Heterogeneity in function of small artery smooth muscle BK_Ca: involvement of the β1-subunit

Abstract

Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage-gated Ca²⁺ channels. To prevent positive feedback and excessive pressure-induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca²⁺-activated K⁺ channels (BK_Ca) provides an opposing hyperpolarizing influence reducing Ca²⁺ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K⁺ currents were determined at pipette [Ca²⁺] of 100 nm or 5 μm in the presence and absence of the BK_Ca inhibitor, iberiotoxin (IBTX; 0.1 μm). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca²⁺ levels. At 5 μm Ca²⁺, cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 ± 1.9 vs 45.5 ± 1.7 pA pF⁻¹ at +70 mV). Studies with IBTX suggested that the differences in K⁺ conductance at 5 μm intracellular [Ca²⁺] were largely due to activity of BK_Ca. 17β-Oestradiol (1 μm), reported to potentiate BK_Ca current via the channel's β-subunit, caused a greater effect on whole cell K⁺ currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the α-subunit-selective BK_Ca opener, NS-1619 (20 μm), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca²⁺ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the β1 subunit and a decrease in the β 1: α ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BK_Ca protein and the β 1: α-subunit ratio. The data support vascular heterogeneity with respect to the activity of BK_Ca in terms of both β-subunit regulation and interaction with SR-mediated Ca²⁺ signalling.

Arteriolar myogenic vasoconstriction occurs when intraluminal pressure-induced stretch, or increases in membrane tension, leads to smooth muscle cell (SMC) depolarization and a subsequent opening of voltage-gated Ca²⁺ channels (Davis & Hill, 1999; Hill & Davis, 2007). While the exact events remain unclear, the depolarization is thought to be mediated via the opening of non-selective cation channels, possibly involving stretch-activated channels (Davis et al. 1992) and/or various Trp channels (Welsh et al. 2002; Earley et al. 2004; Spassova et al. 2006; Earley et al. 2007). The pressure-induced entry of Ca²⁺ into arteriolar SMCs then activates myosin light chain kinase and contraction (Zou et al. 1995, 2000). In addition, evidence exists for involvement of a number of other mechanisms in maintenance of myogenic contraction including roles for integrin activation (Martinez-Lemus et al. 2003; Sun et al. 2008), cytoskeletal remodelling (Gokina & Osol, 2002; Flavahan et al. 2005) and Ca²⁺ sensitization (Lagaud et al. 2002; Schubert et al. 2008).

Historically, the physiological relevance of myogenic constriction has been criticized based on the predicted instability of pressure-induced vasoconstriction due to its predicted feed-forward nature (Folkow, 1964; Koch, 1964). Studies in cerebral artery smooth muscle have suggested that simultaneous activation of the large conductance, Ca²⁺-activated K⁺ channel (BK_Ca) provides an opposing hyperpolarizing stimulus (Brayden & Nelson, 1992; Nelson et al. 1995; Knot et al. 1998) to prevent such a positive feedback situation from producing excessive pressure-induced vasoconstriction. The resultant hyperpolarization thus acts to limit excessive pressure-induced constriction by decreasing the opening of L-type voltage-gated Ca²⁺ channels.

In this schema, activation of BK_Ca is proposed to occur as a result of close proximity between the plasma membrane channel and ryanodine-sensitive Ca²⁺ release sites on the superficial sarcoplasmic reticulum (SR) (Nelson et al. 1995; Knot et al. 1998; Jaggar et al. 2000). Ca²⁺ sparks from the SR promote activation of clusters of BK_Ca channels and the appearance of spontaneously transient outward currents (STOCs) (Perez et al. 1999; Jaggar et al. 2000). In the context of myogenic reactivity, Ca²⁺ sparks are initiated by Ca²⁺ entry resulting from pressure-induced membrane depolarization (Nelson et al. 1995).

Although widely expressed in mammalian tissues, including smooth muscle (Dworetzky et al. 1994; Tseng-Crank et al. 1994), an important question is whether the BK_Ca channel exhibits the same myogenic regulatory function in all vascular beds. Based on apparent differences in the relationship between SMC membrane potential (E_m) and level of active myogenic tone in cannulated cremaster muscle arterioles and small cerebral arteries, we have suggested that this would be unlikely (Kotecha & Hill, 2005). Specifically, it was suggested that under basal conditions, skeletal muscle arterioles maintain a high level of vascular resistance and that the presence of a robust BK_Ca-mediated mechanism to inhibit myogenic tone (i.e. to the same extent as seen in the cerebral circulation) may be counter-productive. In addition to understanding heterogeneity in the physiological contribution of BK_Ca in different vascular beds, such information is important if this K⁺ channel is to be used as a therapeutic target in vascular disease (Lawson, 2000; Gribkoff et al. 2001; Gribkoff & Winquist, 2005).

On the basis of the above considerations, we have carried out electrophysiological and high-speed Ca²⁺ imaging studies on freshly dispersed arteriolar smooth muscle cells as well as isolated arteriole preparations to examine the role of BK_Ca in skeletal muscle arterioles. Specifically, it was hypothesized that relative to the cerebral vasculature, BK_Ca of cremaster muscle arterioles is not tightly coupled to myogenic reactivity. Experiments were conducted to compare and contrast the electrophysiological properties (i.e. current characteristics, STOCs) and expression (protein and mRNA levels) of BK_Ca in vascular smooth muscle from cremaster muscle artery with BK_Ca in small cerebral arteries. The findings of our study demonstrate differences in the BK_Ca properties between these two vessel types, suggesting that the fine-tuning of BK_Ca activity in VSM may contribute to differing degrees of myogenic reactivity and flow resistance amongst tissue vascular beds.

Methods

Studies used male Sprague–Dawley rats weighing 180–280 g in body weight. Prior to studies, animals were housed in a temperature, humidity and light controlled animal facility with free access to standard rat chow and drinking water. All procedures were approved by the Animal Care and Use Committees of the University of Missouri, USA and the University of New South Wales, Australia.

Rats were anaesthetized with sodium pentobarbital (Nembutal, 60 mg (kg body weight)⁻¹) given by intraperitoneal injection. Cremaster muscles were surgically removed, as previously described (Meininger et al. 1991), and placed in a cooled (4°C) dissection chamber. Following killing by anesthetic overdose, a craniotomy was performed and the brain removed and similarly placed in a cooled dissection chamber.

Vessel dissection and cell isolation

Arteriolar myocytes from first- and second-order arterioles (1A/2A) were isolated according to the methods of Wu et al. (2001) and Jackson et al. (1997) with slight modification. In brief, cremaster muscles were excised and pinned flat for vessel dissection at 4°C in Ca²⁺-free physiological saline solution (PSS) containing (mm): 140 NaCl, 5.6 KCl, 1.0 MgCl₂, 1.2 NaH₂PO₄, 5.0 d-glucose, 2.0 sodium pyruvate, 0.02 EDTA, 3 mm Mops, plus 0.1 mg ml⁻¹ bovine serum albumin (BSA, Amersham Life Science, Arlington Heights, IL, USA). Dissected segments of arterioles were transferred to a 1 ml tube of low-Ca²⁺ physiological salt solution (PSS) containing (mm): 144 NaCl, 5.6 KCl, 0.1 CaCl₂, 1.0 MgCl₂, 0.42 Na₂HPO₄, 10 Hepes, 2 sodium pyruvate, and 1 mg ml⁻¹ BSA at room temperature for 10 min. The solution was decanted and replaced with a similar solution containing 26 U ml⁻¹ papain (Sigma, St Louis, MO, USA) and 1 mg ml⁻¹ dithioerythritol. The vessels were incubated for 30 min at 37°C with occasional agitation, then transferred to a new tube containing low-Ca²⁺ PSS containing 1.95 U ml⁻¹ collagenase (FALGPA, Sigma), 1 mg ml⁻¹ soybean trypsin inhibitor (Sigma) and 75 U ml⁻¹ elastase (Calbiochem, La Jolla, CA, USA), and incubated for 7–10 min at 37°C. After further digestion, the remaining fragments were gently rinsed 2–3 times with low-Ca²⁺ PSS without BSA and gently triturated using a fire-polished Pasteur pipette to release single cells. Spindle-shaped arteriolar myocytes were used within approximately 4 h of isolation.

Smooth muscle cells from rat cerebral arteries were enzymatically isolated as previously described (Wu et al. 2007). Briefly, arterial segments were placed in Ca²⁺-free PSS as above (37°C, 10 min). Vessels were then exposed to a two-step digestion process that involved (1) a 15 min incubation in isolation media (37°C) containing 0.6 mg ml⁻¹ papain and 1.8 mg ml⁻¹ dithioerythritol, and (2) a 5–6 min incubation in low-Ca²⁺ PSS with 0.7 mg ml⁻¹ type F collagenase, and 0.4 mg ml⁻¹ type H collagenase. Following enzyme treatment, tissues were washed repeatedly with ice-cold low Ca²⁺ PSS and triturated with a fire-polished pipette. Liberated smooth muscle cells were stored in ice-cold isolation medium for use within 4 h.

The rationale for using different enzymatic procedures for isolating cremaster and cerebral arterial myocytes was based on using methods described in the literature for these specific vessels. Control experiments were performed to ensure that the procedures, per se, did not introduce differences in electrophysiological and Ca²⁺ handling properties. K⁺ currents and Ca²⁺ spark characteristics were unaffected by which enzyme digestion procedure was used (see online Supplemental Material, Fig. S1).

Electrophysiology

Conventional patch-clamp (Hamill et al. 1981) electrophysiology and protocols (Yang et al. 2007; Wu et al. 2008) were used to measure macroscopic whole cell potassium currents (I_k) in isolated cremaster and cerebral smooth muscle cells. The amplifier (EPC-7, HEKA, Germany) was controlled by a Dell XPS computer running pClampex 9.0 software through a Digidata 1322A computer interface (Axon Instruments). Clampfit 9.0 and Sigma Plot 9.0 (Systat Software Inc., San Jose, CA, USA) were used for data analysis. Micropipettes were pulled from borosilicate glass capillaries (Corning 8161; ID, 1.2 mm; OD, 1.5 mm; World Precision Instruments, Sarasota, FL, USA) using a Sutter P-97 electrode puller (Sutter Instrument Co., Novato, CA, USA).

Pipette tip resistances ranged from 3.0 to 5.0 MΩ when filled with standard intracellular solution. Whole-cell K⁺ currents were evoked by voltage steps delivered from a typical holding potential of −60 mV to potentials ranging from −70 to +70 mV, in 20 mV increments. Cell capacitance ranged between 14 and 18 pF and was measured with the cancellation circuitry in the voltage-clamp amplifier. The series resistance (< 10 MΩ) was compensated to minimize the duration of the capacitive surge. Subtraction of leak currents was not performed. Whole cell currents were normalized to cell capacitance and expressed as picoampere per picofarad (pA pF⁻¹). BK_Ca currents were identified as the IBTX-sensitive component of total K⁺ current. All experiments were performed at room temperature.

For whole-cell recordings, the bath solution contained (in mm): 140 NaCl, 5.4 KCl, 1.5 CaCl₂, 1 MgCl₂, 10 glucose, 10 Hepes, 2 sodium pyruvate (pH 7.4). The 140 mm K⁺ pipette solution contained (in mm): 140 KCl, 8 NaCl, 1–2 EGTA, 3 Mg-ATP, 10 Hepes (pH 7.2); CaCl₂ was added to bring free [Ca²⁺] to 100 nm and 5 μm. MgATP was included to inhibit ATP-sensitive K⁺ channels and provide substrate for energy-dependent processes. Where addition of other reagents to the bath and/or pipette solutions was required for specific experiments, details are given in protocols and figure legends.

Measurements of E_m were performed using the whole cell perforated patch technique. Perforated cell patches were produced by the addition of nystatin (15 μg ml⁻¹) to the pipette solution. E_m was measured in current clamp mode.

Ca²⁺ imaging

Dispersed cells were aliquoted into wells of Tissuetek® slide chambers and allowed to adhere to the coverglass bottom (30 min). Cells were then loaded with Fluo-4 AM (5 μm) for an additional 30 min at room temperature after which the cells were washed with physiological salt solution. For fluorescence imaging, the cell chambers were positioned on the stage of an inverted microscope (Olympus IX71) equipped with a Nipkow confocal spinning disk unit (CSU-10; Solamere) and a Stanford Photonics XR Mega-10EX S30 intensified CCD. The microscope was equipped with a water immersion objective lens (60×, NA 1.2; Olympus). The camera was operated under Bin-2 mode (512 × 512 pixel) to allow acquisition speeds of 60 frames s⁻¹. The typical image size was 80 × 125 μm (height × width). Image acquisition was performed using Quad InVivo 3.2.1 (Media Cybernetics, Bethesda, MD, USA).

For analysis of changes in Ca²⁺-related fluorescence a region of interest (ROI) was drawn around the area of an apparent spark. Similarly, an ROI was drawn in area of the cell where fluorescence levels remained constant for the data collection period (20 s). Fluorescence intensity was monitored across each ROI as an indicator of ‘spark-related’ fluorescence (F) and background fluorescence (F_o), respectively. Changes in fluorescence intensity were then expressed as F/F_o. Ca²⁺ sparks were characterized according to rise time, decay time (exponential rate constant τ₁ and τ₂) and full width spread at half maximal intensity (FWHM). Similarly, full circumference spread at half maximal intensity (FCHW) was calculated with the exception that fluorescence intensity was measured within a circumscribed ROI rather than along a line. Where a given cell did not exhibit apparent spark activity the image was divided into 10 regions of interest and fluorescence levels plotted for the 20 s image collection period. Where changes in fluorescence were detected in these areas of interest, the areas were further subdivided in attempt to localize discrete areas showing time-dependent changes in fluorescence. Data analyses were performed using Image Pro (Media Cybernetics) and ImageJ (NIH).

Real-time PCR

Total RNA was extracted immediately from freshly isolated arterioles, using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. RNA was quantified using by measuring absorbance at 260 nm and 280 nm. Equal amounts of RNA samples were reverse-transcribed into cDNA using a Sensiscript RT reverse transcription kit (Qiagen). Quantitative real-time PCR was performed using a Mastercycler EP Realplex rapid thermal cycler system (Eppendorf AG, North Ryde, New South Wales). Primers were designed to amplify BK_Ca-α (forward primer 5′-AAACAAGTAATTCCATCAAGCTGGTG; reverse primer 5′-CGTAAGTGCCTGGTTGTTTTGG), BK_Ca-β₁ (forward primer 5′-GTCTGCATCTTTGGGGATGT; reverse primer 5′-GGGGAAGGTGTGCAGTGTTT) and β-actin (forward primer 5′-AAGTCCCTCACCCTCCCAAAAG; reverse primer 5′-AAGCAATGCTGTCACCTTCCC). The reaction mixture contained 8 μl of Eppendorf Real Sybr Green Mastermix, 1 μl SYBR probe, 2 μl cDNA (300 mm), and 2 μl of the relevant forward and reverse primers and 5 μl RNAse-free water. The PCR conditions were as follows: denaturation, 2 min at 95°C; amplification, 15 s at 95°C, 15 s at 58°C, 20 s at 68°C (40 cycles). Melting curves were routinely performed for analysis of PCR product purity. BK_Ca-subunit expression relative to α-actin (ΔC value) was calculated using the Realplex software, by selecting an individual α-actin sample as an arbitrary calibrator against which all other samples are expressed as fold difference.

Western blotting

First order cremaster arterioles and the main branch of the mid-cerebral artery were removed from Sprague–Dawley rats (between 5 and 10 animals/n; n= 5) as described above. Extraneous tissue was carefully removed and the arteries were stored in liquid nitrogen. Arteries were ground in liquid nitrogen using a pestle and mortar, resuspended in phosphate-buffered saline (PBS) pH7.4 containing complete protease inhibitor cocktail (Roche) and centrifuged (3000 g, 4°C, 5 min). The supernatant was removed and placed on ice and the pellet was snap-frozen in liquid nitrogen and processed again as described above. Following a second centrifugation the supernatants were pooled and centrifuged (25,000 g, 4°C, 1 h) after which the membrane-enriched pellet was carefully resuspended in PBS containing 0.1% Triton X-100 and protease inhibitor cocktail, aliquoted, snap-frozen in liquid nitrogen and stored at −80°C. Protein concentration of the samples was determined using the Bradford protein assay (Bio-Rad).

Aliquots of the protein extracts (5 μg protein unless otherwise indicated) were dissolved in lithium dodecyl sulfate (LDS) sample buffer (0.5% LDS, 62.5 mm Tris-HCl, 2.5% glycerol, 0.125 mm EDTA, pH 8.5) for 10 min at 70°C. The samples were separated by electrophoresis in bis-Tris polyacrylamide gels using MES SDS running buffer and electroblotted onto PVDF membranes overnight at 4°C, according to the manufacturer's recommendations (Invitrogen). Following transfer, blots were thoroughly washed, blocked, probed with primary antibody and specific binding was visualized using alkaline phosphatase-conjugated secondary antibody and chemiluminescence according to the manufacturer's instructions (Invitrogen). See online Supplemental Material for details of antibodies used. The intensity of the band corresponding to each full length protein was quantified by digital densitometry using ImageJ software. Relative intensity for each full length band was determined by comparison with the intensity of actin staining. The resulting ratios were expressed as means ±s.e.m. and analysed by two tailed t-test.

To determine specificity, each antibody was incubated with its cognate peptide in order to block specific binding. Prior to use, peptide was added to antibody in a 1 : 1 ratio (w/w), mixed and incubated at 37°C for 1 h, then overnight at 4°C. The blocked antibody was then used in Western blotting detection as described above (see Supplemental Material).

Functional vessel studies

Cremaster muscle arterioles (1A) were isolated as described above and cannulated on glass micropipettes as previously described (Meininger et al. 1991). Arterioles were pressurized under no flow conditions and studied using video microscopy. Only vessels without pressure leaks and displaying spontaneous myogenic tone were studied. Where appropriate, ryanodine and/or iberiotoxin were added to the superfusate to deplete ryanodine-sensitive Ca²⁺ stores and inhibit BK_Ca, respectively.

Measurements of smooth muscle membrane potential were performed in pressurized arteriolar preparations using glass microelectrodes filled with 2 m KCl (tip resistances of 100–200 MΩ) and an Axoclamp 2B amplifier (Axon Instruments). Impalements were performed using a Leitz precision micromanipulator in a region of the vessel demonstrating typical vasoreactivity and with the pipette approaching the vessel wall from the abluminal surface. Successful recordings were characterized by an abrupt deflection of signal on impalement of smooth muscle cells and an approximate return to pre-impalement values on removal of the microelectrode.

Statistics and data analysis

In general, for analyses of macroscopic, whole-cell recordings, raw current values were normalized to cell capacitance and expressed as current density (pA pF⁻¹). Recording of STOCs was performed in the whole cell mode with a transient outward current of 10 pA set as a threshold value for calculation of event frequency. Group arteriole diameters were normalized relative to the passive diameter of each arteriole measured in 0 mm Ca²⁺/2 mm EGTA containing PSS.

Summary data are expressed as means ±s.e.m. Statistical significance was determined using Student's t test for paired or unpaired data, or ANOVA, as indicated for specific protocols. Significance levels of P < 0.05 were considered significant.

Chemicals and reagents

NS-1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one] was obtained from Biomol International, L.P. (Plymouth Meeting, PA, USA). 17β-oestradiol (E2) and iberiotoxin (IBTX) were purchased from Sigma-Aldrich (St Louis, MO, USA). NS-1619 stock (10 mm) was made with DMSO while a stock solution of E2 (1 mg ml⁻¹) was made in ethanol. IBTX was dissolved in distilled water. Ryanodine was obtained from Sigma and prepared as a stock solution (10 mm) in methanol. All stock solutions were stored frozen at −20°C and final concentrations were obtained by dilution in appropriate physiological salt solutions.

Results

Whole Cell K⁺ currents in VSM cells from cremaster and cerebral arteries

Whole cell K⁺ currents for cremaster and cerebral VSM were measured at pipette [Ca²⁺] of 100 nm and 5 μm over a voltage range of −70 to +70 mV. Representative tracings are shown in Fig. 1 and group data in Figs 1 and 2. When macroscopic current data at 5 μm Ca²⁺ were normalized for cellular capacitance, cremaster VSM showed a significantly (P < 0.05) lower current density compared to that for cerebral VSM (34.5 ± 1.9 vs 49.1 ± 2.3 pA pF⁻¹ at + 70 mV) (Fig. 1C and D). Similar current densities were observed for the two cell preparations at the lower pipette Ca²⁺ level (Fig. 1A and B). Using IBTX (0.1 μm) as a specific inhibitor of BK_Ca current, it appeared that the difference in K⁺ conductance between cremaster and cerebral VSM cells under conditions of 5 μm intracellular [Ca²⁺] was largely due to differences in the activity of BK_Ca (Figs 1C and D and 2A and B). Subsequent addition of 4-AP (1 mm; in the continued presence of IBTX), to inhibit Kv1 and Kv2-type voltage-gated channels, suggested a similar contribution of these channels to total K⁺ current in cremaster and cerebral VSM (Fig. 1). Figure 2B presents mean data (from Fig. 1) calculated as the difference between current density data recorded under low (100 nm) and high (5 μm) Ca²⁺ conditions. The left-shifted curve calculated from the cerebral VSM cell data is suggestive of a BK_Ca current exhibiting a higher Ca²⁺ sensitivity relative to cremaster VSM cells, or possibly a higher density of functional BK_Ca channels in cerebral VSM.

Figure 1. Group data for K⁺ current measured using whole cell patch clamp.

Relative contributions of BK_Ca and Kv were isolated by sequential addition of IBTX (10⁻⁷m) and IBTX + 4AP (1 mm) (see also Fig. 3). A and C, cremaster muscle VSM at 100 nm and 5 μm pipette [Ca²⁺] respectively. B and D, cerebral artery VSM at 100 nm and 5 μm pipette [Ca²⁺]. C and D insets show example recordings for each cell preparation. Group data are shown as means ±s.e.m.; A–C, n= 12–14 cells from 5 arteries and D, n= 18–20 cells from 7 arteries.

Figure 2. Magnitude of Ca²⁺-activated whole cell current is greater in cerebral vs cremaster VSMCs.

A, group data illustrating the IBTX-sensitive, or BK_Ca, component of total whole K⁺ current for cerebral and cremaster artery VSM at pipette [Ca²⁺] of 100 nm and 5 μm and a holding potential of +70 mV. Data are shown as the IBTX-sensitive current density as a fraction of total K⁺ current. Results are presented as mean ±s.e.m.; n= 12–14 cells. B, I–V‘difference’ curves illustrating the magnitude of Ca²⁺-sensitive current present in VSM cells from cremaster and cerebral arteries. Graphs were constructed by subtracting the measured mean (from group data in Fig. 1) currents at 100 nm Ca²⁺ from those measured at 5 μm Ca²⁺ at each test membrane potential. The upper graph shows total whole cell K⁺ current, while the lower shows the IBTX (10⁻⁷m)-sensitive component.

Pharmacological modulation of K⁺ currents in VSM cells from cremaster and cerebral arteries

To provide pharmacological evidence for differences in VSM BK_Ca function between cremaster and cerebral arteries, whole cell K⁺ currents were examined before and after acute exposure to 17β-oestradiol (oestrogen; 1 μm). Oestrogen and related compounds (i.e. tamoxifen) have been reported to enhance BK_Ca current in VSM via an acute, non-genomic effect at the level of the β-subunit of the channel (Valverde et al. 1999; Dick et al. 2001; Dick & Sanders, 2001). In the presence of a pipette [Ca²⁺] of 100 nm cerebral VSM cells showed a substantial increase in K⁺ current in the presence of oestrogen (Fig. 3B). At a test potential of +70 mV, K⁺ current was increased to 160 ± 13% of control (Fig. 4B). In contrast, under the same recording conditions, cremaster cells did not show a significant increase in macroscopic K⁺ current in response to oestrogen (116 ± 11; Fig. 3A and 4B). At a pipette [Ca²⁺] of 5 μm, VSM cells from both cerebral artery and cremaster 1A showed significant enhancement of K⁺ current in response to oestrogen (Figs 3C and D and 4A and B). IBTX (10⁻⁷m) prevented the oestrogen-induced effect in both VSM cell preparations (Fig. 3).

Figure 3. Group data for the effect of oestrogen (E2; 1 μm) on whole cell K⁺ current.

A and C, cremaster VSM at 100 nm and 5 μm pipette [Ca²⁺] respectively; B and D, cerebral artery VSM at 100 nm and 5 μm pipette [Ca²⁺]. Filled circles show control current density–voltage relationships and open circles show the effects of acute E2 exposure. Inverted triangles show that IBTX inhibits effects of E2 consistent with a role for BK_Ca. Results are shown as means ±s.e.m.; n= 4–8 cells.

Figure 4. Group data comparing the relative effects of oestrogen (E2) (A and B) and NS-1619 (C and D) on whole cell K⁺ currents for cerebral and cremaster SMCs.

Data are extracted from the current density–voltage relationships shown in Fig. 3 and Supplemental Fig. S4. A greater potentiating effect of E2 on cerebral SMCs is evident in A and B. The two SMC preparations showed similar degrees of current enhancement by NS-1619 (C and D). The data suggest a greater role for β1-subunit-mediated regulation of the BK_Ca channel in cells from cerebral arteries. Experimental descriptors provided beneath each bar in C and D also apply to the histograms positioned immediately above. Results are shown as means ±s.e.m.; *P < 0.05.

Additional experiments were performed using the BK_Ca channel opener NS-1619 (20 μm), an agent reported to predominately act on the pore-forming α-subunit of the channel (Gribkoff et al. 1996). NS-1619 caused a significant increase in whole-cell K⁺ current in both cremaster and cerebral VSM cells at 100 nm and 5 μm pipette Ca²⁺ concentrations (Fig. 4C and D and Supplemental Material Fig. S4).

Figure 4 shows the comparative effect of oestrogen and NS-1619 stimulation on VSM cells from the cremaster and cerebral arteries. These observed differences in oestrogen responsiveness, in the presence of similar responses to NS-1619, are consistent with functional differences in BK_Ca activity (i.e. enhanced Ca²⁺ sensitivity) being at the level of the channel's β-subunit.

STOCs in VSM cells from cremaster and cerebral arteries

As the opening of clusters of BK_Ca channels has been shown to give rise to STOCs in VSM cells, which increase in frequency and amplitude as holding potential is set at increasingly positive potentials (Benham & Bolton, 1986; Bolton & Imaizumi, 1996; Wang et al. 1997), experiments were performed to compare the characteristics of these currents in VSM cells from cremaster 1A/2A and cerebral arteries. At holding potentials of −30, 0 and +20 mV, STOC frequency and amplitude were significantly (P < 0.001) higher in cells from cerebral vessels compared to those from cremaster 1A (Fig. 5).

Figure 5. Characteristics of STOCs in cremaster and cerebral VSM cells.

A, typical examples of STOCs recorded from a cerebral and a cremaster artery VSM cell at a holding potential of 20 mV and pipette [Ca²⁺] of 100 nm. Recordings were made for 40 s. Greater STOC frequency and amplitude can be appreciated in the recording from the cerebral VSM cell. B and C, group data for frequency and amplitude, respectively, at holding potentials of −30 and 0 mV. Group data are presented as means ±s.e.m.; n= 6–8. *P < 0.05, ***P < 0.001.

The differences in STOCs between cremaster and cerebral VSM cells were not a function of differences in cell size. Cell capacitance was 18.1 ± 0.3 pF in cremaster VSM cells compared to 19.6 ± 0.2 pF in cerebral VSM cells. Further, image-based measurements of freshly prepared cells indicated that VSM cells from cremaster and cerebral vessels were of a similar size (data not shown).

To further define the nature of the STOCs and their dependence on SR Ca²⁺ stores, cells were exposed to ryanodine (10 μm), IBTX (0.1 μm) or caffeine (100 μm) and whole cell recordings performed as above. In both cell preparations STOCs were inhibited by IBTX, confirming their reliance on BK_Ca (Fig. 6). Similarly, ryanodine inhibited STOCs suggesting that the outward currents were dependent on SR function (Fig. 6). Supporting this, acute application of caffeine caused an increase in STOC frequency in both cremaster and cerebral VSM cells (Fig. 7).

Figure 6. Frequency and amplitude of STOCs are greater in cerebral vs cremaster VSMCs.

A, effect of ryanodine (10 μm) on the appearance of STOCs. Left show example tracings for cremaster and cerebral VSM cells at a holding potential of +20 mV. A positive E_m was used to increase likelihood of STOC generation in cremaster cells. Right hand show group data analysed in terms of frequency and amplitude. Group data are shown as means ±s.e.m. for 10 cells for each of the cremaster and cerebral VSM preparations. Ryanodine decreased the appearance of STOCs in both preparations. B, effect of IBTX (0.1 μm) on the appearance of STOCs. Holding potential =+20 mV. Data are shown as means ±s.e.m. for 6 cells for each of the cremaster and cerebral VSM preparations. IBTX decreased the appearance of STOCs in both preparations.

Figure 7. Caffeine-induced stimulation of STOCs in cremaster and cerebral artery VSM cells.

STOCs were determined at a holding potential of +20 mV. Results are shown as means ±s.e.m.; n= 8 per group.

Ca²⁺ sparks in VSM cells from cremaster and cerebral arteries

Freshly isolated VSM cells from cerebral vessels showed spontaneous, transient, spatially restricted and non-propagating Ca²⁺ release events similar to those previously defined as sparks (Jaggar et al. 2000; Fig. 8A and C). Characteristics (rise time, decay time, FWHM, FCHM) of these events are described in Table 1. While Ca²⁺ sparks were identified in 73% of cerebral VSM cells studied (76 of 105 cells), similar events were not seen in VSM cells from cremaster 1A vessels (0 of 76 cells) under comparable recording conditions (Fig. 8B and Table 1). As positive controls, cremaster VSM cells showed detectable increases in global fluorescence in response to phenylephrine (1 μm) and a high concentration of caffeine (2 mm) (data not shown).

Figure 8. Recording of Ca²⁺ sparks in isolated SMCs.

A, a localized, spontaneous and transient increase in [Ca²⁺]_i, in an isolated VSM cell from cerebral artery, consistent with the occurrence of a Ca²⁺ spark, as previously described by Jaggar et al. 2000 in cerebral artery SMCs (see also Table 1 for group statistical data describing measured Ca²⁺ events). The figure provides both a top view of the whole cell and a surface plot of a region of interest drawn around the area of the apparent spark. B, an image series (whole cell, top view) collected over the same time period as that shown for the cerebral VSM cell in A. C, fluorescence vs. time occurring along a line superimposed on the spark site for the cerebral cell shown in A or the 10 regions of interest (ROI) drawn across the cremaster VSM cell in B. The cerebral VSM cell exhibited 3 sparks during the 20 s observation period with minimal change being observed in total cellular fluorescence. Similar spontaneous Ca²⁺ events were not seen in the cremaster cells despite being imaged under the same conditions and despite the cells exhibiting similar E_m to that of the SMCs isolated from cerebral arteries (see text for details) The right hand graph in C shows fluorescence signals versus time for the 10 ROIs covering the typical cremaster SMC.

Table 1.

Characteristics of Ca²⁺ Sparks

	Cremaster	Cerebral	Jaggar et al. (2000)
Frequency	0% (0 of 76 cells†)	73% (76 of 105 cells††)
Rise Time	—	34.1 ± 2.7 ms	20–95 ms
Decay Time	—	Total = 341 ± 26 ms	τ1= 27–60 ms
		τ1= 42.5 ± 7.5 ms	τ2= 275 ms
		τ2= 298 ± 26 ms
Duration	—	382 ± 23 ms
FWHM	—	2.1 ± 0.4	1.4–1.5
FCHM	—	10.2 ± 0.5

^†From 6 vessel segments;

^††from 9 vessel segments.

To determine whether isolated smooth muscle cells from cremaster and cerebral vessels exhibited comparable resting E_m levels, cells were studied using the perforated patch technique in current clamp mode. Cells from both vessels showed comparable resting E_m (−38.2 ± 2.8 vs. −39.8 ± 2.3; n= 9 and 12, cremaster and cerebral respectively) and depolarizing responses when exposed to 60 mm KCl.

To examine whether Ca²⁺ sparks could be induced in the cremaster VSM cells, studies were conducted with a low concentration of caffeine (100 μm) as previously described by Janiak et al. (2001) and Borisova et al. (2007). On acute exposure to caffeine, cerebral VSM cells (n= 17) showed a significant increase in spark frequency and the number of sites in an image plane of a given cell displaying Ca²⁺ sparks (Fig. 9). Consistent with the data described above, none of the cremaster VSM cells (n= 24) showed detectable sparks prior to stimulation while only two cells showed sparks on addition of caffeine (100 μm) (see Supplemental Fig. S5).

Figure 9. Caffeine (100 μm)-induced increases in the generation of Ca²⁺ sparks in isolated cerebral VSM cells (n= 17).

A, an example trace illustrating increased spark frequency, at two sites, upon addition of caffeine. In this case an eventual increase in global Ca²⁺ was observed. An increase in the number of spark sites per cell (B) was observed, in addition to an increase in frequency (C). Group data are presented as means ±s.e.m.; n= 17 cells, *P < 0.05.

Detection of α and β BK_Ca subunits by real-time PCR

The expression of mRNA for BK_Caα- and β1-subunits in cremaster arterioles and small cerebral arteries was examined using quantitative, real-time RT-PCR. This process was selective for the relevant subunits as demonstrated by the appearance of a single product by melting-curve analysis. Expression of mRNA encoding for the α-subunit, relative to β-actin expression, was not significantly different between the arteries while expression of the β1-subunit mRNA was significantly less in the cremaster arteriole (Fig. 10A). When β1 mRNA levels were expressed as a ratio relative to α subunit mRNA, a significant difference in expression was also apparent between cremaster and mid-cerebral vessels (Fig. 10B). Specifically, the ratio of β1 : α subunit mRNA was approximately 2.8× higher in the mid-cerebral arteries compared to that measured in cremaster arteriole.

Figure 10. BK_Ca subunit expression.

A, mRNA expression levels for the α- and β1-subunits of BK_Ca for cremaster (n= 5) and cerebral (n= 5) arteries. Results have been normalized to β-actin mRNA expression levels. B, ratio of β1 : α BK_Ca subunit mRNA expression for cremaster and cerebral vessels. Data are from samples described in A. C, representative immunohistochemical staining for BK_Caβ1 (green) in intact cremaster and cerebral arteriess. Paraformaldehyde-fixed vessel segments were stained with rabbit polyclonal anti-β1 BK_Ca (Alomone Laboratories, Jerusalem, Israel) followed by AlexaFuor-488 conjugated goat anti-rabbit IgG. Cell nuclei were counterstained with propidium iodide (red). D, representative Western blots for membrane-associated BK_Caα, BK_Caβ1 and actin. Cremaster and cerebral arteriole membrane samples (approximately 5 μg of protein each) were resolved by SDS-PAGE and transferred to PVDF membranes. Lanes 1–5 represent independent membrane preparations from mid-cerebral or cremaster arteries. Group data show calculated amounts of the individual channel subunits (relative to actin) and the ratio of β1 : α subunit. Values are presented as means ±s.e.m., n= 5, *P < 0.05.

Protein expression of α and β BK_Ca subunits

In situ immunohistochemical identification for BK_Caβ1 (anti-BK_Caβ1 antibody, sc-14749, Santa Cruz Biotechnology) showed more intense staining in VSM cells of cerebral arteries compared to those from cremaster muscle (Fig. 10C).

Western blotting was subsequently performed to determine whether differences in BK_Ca channel activity between the cremaster and mid-cerebral vessels were a reflection of quantitative differences in protein expression of the channel. Blots of isolated membrane fractions from pooled artery samples were probed with specific antibodies against the α (pore-forming) subunit and the β1 (regulatory) subunit. The α-subunit protein appeared as a clear, well defined band of approximately 100 kDa in both arteries (Fig. 10D). After normalization with respect to actin expression, the expression of BK_Caα was calculated to be 3.27-fold higher in the membrane protein fraction from mid-cerebral compared with cremaster arterioles (Fig. 10D and Table 2, P < 0.0001).

Table 2.

BK_Caα and β1-subunit protein expression relative to actin (mean ±s.e.m.; n= 5) in cremaster and mid-cerebral arteries

Cremaster membrane			Mid-cerebral membrane
α	β1†	Ratio	α	β1†	Ratio
0.3289	0.1187	0.3568	1.0767	0.7396	0.6847
± 0.0678	± 0.0304	± 0.1060	± 0.0745	± 0.0651	± 0.0237

^†Values for β1 expression were calculated using anti-β1_118–132.

Expression of the BK_Caβ1-subunit was examined using two separate antibodies raised against peptides corresponding to residues 61–75 (anti-β1_61–75) and residues 118–132 (anti-β1_118–132). In each case, the whole antiserum recognized a series of bands including one of 32.5 kDa, the size of the glycosylated β1-subunit protein that has been previously characterized using these antibodies (Knaus et al. 1994; Matharoo-Ball et al. 2003). Compared with cremaster arterioles, BK_Caβ1 protein was more highly expressed in mid-cerebral vessels as determined by anti-β1_118–132 (Fig. 10D, 6.23-fold P < 0.0001) and anti-β1_61–75 (see Supplemental Figure S3, 3.87-fold P < 0.005). Differences in the titre and affinity of the anti-BK_Caβ1 antisera may account for the modest difference in the calculated expression level of the β1-subunit detected by these two polyclonal antibodies. Furthermore, consistent with the mRNA results, the ratio of β1 protein relative to BK_Caα-subunit protein was approximately 1.9-fold higher in the membrane extracts from mid-cerebral compared with cremaster vessels (Table 2).

Prior incubation of the primary antibodies with their cognate peptide antigens abolished immunodetection of the bands corresponding to the expected sizes of the BK_Caα (Fig. S2) and β1-subunit (Fig. S3) proteins.

Functional vessel experiments

Cannulated cremaster muscle arterioles showed concentration-dependent constriction to ryanodine (Fig. 11A) consistent with the presence of functional ryanodine-sensitive intracellular Ca²⁺ stores. Despite the presence of a maximally effective concentration of ryanodine (10⁻⁵m), the BK_Ca inhibitor, iberiotoxin (10⁻⁷m), elicited a further vasoconstrictor response (Fig. 11B). The extent of iberiotoxin-induced constriction was similar to that occurring in the absence of ryanodine suggesting that tonic BK_Ca activity in the pressurized cremaster arteriole was not solely dependent on Ca²⁺ availability via ryanodine-sensitive stores. Reversing the order of iberiotoxin and ryanodine additions did not alter the net vasoconstrictor effect of the two agents, consistent with these inhibitors having independent actions (Fig. 11C).

Figure 11. Effects of ryanodine and iberiotoxin on cannulated and pressurised (70 mmHg) cremaster 1A arterioles.

A, concentration-dependent effects of ryanodine on arteriolar diameter. B, a maximally effective concentration of ryanodine (10⁻⁵m) does not prevent subsequent IBTX-induced vasoconstriction as reported for cerebral vessels (Nelson et al. 1995). C, a similar diameter is achieved regardless of the order of IBTX (0.1 μm) and ryanodine addition, suggesting that a component of BK_Ca activation in cremaster vessels occurs independently of ryanodine-sensitive SR function. Results are shown as means ±s.e.m. for an n= 4 for each condition.

Consistent with our previous studies (Kotecha & Hill, 2005) cannulated cremaster arterioles demonstrated pressure-dependent smooth muscle membrane depolarization (Fig. 12). Iberiotoxin caused further membrane depolarization, and constriction, with this effect being more apparent at lower intraluminal pressures (Fig. 12).

Figure 12. Effect of IBTX (10⁻⁷m) on the basal E_m in smooth muscle of cannulated cremaster 1A arterioles, as measured by glass microelectrodes.

A, group data (n= 3–7 measurements/vessels per intraluminal pressure); B, representative recordings of E_m. Group data are shown as means ±s.e.m.

Discussion

The smooth muscle large conductance, Ca²⁺-activated, potassium channel, BK_Ca, has been implicated as a significant contributor in the control of arteriolar tone. In particular, BK_Ca has been suggested to act as a negative feedback mechanism to limit pressure-induced, or myogenic, vasoconstriction (Nelson et al. 1995). Activation and opening of BK_Ca and the appearance of STOCs are suggested to result from pressure-induced membrane depolarization, Ca²⁺ entry via voltage-dependent mechanisms, and the subsequent generation of Ca²⁺ sparks from ryanodine-sensitive stores. Ca²⁺ sparks provide a localized increased in Ca²⁺ sufficient to activate BK_Ca. While evidence for this mechanism is strong in cerebral artery smooth muscle, it is currently uncertain whether it is dominant in all vascular beds. In particular, based on apparent differences in membrane potential–myogenic constriction relationships in arterioles from skeletal muscle and the cerebral vasculature (Kotecha & Hill, 2005), we have hypothesized that differences exist in the role of BK_Ca between these two functionally distinct vascular beds. The results of the present study provide electrophysiological data to support this hypothesis and further suggest that these differences may result from a differing level of contribution from the regulatory β1-subunit of the channel and the coupling of the channel to SR Ca²⁺ release.

Measurements of macroscopic K⁺ currents using the whole cell patch clamp technique showed a lower current density in smooth muscle cells isolated from cremaster arterioles compared to those from small cerebral arteries (Figs 1 and 2). This difference was apparent in the IBTX-sensitive component of the total K⁺ current (Fig. 2) consistent with a difference at the level of BK_Ca. Further, this difference was seen at a pipette Ca²⁺ concentration of 5 μm, but not at 100 nm (Fig. 2), suggesting that a difference in the Ca²⁺ sensitivity of the BK_Ca channel between the two cell types, rather than a difference in the absolute number of functional channels, may account for this primary observation. This conclusion assumes similar current amplitudes under conditions where BK_Ca channels are maximally active, an assumption that will require single channel measurements.

The BK_Ca channel generally exists as a heteromeric structure comprised of four pore-forming α-subunits and four accessory β-subunits (Salkoff et al. 2006). Although four (β1–β4) isoforms have been described, the predominant subunit expressed in vascular smooth muscle is β1 (Knaus et al. 1994). The four α-subunits form the basic channel pore, which in the absence of accessory β-subunits still acts as a voltage- and Ca²⁺-dependent K⁺ channel While the α-subunit has been reported to exhibit Ca²⁺ sensitivity, the β1-subunit confers additional voltage and Ca²⁺ sensitivity (Cox & Aldrich, 2000). Thus, at a cytosolic Ca²⁺ concentration of approximately 5 μm, Cox & Aldrich (2000) demonstrated that the half-maximal voltage of activation (V_1/2) of BK_Ca channels consisting of both α- and β-subunits is −6 mV compared to a V_1/2 of 66 mV for channels consisting of only the α-subunit. As a result, in the presence of 5 μm cytosolic Ca²⁺, BK_Ca channels with non-stoichiometric amounts of β1-subunit would be expected to be less active at a given holding potential compared to channels with a full complement of α- and β1-subunits. As a result, the lower current density we observed for cremaster BK channels (Fig. 2) could reflect less channel subunit protein, as directly quantified by our Western blotting data and indirectly by mRNA levels (refer to Fig. 10), but could also be accounted for by channels with lower Ca²⁺ sensitivity, as suggested by the observed difference in oestrogen responsiveness between cremaster and cerebral VSM cells (Fig. 4). Thus, the weak/absent E2 effect on cremaster BK_Ca channels is consistent electrophysiologically with a limited complement of β-subunits per channel (i.e. < 4), and is supported by the mRNA and protein data demonstrating weaker BK_Caβ1-subunit expression in cremaster VSM. Also consistent with the data from the present studies Cox & Aldrich (2000) reported no difference in BK_Ca channel gating, with or without co-expressed β-subunits at cytosolic Ca²⁺ concentrations of 100 nm.

Several of the results obtained argue for a functional similarity between cremaster and cerebral VSM at the level of the BK_Caα-subunit, despite differences in detected protein levels. Both VSM cell types respond similarly to the BK_Ca opener NS-1619, which has been reported to exert its action primarily via the pore forming α-subunit (Gribkoff et al. 1996). In addition, as stated above, no differences were observed, with respect to the measured IBTX-sensitive BK_Ca current, between the two cell preparations at 100 nm Ca²⁺, where β-subunit contribution to channel activity is minimal. Taking together, these findings support the likelihood that equivalent levels of functional BK_Caα-subunits exist in these two cell types. These data, however, do not rule out differences in other regulatory mechanisms, including those utilizing cyclic nucleotides, as the potential exists for the alternate expression of splice variants, a number of which have been described for the α-subunit (Shipston, 2001; Tian et al. 2001; Chen et al. 2005).

One component of the data suggesting that the differences in BK_Ca function between the two VSM preparations may be more complex is that, while the normalized current densities (pA pF⁻¹) were similar at 100 nm pipette Ca²⁺, Western blotting showed a significantly lower absolute α-subunit protein expression in cremaster VSM cells. While measurements of protein expression were performed on membrane fractions, these data may reflect differences in the contribution of protein located on intracellular membranes and are not necessarily limited to differences at the plasma membrane level. It remains to be examined whether differences exist in factors such as the handling of the channel (for example, trafficking and membrane insertion of both subunits). Alternatively, the measured electrophysiological and pharmacological properties of the α-subunit may require lower levels of protein than are expressed, suggesting a relative ‘channel reserve’, particularly in VSM from cerebral arteries.

While the electrophysiological and expression data suggest that differences in BK_Ca function between cremaster and cerebral vascular smooth muscle may be explained by differences in β1-subunit involvement, argument can also be made for a contribution of a lack of Ca²⁺ spark production. As stated earlier, the occurrence of Ca²⁺ sparks has been shown to be necessary to increase Ca²⁺ to sufficiently high levels in the vicinity of the cytoplasmic surface of BK_Ca to enhance the open probability of the channels and generate the appearance of STOCs. It has been estimated that localized Ca²⁺ concentrations of approximately 10 μm are required to serve this function (Zhuge et al. 2002). The difference in Ca²⁺ spark activity between the cremaster and cerebral vascular smooth muscle cells was indeed striking. Despite being prepared in the same laboratory, studied using the same optical system, and the cell preparations exhibiting comparable resting E_m, Ca²⁺ sparks were not seen in the cells from cremaster muscle arterioles, under unstimulated conditions, while they were readily observed in cerebrovascular smooth muscle cells (Fig. 8 and Table 1). While cremaster VSM cells appeared to show variation in [Ca²⁺]_i across a given cell, as indicated by variation in fluorescence intensity, the signal did not show spatio-temporal characteristics consistent with those published for sparks in VSM cells from other vascular beds (for review see Jaggar et al. 2000). Importantly, the characteristics (Table 1) of the Ca²⁺ sparks we observed in cerebrovascular smooth muscle cells were largely similar to those previously published (Jaggar et al. 2000). This observation, in conjunction with our capacity to observe stimulated changes in Fluo-4 fluorescence in the cells isolated from cremaster muscle, suggests that the lack of detection of sparks was not due to a technical limitation.

In an attempt to stimulate production of Ca²⁺ sparks, additional experiments were performed in the presence of caffeine (100 μm). This ‘low’ concentration of caffeine has previously been used by several investigators, including Janiak et al. (2001) in gastrointestinal smooth muscle and Borisova et al. (2007) in ureter smooth muscle to increase spark frequency. Consistent with these studies, VSM cells from cerebral arteries responded with an increased spark frequency, as well as an increase in the number of sites exhibiting sparks within a given cell (Fig. 9). In contrast, caffeine was relatively ineffective in inducing Ca²⁺ sparks in cremaster VSM cells, with only 2 of 24 cells exhibiting sparks following caffeine exposure (see Supplemental Material). In apparent contradiction, however, cremaster VSM cells exhibited STOCs that were inhibited by ryanodine and stimulated by caffeine, consistent with a role for the SR in the generation of these BK_Ca-mediated events. Several explanations may underlie these observations. For example, it is conceivable that smaller sparks exist in cremaster VSM cells that are below threshold for detection. This possibility is consistent with the suggestions of Perez et al. (1999), who discussed potential mechanisms underlying their observations of ‘sparkless STOCs’. Nevertheless, it is clear that cremaster muscle VSM cells do not typically exhibit the Ca²⁺ sparks that were evident in the cerebral VSM cells (Fig. 8 and Table 1). Alternatively STOCs in the cremaster VSM cells might be stimulated by a ryanodine-sensitive mechanism not requiring a focal increase in [Ca²⁺]_i.

Additional support for a difference between cremaster and cerebral artery coupling of ryanodine-sensitive Ca²⁺ stores was provided by the intact vessel studies. While ryanodine caused significant vasoconstriction of cremaster arterioles (indicating the presence of functional ryanodine-sensitive Ca²⁺ stores) a maximally effective concentration of the alkaloid did not prevent subsequent constriction to IBTX (Fig. 11). This is in contrast to earlier studies in small cerebral arteries where ryanodine prevented both Ca²⁺ sparks and IBTX-mediated vasoconstriction (Nelson et al. 1995; Jaggar et al. 1998). The fact that cremaster vessels constricted in response to IBTX suggests that an alternative pathway to the Ca²⁺ spark-mediated mechanism demonstrated for small cerebral arteries (Nelson et al. 1995; Jaggar et al. 1998) may also contribute to BK_Ca activation. Consistent with this suggestion, Jackson & Blair (1998) previously proposed that BK_Ca channels in cremaster arterioles exist in a quiescent state, requiring a stimulus such as an agonist or hypoxia for their activation. Such activation may involve cyclic nucleotide signalling, as BK_Ca open probability has been shown to increase by cAMP and cGMP-mediated mechanisms, such as protein phosphorylation (Schubert & Nelson, 2001). When the order of addition of the drugs was reversed (i.e. IBTX first), ryanodine caused vasoconstriction of cremaster arterioles to a diameter not significantly different from that when IBTX followed ryanodine treatment. In this case it is assumed that the ryanodine-induced constriction was due to SR Ca²⁺ release.

For BK_Ca to act in a negative feedback role to limit myogenic vasoconstriction, as suggested for the cerebral circulation, it would be expected that as intraluminal pressure is raised, an increase in BK_Ca activity would also be evident. As a consequence, IBTX would be expected to cause a greater membrane depolarization at those higher pressures. However, the present studies did not support this hypothesis and, in fact, tended to show a greater IBTX depolarization as pressure was decreased (Fig. 12). Such a situation is therefore more consistent with a pressure-dependent closure of K⁺ channels as proposed by Harder, Roman and colleagues (Zou et al. 1996; Gebremedhin et al. 2000). Those investigators, primarily in studies of renal and cerebral vasculature, have suggested that mechanical forces elicited by intraluminal pressure cause the production of the cytochrome P450-dependent arachidonic acid metabolite, 20-HETE, which causes membrane depolarization by a mechanism involving closure of BK_Ca channels (Zou et al. 1996; Gebremedhin et al. 2000).

A question remains as to what is the most important factor in the difference in STOC appearance between the two vascular beds. Is the relative lack of STOCs a result of the electrophysiological properties of BK_Ca in cremaster vascular smooth muscle, the apparent absence of sparks, or a combination of both? In addition to these factors, coupling of sparks to BK_Ca activation also requires a favourable cellular architecture such that the plasma membrane associated BK_Ca channels are in close proximity to ryanodine-sensitive Ca²⁺ release channels on the superficial SR (Lohn et al. 2001; Lu et al. 2006). As the cremaster vascular smooth muscle did not exhibit spark activity this latter point may not be an important consideration unless spark production can be activated. Further studies are required to assess these factors.

Given that the results of this study show that the BK_Ca channel of cremaster VSM cells more closely resembles an α-subunit dominated stoichiometry than the presumed 1 : 1 α/β1 subunit arrangement in VSM from cerebral arteries, it might be expected that the cremaster cells would functionally resemble cerebral cells from a BK_Caβ1 knockout model. Consistent with this possibility, STOC frequency is diminished in cerebral VSM cells isolated from BK_Caβ1^–/– mice compared to those from BK_Caβ1^+/+ mice (at equivalent E_m) (Brenner et al. 2000; Pluger et al. 2000). Similarly to that observed for cremaster VSM cells in the present study, the appearance of STOCs in cerebral VSM cells from BK_Caβ1^–/– mice required a relatively more positive E_m (Pluger et al. 2000). A difference between cerebral VSM cells of BK_Caβ1^–/– mice and the rat cremaster VSM cells we studied (at resting E_m), however, is that the former maintain their ability to generate Ca²⁺ sparks. Further studies will be required to examine the contributions of a number of factors, including the relative Ca²⁺ sensitivity at the single channel level, the cellular architecture and whether differences in channel subunit composition alter plasma membrane localization of BK_Ca channels expressed in each VSM.

In summary, BK_Ca function differs significantly in small artery smooth muscle cells from the vasculature of skeletal muscle and brain. Both functional and expression data support the hypothesis that the BK_Ca channel in cremaster arteriolar smooth muscle shows a decreased contribution (relative to cerebral vascular smooth muscle) of the BK_Caβ1 regulatory subunit which, in part, confers Ca²⁺ sensitivity of the channel. Further complexity arises from differences in the absolute levels of channel protein expression and in functional coupling of the channel to the SR. Collectively, these difference lead to STOC generation (a functional correlate of BK_Ca activity) requiring relatively more positive levels of E_m in cremaster compared with cerebral VSM cells. The physiological relevance of this heterogeneity may relate to an advantage in maintaining a high vascular resistance in resting skeletal muscle, while at the same time having a relatively lower vascular resistance in cerebral tissue. The data have further relevance to the potential use of BK_Ca as a therapeutic target in vascular disorders.

Glossary

Abbreviations

4AP

4-aminopyridine

BK_Ca

large conductance Ca²⁺-activated K⁺ channel

DMSO

dimethylsulphoxide

E2

17β-oestradiol

E_m

membrane potential

FCHW

full circumference spread at half maximal intensity

FWHW

full width spread at half maximal intensity

IBTX

iberiotoxin

LDS

lithium dodecyl sulfate

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

PSS

physiological salt solution

PVDF

polvinylidene difluoride

ROI

region of interest

SDS

sodium dodecyl sulfate

SMC

smooth muscle cell

SR

sarcoplasmic reticulum

STOC

spontaneous transient outward current

VSM

vascular smooth muscle

Author contributions

Experiments detailed in this manuscript were performed at the Dalton Cardiovascular Research Center, University of Missouri or the School of Medical Sciences, University of New South Wales. All authors approved the final version of the manuscript for publication. M.A.H., M.J.D., Y.Y. and A.P.B. were responsible for study conception and design, analysis and interpretation of data. T.V.M., T.H.G., S.R.E., R.H., Y.T.H., G.P. and R.J.K. contributed to data collection and analysis and interpretation of data.