^*Significant difference from control.

Figure 3.

Enhanced BK_Ca currents elicited by extracellular application of membrane-permeable cGMP and cAMP analogs 8-Br-cGMP (1 μM) and 8-Br-cAMP (1 μM). (a) Representative recordings of BK_Ca currents under control conditions and in the presence of 8-Br-cGMP or 8-Br-cGMP+ChTX. (b) Average I–V relationships under control conditions and in the presence of 8-Br-cGMP, 8-Br-cGMP+ChTX (100 nM) or 8-Br-cGMP+IbTX (100 nM) (n=6). (c) Average I–V relationships under control conditions and in the presence of 8-Br-cAMP, 8-Br-cAMP+ChTX or 8-Br-cAMP+IbTX (n=6). ^*Significant difference from control.

KMUP-1 activates BK_Ca currents via sGC/cGMP- and AC/cAMP-dependent mechanisms

To investigate further the signaling mechanisms that lead to BK_Ca channel activation, KMUP-1 was applied to voltage-clamped cells in the presence of ODQ, a soluble guanylate cyclase (sGC) inhibitor (+40 mV: 2.6±0.2 to 2.1±0.1 pA pF⁻¹, n=6, P<0.05). ODQ (10 μM) markedly blocked the KMUP-1-induced increases in BK_Ca activity, indicating that the modulatory effect of this compound involved the sGC/cGMP pathway (Figure 4). The KMUP-1-induced increase in BK_Ca activity was also partially attenuated (40±5.3% at +40 mV) by the AC inhibitor SQ 22536 (10 μM; +30 mV: 2.0±0.2 to 1.1±0.2 pA pF⁻¹; +40 mV: 3.2±0.3 to 2.0±0.1 pA pF⁻¹, n=7, P<0.05), while the cells superfused with ODQ (10 μM) and SQ 22536 (10 μM) inhibited but did not abolish the channel activity (+40 mV: 2.4±0.2 to 2.0±0.1 pA pF⁻¹, n=7, P<0.05) (Figure 4). This result suggests that KMUP-1 appears to have a direct action on BK_Ca channels in addition to activation of the sGC/cGMP and AC/cAMP pathways.

Figure 4.

BK_Ca current activation by KMUP-1 (1 μM) is dependent on soluble guanylate cyclase and adenylate cyclase. (a) Representative recordings of BK_Ca currents under control conditions and in the presence of KMUP-1, KMUP-1+ODQ (10 μM), KMUP-1+SQ 22536 (10 μM) or KMUP-1+SQ 22536+ODQ. (b) Average I–V relationships under control conditions and in the presence of KMUP-1 or KMUP-1+ODQ (n=6). (c) Average I–V relationships under control conditions and in the presence of KMUP-1, KMUP-1+SQ 22536, or KMUP-1+SQ 22536+ODQ (n=7). ^*Significant difference from control.

KMUP-1 activates BK_Ca currents via PKG- and PKA-dependent pathways

The KMUP-1-induced increase in BK_Ca channel activity was inhibited in the presence of cGMP- and cAMP-dependent protein kinase inhibitors KT5823 (300 nM; +40 mV: 2.6±0.2 to 2.0±0.2 pA pF⁻¹, n=6, P<0.05) and KT5720 (300 nM; +30 mV: 1.7±0.2 to 1.1±0.2 pA pF⁻¹; +40 mV: 3.1±0.2 to 1.9±0.2 pA pF⁻¹, n=6, P<0.05), respectively (Figure 5). The competitive antagonist of cGMP, Rp-cGMP (100 μM), prevented the stimulatory effect of KMUP-1 on BK_Ca when dialyzed into the cell via the patch pipette, but not at 40 mV (Figure 6a and c; from 2.6±0.2 to 2.0±0.2 pA pF⁻¹, n=6, P<0.05). As in the KMUP-1 experiments, Rp-cGMP (100 μM) fully prevented 8-Br-cGMP (1 μM) from enhancing BK_Ca activity (Figure 6c). Interestingly, the inclusion of Rp-cGMP in the pipette solution did not prevent the β-adrenoceptor agonist isoproterenol (1 μM) from stimulating the BK_Ca current (+30 mV: 1.7±0.2 to 1.1±0.2 pA pF⁻¹; +40 mV: 3.0±0.2 to 1.9±0.2 pA pF⁻¹, n=6, P<0.05). The stimulatory effects of isoproterenol were not fully reversible during washout (+40 mV: 2.7±0.1 to 1.9±0.2 pA pF⁻¹, n=6, P<0.05), indicating that this agonist might have a high binding affinity to its own receptors in basilar artery myocytes (Figure 6b and d).

Figure 5.

BK_Ca current activation by KMUP-1 (1 μM) is dependent on PKG and PKA. (a) Representative recordings of BK_Ca currents under control conditions and in the presence of KMUP-1, KMUP-1+KT5823 (300 nM) or KMUP-1+KT5720 (300 nM). (b) Average I–V relationships under control conditions and in the presence of KMUP-1 or KMUP-1+KT5823 (n=6). (c) Average I–V relationships under control conditions and in the presence of KMUP-1 or KMUP-1+KT5720 (n=6). ^*Significant difference from control.

Figure 6.

BK_Ca current activation by KMUP-1 (1 μM) is dependent on cGMP/PKG activity. (a) Representative recordings demonstrating the effect of KMUP-1 (1 μM) on BK_Ca currents with Rp-cGMP (100 μM) in the pipette solution. (b) Representative recordings demonstrating the effect of isoproterenol (1 μM) on BK_Ca currents with Rp-cGMP (100 μM) in the pipette solution. (c) Average I–V relationships in the presence of Rp-cGMP, Rp-cGMP+KMUP-1 or Rp-cGMP+8-Br-cGMP, and wash out (n=6). (d) Average I–V relationships in the presence of Rp-cGMP or Rp-cGMP+isoproterenol, and wash out (n=6). ^*Significant difference from control.

Further experiments revealed that Rp-cAMP (100 μM in the pipette), a competitive antagonist of cAMP, attenuated the effect of KMUP-1-induced increases in BK_Ca activity, but did not prevent the effect of KMUP-1 at ⩾30 mV (Figure 7a and c; +30 mV: 1.7±0.2 to 1.2±0.2 pA pF⁻¹; +40 mV: 2.7±0.2 to 2.0±0.2 pA pF⁻¹, n=6, P<0.05). The addition of Rp-cAMP to the pipette solution prevented 8-Br-cAMP (1 μM) from activating the BK_Ca current (Figure 7c). In contrast, in the presence of Rp-cAMP, the cGMP-dependent NO donor, SNP (100 μM), stimulated BK_Ca (Figure 7b and d; +30 mV: 1.8±0.2 to 1.2±0.1 pA pF⁻¹; +40 mV: 2.9±0.3 to 2.0±0.1 pA pF⁻¹, n=6, P<0.05). From these results, this study cannot exclude the involvement of a PKA-dependent signaling pathway in the activation of BK_Ca channels by KMUP-1.

Figure 7.

BK_Ca current activation by KMUP-1 (1 μM) is dependent on cAMP/PKA activity. (a) Representative recordings demonstrating the effect of KMUP-1 (1 μM) on BK_Ca currents with Rp-cAMP (100 μM) in the pipette solution. (b) Representative recordings demonstrating the effect of SNP (1 μM) on BK_Ca currents with Rp-cAMP (100 μM) in the pipette solution. (c) Average I–V relationships in the presence of Rp-cAMP, Rp-cAMP+KMUP-1 or Rp-cAMP+8-Br-cAMP, and wash out (n=6). (d) Average I–V relationships in the presence of Rp-cAMP or Rp-cAMP+SNP, and wash out (n=6). ^*Significant difference from control.

KMUP-1 activates BK_Ca channels in excised membrane patches

Since BK_Ca channel activation by KMUP-1 was caused by stimulation of the sGC/PKG and AC/PKA pathways, we sought to investigate whether KMUP-1 could activate BK_Ca channels in the complete absence of intracellular signaling factors. KMUP-1 regulation of BK_Ca channel activity was measured in excised inside-out membrane patches with 300 nM free Ca²⁺ present in the bathing solution. At 0 mV, KMUP-1 (1 μM) increased the mean BK_Ca channel open probability ∼2.5-fold (NP₀ from 0.024±0.007 to 0.061±0.015, n=7; Figure 8). These data suggest that KMUP-1 could directly activate BK_Ca channels located on the cerebral artery myocyte membrane, and is not required for stimulating the enzyme systems contained in cytosol.

Figure 8.

KMUP-1 activates BK_Ca channels in excised inside-out membrane patches. (a) Original current recording illustrating BK_Ca channels activation by KMUP-1 (1 μM), voltage-clamped at 0 mV. (b) Current amplitude histograms constructed from the traces shown in a. (c) Bar graph showing the control and in the presence of KMUP-1 on the relative open probability of BK_Ca channels (n=7). ^*Significant difference from control.

Inhibition of L-type Ca²⁺ channels by KMUP-1

The experiment was conducted with a Cs⁺-containing solution. Perfusion with KMUP-1 (1 μM) was found to suppress significantly the voltage-dependent L-type Ca²⁺ currents (I_Ca,L), and the perfusate with a calcium channel blocker nifedipine (1 μM) nearly abolished the currents (Figure 9). KMUP-1 reduced the amplitude of I_Ca,L to 60±7 pA from a control value of 100±9 pA (P<0.05, n=7) when cells were depolarized from −40 to 10 mV, but it did not modify the I–V relationship of I_Ca,L in these cells (unpublished data).

Figure 9.

Effects of KMUP-1 on voltage-dependent L-type Ca²⁺ currents (I_Ca,L) in rat basilar artery myocytes. (a) Voltage protocol. (b) Representative recordings of I_Ca,L before and after the addition of KMUP-1 or nifedipine (1 μM). (c) Bar graph showing the control and in the presence of KMUP-1 or nifedipine (n=6). ^*Significant difference from control.

Discussion

KMUP-1 has been shown to relax aortic, corporeal carvenosa, and tracheal smooth muscles, and elevate cAMP and cGMP levels through inhibition of PDEs. We suggested that the smooth muscle relaxant effects of KMUP-1 are mediated via cyclic nucleotide elevation and K⁺ channel activation (Wu et al., 2001; 2004; Lin et al., 2002). In this study, we first investigated the ability of KMUP-1 to activate BK_Ca channels through cyclic nucleotide-dependent and -independent pathways using patch-clamp electrophysiology.

Potassium channels are the main determinant of resting membrane potential, and their activation causes hyperpolarization, the inhibition of voltage-gated calcium channels, and vascular relaxation. Although multiple classes of K⁺ channels are expressed in a variety of vascular beds, the BK_Ca channel is a particularly important target for physiological regulation (Faraci & Heistad, 1998). The BK_Ca channel is a large conducting channel with conductance values measured at physiological levels of K⁺, ranging from 100 to 250 pS (Faraci & Heistad, 1998; Barman et al., 2003). Similar values were also found in this study in the rat basilar artery. The BK_Ca channel is voltage- and calcium-dependent, and does not display voltage-dependent inactivation (Latorre et al., 1989; Holland et al., 1996). To study the BK_Ca in basilar artery myocytes, we first inactivated K_DR channels through a step depolarization to 0 mV. Under these conditions, BK_Ca is the dominant outward current and KMUP-1 significantly increases the magnitude of this ChTX/IbTX-sensitive conductance (Figure 2). In contrast, KMUP-1 only slightly affected the voltage-sensitive K_DR channels.

To explore whether KMUP-1 could directly affect the BK_Ca channel or a closely associated site, inside-out patch-clamp electrophysiology was used in a number of experiments. In general, we observed that KMUP-1 applied to the cytoplasmic face of inside-out patches could directly activate these channels. These findings are consistent with our previous reports that KMUP-1 rapidly relaxes smooth muscle (Wu et al., 2001; 2004; Lin et al., 2002) presumably through a mechanism that involves the modulation of a K⁺ channel.

Under whole-cell recording conditions, there was a substantial delay (∼5 min) between the addition of KMUP-1 and the activation of BK_Ca currents. This delay contrasts sharply with the very rapid activation of BK_Ca channels in excised patches. A possible explanation for the discrepancy is that the primary site of action of KMUP-1 is intracellular. Thus, the delayed activation of BK_Ca channels by KMUP-1 in whole-cell preparations might be rationally explained by the additional time required to penetrate the cell membrane and to activate the second messenger cascades leading to the stimulation of K⁺ effluxes.

Alterations in BK_Ca channel activity play a central role in mediating vasoconstriction and vasodilatation. For example, studies have noted the involvement of BK_Ca channels in mediating vascular relaxation to agents that elevate cGMP (Zhao et al., 1997). In addition, the relaxation induced by 8-Br-cGMP has been demonstrated to be attenuated by IbTX (Tanaka et al., 1998), a finding consistent with BK_Ca channels being a target of cGMP signaling. This study confirmed that 8-Br-cGMP does indeed increase the magnitude of the BK_Ca current in basilar artery myocytes. Agents that elevate cAMP have also been shown to modulate this current and initiate vascular relaxation (Paterno et al., 1996). In basilar artery myocytes, superfused 8-Br-cAMP did enhance the BK_Ca current; this result is consistent with the work of White et al. (2000) in coronary artery smooth muscle cells. Taken together, PKA and PKG pathways were shown to participate in the regulation of BK_Ca activity in the rat basilar artery because 8-Br-cAMP and 8-Br-cGMP caused a marked augmentation in BK_Ca currents, which was reversed by the competitive antagonists of cAMP (Rp-cAMP) and cGMP (Rp-cGMP). These findings were consistent with previous studies in rabbit cerebral artery (Robertson et al., 1993) and rat tail artery (Schubert et al., 1996). Torphy (1994) also documented that increases in cAMP and cGMP simultaneously activated the PKA and PKG pathways resulting in the opening of BK_Ca channels.

Recently, we have demonstrated that KMUP-1, like the representative sGC activator YC-1, possesses multiple pharmacological activities including sGC activation, inhibition of PDEs, elevation of cyclic nucleotide levels, enhancement of the expression of PKA and PKG, and increase in associated K⁺ channels opening (Wu et al., 2001; 2004). In this study, KMUP-1-induced increases in BK_Ca currents are extremely inhibited, but not abolished, by ODQ together with SQ 22536. This finding indicates that activation of the BK_Ca channel by KMUP-1 is predominantly dependent on both cGMP- and cAMP-mediated signaling mechanisms. However, at least part of the response to KMUP-1 is cGMP- and cAMP-independent mechanisms, and may be due to the direct BK_Ca channel opening. Additionally, enhanced BK_Ca currents by KMUP-1 are reduced by competitive antagonists of cGMP (Rp-cGMP) and cAMP (Rp-cAMP), and by inhibitors of PKG (KT5823) and PKA (KT5720). Compared with KT5823, KT5720 attenuated the KMUP-1-induced increases in BK_Ca currents to a lesser extent. These results suggest that BK_Ca currents activation by KMUP-1 is principally via PKG and partially via PKA.

In summary, we provide the first direct evidence that KMUP-1 activates BK_Ca currents in rat basilar artery. We suggest that KMUP-1 could reduce the activity of Ca²⁺ channels by two routes; directly, by its Ca²⁺ channel-blocking action, and indirectly by activating BK_Ca channels producing hyperpolarization, thus decreasing the open probability of Ca²⁺ channels through their voltage dependence. Functionally, both these effects could contribute to its vasorelaxant property, and the hypothesis is confirmed in rat aorta (Wu et al., 2001). Finally, the stimulatory effect of KMUP-1 on BK_Ca channels is mediated by PKA and PKG, which phosphorylate the channels or associated regulatory proteins, enhancing the K⁺ effluxes, and could lead to membrane hyperpolarization and closure of voltage-dependent L-type Ca²⁺ channels.

Abbreviations

AC

adenylate cyclase

8-Br-cAMP

8-bromo-adenosine 3′,5′-cyclic monophosphate

8-Br-cGMP

8-bromo-guanosine 3′,5′-cyclic monophosphate

BK_Ca channels

large-conductance Ca²⁺-activated potassium channels

ChTX

charybdotoxin

IbTX

iberiotoxin

K_DR channels

delay-rectifying potassium channels

ODQ

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one

PKA

protein kinase A

PKG

protein kinase G

sGC

soluble guanylate cyclase

SQ 22536

9-(terahydro-2-furanyl)-9H-purin-6-amine