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Am J Physiol Heart Circ Physiol 292: H1085-H1094, 2007. First published October 20, 2006; doi:10.1152/ajpheart.00926.2006
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Hyposmotic challenge inhibits inward rectifying K⁺ channels in cerebral arterial smooth muscle cells

¹Department and Graduate Institute of Pharmacology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; ²Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada; and ³Department of Pharmacology, University of Vermont, Burlington, Vermont

Submitted 25 August 2006 ; accepted in final form 13 October 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study sought to define whether inward rectifying K⁺ (K_IR) channels were modulated by vasoactive stimuli known to depolarize and constrict intact cerebral arteries. Using pressure myography and patch-clamp electrophysiology, initial experiments revealed a Ba²⁺-sensitive K_IR current in cerebral arterial smooth muscle cells that was active over a physiological range of membrane potentials and whose inhibition led to arterial depolarization and constriction. Real-time PCR, Western blot, and immunohistochemical analyses established the expression of both K_IR2.1 and K_IR2.2 in cerebral arterial smooth muscle cells. Vasoconstrictor agonists known to depolarize and constrict rat cerebral arteries, including uridine triphosphate, U46619 [GenBank] , and 5-HT, had no discernable effect on whole cell K_IR activity. Control experiments confirmed that vasoconstrictor agonists could inhibit the voltage-dependent delayed rectifier K⁺ (K_DR) current. In contrast to these observations, a hyposmotic challenge that activates mechanosensitive ion channels elicited a rapid and sustained inhibition of the K_IR but not the K_DR current. The hyposmotic-induced inhibition of K_IR was 1) mimicked by phorbol-12-myristate-13-acetate, a PKC agonist; and 2) inhibited by calphostin C, a PKC inhibitor. These findings suggest that, by modulating PKC, mechanical stimuli can regulate K_IR activity and consequently the electrical and mechanical state of intact cerebral arteries. We propose that the mechanoregulation of K_IR channels plays a role in the development of myogenic tone.

cerebral arteries; potassium channels; vasoconstrictor stimuli

In vascular smooth muscle, V_M is determined by a dynamic interplay between depolarizing inward and hyperpolarizing outward currents. While inward current is governed by chloride and sodium conductances, outward current is principally driven by voltage-gated (K_V), Ca²⁺-activated (BK_Ca), ATP-sensitive (K_ATP), and inward rectifying (K_IR) K⁺ channels (27, 34). Past studies have carefully characterized the regulatory and compositional properties of several smooth muscle conductances. Despite this work, a number of conductances including K_IR have received limited experimental analysis. K_IR channels are aptly named for their ability to pass current more readily in the inward direction. To date, molecular approaches have identified seven subfamilies, including those that encode for weakly rectifying (K_IR1.x), strongly rectifying (K_IR2.x), G protein-coupled (K_IR3.x), and K_ATP [K_IR6.x + sulfonylurea receptor (SUR) subunit] K⁺ channels (2, 5). In the cerebral circulation, smooth muscle K_IR channels are potently blocked by micromolar Ba²⁺, activated by extracellular K⁺, and thought to be principally composed of K_IR2.1 subunits (3, 33). Since their initial identification (7), cerebral arterial K_IR channels generally have been regarded as a tonic background conductance (27, 40). As such, they are presumed to be unresponsive to a range of agonists and mechanical perturbations that constrict arteries through mechanisms that partly depend on electromechanical coupling. While recent studies have started to challenge this perception (29, 30), experimental observations supporting the concept of smooth muscle K_IR channel regulation by vasoactive stimuli remain limited.

The present study examined whether vasoactive stimuli known to depolarize and constrict cerebral arteries suppress K_IR channel activity. Using functional, electrical, and molecular approaches, we confirmed that K_IR channels are 1) present in cerebral arterial smooth muscle cells, 2) active over a physiological V_M range, and 3) likely composed of both K_IR2.1 and K_IR2.2 subunits. Subsequent experiments demonstrated that whole cell K_IR activity was unaffected by several vasoconstrictor agonists including uridine triphosphate (UTP), U46619 [GenBank] , and 5-HT. In contrast, K_IR currents were potently suppressed by hyposmotic challenge, a stimulus known to activate mechanosensitive ion channels in the resistance vasculature (11, 43, 44). Further investigation revealed that this hyposmotic-induced suppression was dependent on protein kinase C (PKC) signaling. On the basis of the observations, we propose that smooth muscle K_IR channels are mechanically sensitive and that this property facilitates the participation of this conductance in myogenic tone development.

	MATERIALS AND METHODS

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Animal and dissection procedures. Animal procedures were approved by the Animal Care and Use Committee at the University of Calgary or the University of Vermont. Briefly, female Sprague-Dawley rats (10–12 wk of age) were euthanized via carbon dioxide asphyxiation. The brain was carefully removed and placed in cold phosphate-buffered (pH 7.4) saline solution containing (in mM) 138 NaCl, 3 KCl, 10 Na₂HPO₄, 2 NaH₂PO₄, 5 glucose, 0.1 CaCl₂, and 0.1 MgSO₄. Cerebral and basilar arteries were carefully dissected out of surrounding tissue and cut into 2-mm segments.

Intact cerebral arteries. Cerebral arterial segments were mounted in a customized arteriograph and superfused with warm (37°C) physiological salt solution (PSS; pH 7.4) containing (in mM) 119 NaCl, 4.7 KCl, 20 NaHCO₃, 1.7 KH₂PO₄, 1.2 MgSO₄, 1.6 CaCl₂, and 10 glucose. Endothelial cells were removed by passing air bubbles through the vessel lumen (2 min); successful removal was confirmed by the loss of acetylcholine-induced dilations. Cerebral arteries were equilibrated for 60 min, and contractile responsiveness was then assessed by briefly (10 s) exposing the tissue to a 60 mM KCl challenge. Following equilibration, cerebral arteries were maintained at low (15 mmHg) or high (80 mmHg) intravascular pressure in the absence or presence of 30 µM Ba²⁺ (K_IR channel inhibitor). Arterial diameter was monitored using an automated edge detection system (IonOptix, Milton, MA). Smooth muscle V_M was assessed by inserting a glass microelectrode backfilled with 1 M KCl (tip resistance = 120–150 M) into the vessel wall. The criteria for successful cell impalement included 1) a sharp negative V_M deflection on entry, 2) a stable recording for at least 1 min following entry, and 3) a sharp return to baseline on electrode removal.

Isolation of arterial smooth muscle cells. Smooth muscle cells from rat basilar arteries were enzymatically isolated as previously described (43). Briefly, arterial segments were placed in an isolation medium (37°C, 10 min) containing (in mM) 60 NaCl, 80 Na-glutamate, 5 KCl, 2 MgCl₂, 10 glucose, and 10 HEPES with 1 mg/ml albumin (pH 7.4). 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 papain and 1.8 mg/ml dithioerythritol, and 2) a 10-min incubation in isolation medium containing 100 µM Ca²⁺, 0.7 mg/ml type F collagenase, and 0.4 mg/ml type H collagenase. Following treatment, tissues were washed repeatedly with ice-cold isolation medium and triturated with a fire-polished pipette. Liberated smooth muscle cells were stored in ice-cold isolation medium for use the same day.

Electrophysiology. Conventional patch-clamp electrophysiology was used to measure whole cell currents in isolated smooth muscle cells. Briefly, recording electrodes (resistance of 4–7 M) were fashioned from borosilicate glass, covered in sticky wax to reduce capacitance, and backfilled with solution containing (in mM) 5 NaCl, 35 KCl, 100 K-gluconate, 1 CaCl₂, 0.5 MgCl₂, 10 HEPES, 10 EGTA, 2.5 Na₃-ATP, and 0.2 GTP (pH 7.2). This pipette was then gently lowered onto an isolated cell and negative pressure applied to rupture the membrane. Cells with a seal resistance >10 G were then voltage clamped (–60 mV) and equilibrated for 15 min in a bath solution containing (in mM) 90 NaCl, 5 KCl, 0.5 MgCl₂, 10 HEPES, 10 glucose, 0.1 CaCl₂, and 100 D-mannitol (pH 7.4). K_IR activity was assessed by ramping cells between –120 and 20 mV (0.047 mV/ms) and quantifying the component of the whole cell current that was sensitive to 30 µM Ba²⁺. In contrast, voltage-dependent delayed rectifier K⁺ (K_DR) current activity was assessed by stepping cells to voltages between –70 and +40 mV (300 ms) and monitoring peak outward current. Following this voltage pulse, cells were stepped back to –40 mV (500 ms) to determine whether tail currents were present. Control experiments confirmed that the pipette solution did minimize K_ATP and K_Ca channel activity. Whole cell currents were recorded on an Axopatch 200B amplifier (Axon Instruments, Union City, CA), filtered at 1 kHz, digitized at 5 kHz, and stored on a computer for subsequent analysis with Clampfit 8.1 software. Cell capacitance ranged between 14 and 18 pF and was measured with the cancellation circuitry in the voltage-clamp amplifier. Cells that displayed a noticeable shift in capacitance (>0.3 pF) during experiments were excluded from analysis. A 1 M NaCl-agar salt bridge between the reference electrode and the bath solution was used to minimize offset potentials (<2 mV). All experiments were performed at room temperature (20–22°C).

Ba²⁺-sensitive K_IR currents were first assessed in bath solutions containing 5, 20, and 60 mM K⁺. To elevate the bath concentration of K⁺, KCl was substituted for NaCl; osmolarity was maintained at 305 mosM, as confirmed with a bioosmometer. In all subsequent K_IR experiments, extracellular K⁺ was maintained at 20 mM, and the magnitude of the Ba²⁺-sensitive current was monitored in response to UTP (30 µM), U46619 [GenBank] (0.1 µM), 5-HT (0.1 µM), or phorbol-12-myristate-13-acetate (PMA; 50 nM) or to hyposmotic challenge ± calphostin C (300 nM). To render bath solutions hyposmotic (205 mosM), D-mannitol was removed. K_DR activity was also assessed in the absence and presence of UTP, U46619 [GenBank] , and hyposmotic challenge.

Real-time PCR analysis of K_IR subtypes. Whole basilar arteries or 300 isolated smooth muscle cells were placed in RNase- and DNase-free collection tubes. Total RNA was extracted (RNeasy Mini kit with DNase treatment; Qiagen, Valencia, CA), and first-strand cDNA was synthesized using the Sensi-script RT kit (Qiagen) with oligo d(T) primer. To optimize reaction specificity, real-time PCR was initially performed on each primer set using rat brain cDNA, SYBR green (Bio-Rad, Hercules, CA), and a range of annealing temperatures (52–62°C). On the basis of melt curve analysis, 1 µl of each reaction product was placed on a DNA 500 lab chip and examined using a bioanalyzer (model no. 2100, Agilent Technologies). A second aliquot of product was electrophoresed on a 1.5% (wt/vol) agarose gel, extracted using a gel extraction kit (Qiagen), and sequenced at the University of Calgary Core DNA facility. Having ascertained an ideal annealing temperature, real-time PCR efficiency was determined for all primer sets, with serial dilutions of brain cDNA used as template. Reaction efficiencies were as follows: K_IR2.1, 94.4%; K_IR2.2, 94.9%; K_IR2.3, 98.8%; K_IR2.4, 98.0%. The optimal real-time PCR reaction consisted of a hot start (95°C for 3 min) followed by 40 cycles of 95°C for 15 s, 55.1°C for 30 s, and 72°C for 30 s. A melt curve analysis was performed on each reaction, and the threshold cycle was determined using software provided with the Bio-Rad iCycler. Forward (F) and reverse (R) primers specific to rat K_IR2.1–2.4 were as follows: K_IR2.1 (accession no. NM_017296) (F) 5'-AGAGGAAGAGGACAGTGAGAAC-3', K_IR2.1 (R) 5'-TCGCCTGGTTGTGGAGATC-3'; K_IR2.2 (accession no. NM_053981) (F) 5'-GCAGCCTTTCTCTTCTCCATTGA-3', K_IR2.2 (R) 5'-GACTGAGCCACCACCATGAAG-3'; K_IR2.3 (accession no. NM_053870) (F) 5'-CCTGGACCGCATCTTCTTGG-3', K_IR2.3 (R) 5'-CAGGATGACCACAATCTCAAAGTC-3'; K_IR2.4 (accession no. NM_170718) (F) 5'-ATGAGGTTGACTATCGACACTTCC-3', K_IR2.4 (R) 5'-GGGAGCCAGGAAAACTTGA CTTA-3'. K_IR mRNA levels were expressed relative to K_IR2.1.

All smooth muscle cell samples were screened a priori for template and endothelial cell contamination using conventional PCR techniques. In these control experiments, 2 µl of first-strand cDNA were added to a PCR reaction containing 1.5 mM MgCl₂, 0.25 µM forward and reverse primers (University of Calgary, Core DNA facility), 0.2 mM deoxynucleotide triphosphates, and 2.5 units of recombinant Taq DNA polymerase (Qiagen). PCR reactions were hot started (94°C for 3 min) and underwent 35 cycles of 94°C for 1.0 min, 60°C for 0.5 min, and 72°C for 0.75 min. PCR samples were then exposed to a final extension period at 72°C for 10 min. Forward and reverse primers specific to RhoA (accession no. NM_057132) were as follows: (F) 5'-CGGGATCCCGATGGCTGCCATCCAGGAAG-3'; (R) 5'-GGAATTCCTCACAAGATCGAAGGCAAC-3'. Nested primers (F1/R1, F2/R2) for endothelin-1 (accession no. NM_012548) were as follows: (F1) 5'-GAGCTGAGAAGGAAGTGCAGAG-3'; (R1) 5'-GGTCTTGATGCTGTTGCTGATG-3'; (F2) 5'-TGTGTCTACTTCTGCCACCTG-3'; (R2) 5'-GCCTCCAACCTTCTTAGTTTTCTT-3'. The expected amplicon sizes for RhoA and endothelin-1 were 550 and 350, respectively. DNA sequencing identified each reaction product, and control experiments confirmed the absence of genomic DNA contamination.

Western blotting. Whole basilar arteries were placed in 100 µl of lysis buffer (pH 7.4) containing (in mM) 150 NaCl, 1 CaCl₂, 1 MgCl₂, and 10 HEPES with 0.5% Tween and 10% mammalian protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Samples were mechanically disrupted, exposed to three liquid nitrogen freeze-thaw cycles, and then centrifuged (10 min, 13,000 rpm). Supernatant was placed in a clean tube, assayed for total protein, and stored at –20°C for up to 1 wk. Samples were prepared for electrophoresis by placing 15 µl of supernatant in 5 µl of 4x sample buffer and 2 µl of DTT. After heating (10 min, 90°C), 20 µg of protein were loaded to run on a 10% polyacrylamide gel. Protein was transferred to a polyvinylidene difluoride (PVDF) membrane, blocked for 2 h (5% nonfat milk in Tris-buffered saline), and incubated overnight (4°C) with a primary antibody (K_IR2.1 or K_IR2.2, 1:200 dilution; Sigma-Aldrich) diluted in milk-Tris buffer. The PVDF membrane was subsequently washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:1,000; Jackson Laboratories, West Grove, PA) diluted in milk-Tris buffer (1 h, 20–22°C). Washing was repeated, and the blot was developed using chemiluminescent substrate (Pierce Biochemicals, Rockford, IL).

Immunohistochemistry. K_IR2.1 and K_IR2.2 protein expression was detected in smooth muscle cells isolated from basilar myocytes. Isolated cells were air dried onto poly-L-lysine-coated slides and washed with Tris-buffered saline (pH 7.4) containing 50 mM Tris, 0.9% NaCl, 0.1% BSA, and 0.01% Triton X. Sections were lightly fixed in 2% paraformaldehyde-Tris-buffered saline (20 min) and incubated (4 h) with a quench solution containing 100 mM Tris, 1.8% NaCl, 5% goat serum, 2% BSA, and 0.2% Triton X. Sections were subsequently incubated overnight (4°C) with a polyclonal (K_IR2.1 or K_IR2.2; 1:200) and a monoclonal (endothelial nitric oxide synthase, 1:2,500; Chemicon, Ternecula, CA) antibody diluted in quench solution. Slides were then washed with Tris-buffered saline and incubated for 4 h (20–22°C) with an anti-rabbit-Cy3 and an anti-mouse-Cy5 IgG-specific secondary antibody (Molecular Probes, Eugene, OR). After further washing, slides were air dried and mounted in anti-fade media (Molecular Probes). Smooth muscle cells were viewed and photographed using a Zeiss fluorescent microscope coupled to a x63 oil immersion lens.

Chemicals, drugs, and enzymes. Buffer reagents, collagenases (types F and H), UTP, and 5-HT were obtained from Sigma-Aldrich. Papain was acquired from Worthington (Lakewood, NJ). PMA, calphostin C, and U46619 [GenBank] were purchased from Calbiochem (La Jolla, CA). PMA was dissolved in DMSO, with a final solvent concentration 0.05%.

Statistical analysis. Data are expressed as means ± SE, and n indicates the number of vessels or cells. Paired t-tests were performed to statistically compare the effects of a given condition/treatment on arterial diameter, V_M, or whole cell current. P values 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
K_IR in intact cerebral arteries and isolated smooth muscle cells. To examine whether K_IR channels were functionally active over a physiological V_M range, initial experiments examined the Ba²⁺ responsiveness of denuded cerebral arteries at low (15 mmHg) and high (80 mmHg) intravascular pressure (Fig. 1). At low intravascular pressure, where myogenic tone was limited and V_M negative, 30 µM Ba²⁺ elicited a profound constriction (235 ± 7 to 195 ± 9 µm) and arterial depolarization (–54 ± 3 to –42 ± 2 mV). Elevating intravascular pressure to 80 mmHg depolarized and constricted cerebral arteries. In this myogenically active state, Ba²⁺ superfusion continued to constrict (187 ± 5 to 180 ± 6 µm) and depolarize (–38 ± 1 to –36 ± 1 mV) arteries, albeit in a more modest manner.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Inward rectifying K+ (KIR) channels regulate cerebral arterial diameter and membrane potential (VM). A and C: representative traces demonstrating the effects of Ba2+ (KIR channel inhibitor; 30 µM) on arterial diameter and VM at an intravascular pressure of 15 or 80 mmHg. B and D: summary data (n = 6/group) illustrating the effects of Ba2+ on arterial diameter and VM at 15 and 80 mmHg. Data are means ± SE. *Significant difference from control.

View larger version (19K): [in this window] [in a new window]   Fig. 2. KIR current in cerebral arterial smooth muscle cells. A: representative traces demonstrating the effect of Ba2+ (30 µM) on whole cell current. B: representative traces demonstrating the effects of extracellular K+ concentration ([K+]) on the Ba2+-sensitive KIR current. Inset: outward KIR current in a smooth muscle cell bathed in the 5 mM K+ solution. C: summary data illustrating the effects of extracellular [K+] on the Ba2+-sensitive KIR current at –120 mV (n = 6). D: effects of time on the Ba2+-sensitive KIR current at –120 mV (n = 6). Data are means ± SE.

View larger version (21K): [in this window] [in a new window]   Fig. 3. PCR analysis of KIR2.1–2.4. A: conventional RT-PCR analysis of whole basilar arteries and isolated smooth muscle cells for RhoA (product marker) and endothelin-1 (ET-1; endothelial cell-specific marker) mRNA. Ladder markers are measured in bp. B: real-time PCR reaction plot and mRNA expression of KIR2.1–2.4 in whole arteries (n = 4). C: real-time PCR reaction plot and mRNA expression of KIR2.1–2.4 in isolated smooth muscle cells (n = 4). Data are expressed relative to KIR2.1. RFU, relative fluorescence units.

View larger version (35K): [in this window] [in a new window]   Fig. 4. KIR2.1 and KIR2.2 protein expression. A: Western blot analysis of whole basilar artery probed with an anti-KIR2.1 (left) or anti-KIR2.2 (right) polyclonal antibody. B: smooth muscle cells isolated from the basilar artery were treated with a KIR2.1 or KIR2.2 polyclonal antibody and IgG secondary antibody conjugated to Cy3 (left). Staining was absent when the antigenic peptide was incubated with the primary antibody (right).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Vasoconstrictor agonists do not affect the KIR current. A–C: representative traces of the Ba2+-sensitive KIR current before and after a 20-min exposure to uridine triphosphate (UTP; 30 µM), U46619 (0.1 µM), and 5-HT (0.1 µM). D: summary data illustrating the effects of UTP (n = 6), U46619 (n = 6), and 5-HT (n = 6) on the Ba2+-sensitive KIR current at –120 mV. Data are means ± SE.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Vasoconstrictor agonists inhibit the voltage-dependent delayed rectifier K+ (KDR) current. A and B: representative traces of the KDR current before and after a 20-min exposure to UTP (30 µM) and U46619 (0.1 µM). C: summary data illustrating the effects of UTP (n = 6) and U46619 (n = 6) on the KDR current. Data are means ± SE. *Significant difference from control.

View larger version (10K): [in this window] [in a new window]   Fig. 7. Hyposmotic challenge inhibits the KIR current. A: representative traces of the Ba2+-sensitive KIR current before and after 6 min of hyposmotic challenge. Bath osmolarity was reduced from 305 (isosmotic) to 205 (hyposmotic) mosM. B: time-course data documenting the effect of hyposmolarity on the Ba2+-sensitive KIR current at –120 mV (n = 6). Data are means ± SE. *Significant difference from isosmotic conditions.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Hyposmotic challenge does not inhibit the KDR current. A: representative traces of the KDR current before and after 20 min of hyposmotic challenge. Bath osmolarity was reduced from 305 (isosmotic) to 205 (hyposmotic) mosM. B: summary data illustrating the effects of hyposmotic stimuli (n = 6) on the KDR current. Data are means ± SE.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Hyposmotic challenge inhibits the KIR current through a transduction process involving PKC. A: representative traces of the Ba2+-sensitive KIR current before and after a 6-min exposure to phorbol-12-myristate-13-acetate (PMA; 50 nM). B: representative traces of the Ba2+-sensitive KIR current before and after 6 min of hyposmotic challenge + calphostin C (300 µM). C: summary data illustrating the effects of PMA (n = 6), hyposmolarity (n = 6), and hyposmolarity ± calphostin C (n = 6) on the Ba2+-sensitive KIR current at –120 mV. Data are means ± SE. *Significant difference from isosmotic conditions.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

K_IR channels in intact cerebral arteries. Cerebral arteries are coupled in series and parallel to one another, forming an arterial network that controls the magnitude and distribution of tissue blood flow. Under dynamic conditions, tone within this network is regulated by changes in tissue metabolism (14), neuronal activity (37), blood flow (12), and intraluminal pressure (15, 22, 43). Many of these stimuli initiate vasomotor responses by activating transduction pathways that alter resting V_M and the influx of Ca²⁺ through voltage-operated Ca²⁺ channels (17, 25, 41). In vascular smooth muscle, steady-state V_M is set by an array of depolarizing inward and hyperpolarizing outward currents including K_IR (23, 27, 34). K_IR channels are aptly named for their distinctive ability to pass current more readily in the inward direction. Structurally, these channels consist of four -subunits, each of which is comprised of two transmembrane domains and a GYG-containing "P-loop" that confers K⁺ selectivity (2). Since their initial isolation in arterial smooth muscle cells (10), vascular studies have consistently asserted that K_IR channels principally function as a background conductance (27, 40). To operate in this manner, K_IR channels would have to display activity over a range of resting membrane potentials. This functional activity is evident in Fig. 1, where micromolar Ba²⁺ elicited electrical and mechanical responses in arteries maintained in a depolarized or hyperpolarized state through changes in intravascular pressure. This broad range of activity ensures that K_IR can function in a housekeeping manner. However, it also invites the suggestion that these channels may serve as a target for vasoactive stimuli to initiate arterial depolarization and constriction.

K_IR regulation in cerebral arterial smooth muscle cells. To address whether K_IR channels are actively regulated by vasoconstrictor stimuli, it is important to first establish the basic electrical and molecular properties of this current in cerebral arterial smooth muscle cells. Consistent with past vascular investigations (32, 33, 35), this study observed a prominent K_IR current that was blocked by micromolar Ba²⁺, potentiated by extracellular K⁺, and displayed sustained activity over a 40-min recording period (Fig. 2). While inward rectification is the most distinctive feature of this current, it is the outward current between –20 to –60 mV that is physiologically important. In this study, a small outward component was observed in a limited number of cells, and it was generally no greater than 2 pA. Although subtle, modeling studies have shown that such currents can clearly alter V_M as long as the input resistance of a smooth muscle cell remains sufficiently high (7, 19). An analysis of mRNA and protein (Figs. 3 and 4) revealed the expression of both K_IR2.1 and K_IR2.2 in cerebral arterial smooth muscle cells, raising the possibility that arterial K_IR channels are perhaps heteromultimeric in nature. As such, it is worthwhile to note that K_IR2.1 and K_IR2.2 can coassociate and that the resulting heteromultimer displays electrophysiological properties including a Ba²⁺ sensitivity that corresponds well with the native cerebral arterial K_IR current (24, 36). These findings are in contrast to a previous study (3) that found little evidence for K_IR2.2 expression in vascular smooth muscle. Clarification of this issue awaits further detailed molecular characterization studies.

With K_IR activity prominent at negative potentials and in elevated extracellular K⁺, the influence of vasoactive stimuli on cerebral arterial K_IR currents was quantified at –120 mV and in bath solutions containing 20 mM K⁺. Initial trials focused on vasoconstrictor agonists including UTP and U46619 [GenBank] , agents previously shown to strongly depolarize and constrict intact cerebral arteries (25). As illustrated in Fig. 5, K_IR activity was not substantively altered by these vasoconstrictors; 5-HT applied at concentrations known to constrict cerebral arteries was also ineffective (20). This absence of modulation could not be ascribed to enzymatic digestion or to the whole cell recording conditions, since control experiments confirmed that UTP and U46619 [GenBank] suppressed whole cell K_DR activity (Fig. 6). Agonist modulation of K_IR has, in general, been poorly examined in vascular tissue. The recent work of Park and colleagues (29, 30) is a notable exception, with these limited studies demonstrating that smooth muscle K_IR activity can be suppressed if agonists mobilize PKC. While UTP and U46619 [GenBank] are often presumed to mobilize PKC in all resistance arteries, the cerebral circulation appears to be an exception (25, 41). This view is based on recent findings showing that PKC blockers do not prevent these agonists from depolarizing cerebral arteries or from suppressing K_DR activity (25). Indeed, in the cerebral circulation, vasoconstrictors like UTP and U46619 [GenBank] appear to elicit electrical and mechanical responses by mobilizing RhoA and Rho kinase (25).

The preceding results should not be interpreted in a manner that suggests all vasoactive stimuli initiate cerebral vascular responses independent of PKC. Indeed, several studies have reported an important role for this kinase in enabling intravascular pressure to depolarize and constrict intact cerebral arteries (13, 28, 38). In light of these observations, the present study examined whether mechanical stimuli could inhibit K_IR and whether this resulting suppression arose from PKC activation. To mechanically stimulate cerebral arterial smooth muscle, isolated cells were exposed to a hyposmotic challenge. We and others have previously employed this swelling stimulus to activate mechanically sensitive inward currents and initiate myogenic-like responses in intact cerebral arteries (8, 11, 38, 43, 44). Hyposomotic challenge induced a rapid inhibition of K_IR that was sustained over time (Fig. 7). This suppression displayed a degree of specificity, since whole cell K_DR activity was unaffected by the swelling stimulus (Fig. 8). Like past vascular studies that have employed hyposmolarity to modulate mechanically sensitive inward currents (38, 45), PKC mobilization appears essential to initiating K_IR suppression (Fig. 9). This was principally revealed though the ability of 1) PMA to modulate K_IR, and 2) calphostin C (a PKC inhibitor) to prevent hyposmotic bath solutions from inhibiting this K⁺ conductance. At present, the PKC isoform responsible for inhibiting smooth muscle K_IR channels remains unclear. However, given that free pipette Ca²⁺ concentration was 20 nM in the present study, a role for a Ca²⁺-independent isoform of PKC is likely. This view is consistent with past vascular work noting that the activation of swelling-activated Cl^– currents is dependent on PKC (45). While this study is the first in smooth muscle to indicate that K_IR channels are mechanically sensitive, these observations are not without broader precedent. Indeed, both stretch and shear stress have been reported previously to alter current flow through K_IR channels in ventricular myocytes and endothelial cells, respectively (4, 16).

Functional implications. A century ago, Bayliss (1) described the ability of resistance arteries to constrict to elevated intravascular pressure. In the cerebral circulation, pressure-induced vasoconstriction is an essential regulatory response that fundamentally depends on smooth muscle cell depolarization and influx of Ca²⁺ through voltage-operated Ca²⁺ channels (15, 22, 43). Traditionally, studies probing the basis of pressure-induced depolarization have concentrated on inward conductances. This includes a swelling-activated Cl^– current (11, 44) as well as nonselective cation currents comprised of various members of the transient receptor potential channel family (9, 26, 42). Such studies have, however, generally overlooked the potential contribution of K⁺ conductances except for those functioning in a negative-feedback mode (6, 18, 21). By illustrating the mechanosensitivity of the K_IR current, this study suggests for the first time that these channels could play a role in initiating pressure-induced depolarization. Designing a functional experiment to definitively link K_IR modulation with myogenic tone development is difficult and limits the present investigation. One must contend with the fact that K_IR inhibition substantively changes V_M and diameter, which makes group comparisons problematic. Likewise, agents that prevent hyposmolarity from inhibiting K_IR channels (i.e., PKC inhibitors) also impair the activation of mechanosensitive inward currents (8, 38, 45).

In summary, this study explored whether K_IR channels in cerebral arterial smooth muscle cells were suppressed by vasoconstrictor stimuli. Vasoconstrictor agonists previously shown to depolarize and constrict cerebral arteries did not inhibit K_IR channels likely composed of K_IR2.1 and K_IR2.2. In contrast, K_IR activity was suppressed by a hyposmotic challenge, a stimulus that activates mechanically sensitive currents in vascular smooth muscle cells. Subsequent experiments confirmed the dependency of current suppression on PKC activation. Given the apparent mechanosensitivity of K_IR, we propose a role for K_IR in the development of myogenic depolarization and constriction.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by the Canadian Institute for Health Research (D. G. Welsh), the National Institutes of Health (J. E. Brayden), and the Alberta Heritage Foundation for Medical Research (AHFMR). D. G. Welsh is a Senior Scholar with AHFMR.

	ACKNOWLEDGMENTS

 
We acknowledge the technical assistance of Suzanne Brett Welsh and Laura MacAuley.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. G. Welsh, Smooth Muscle Research Group, HMRB-G86, Heritage Medical Research Bldg., Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada, T2N-4N1 (e-mail: dwelsh{at}ucalgary.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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