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1Smooth Muscle Research Group and Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1; and 2Department of Pharmacology, Kaohsiung Medical University, Kaohsiung, Taiwan
Submitted 22 September 2003 ; accepted in final form 29 October 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vasoconstrictors; membrane potential; potassium channels; protein kinase C
Pyrimidine nucleotides such as UTP and UDP are released from cells that are part of, or pass through the lumen of, resistance arteries (23, 31). These sources include activated platelets and endothelial cells (25, 36). In the cerebral circulation, the binding of pyrimidine nucleotides to P2Y receptors can both dilate and constrict resistance arteries, depending on which population of receptors is recruited (17, 27). Endothelial P2Y receptors mediate cerebral artery dilation through the enhanced production of endothelium-derived factors including nitric oxide (26, 27, 31). In contrast, it is P2Y receptors on smooth muscle that are responsible for cerebral artery constriction (17, 27).
The mechanisms that enable smooth muscle P2Y receptors to constrict the cerebral vasculature have been poorly defined. Jaggar and Nelson (18), in one of the few investigations to address the issue, theorized a prominent role for electromechanical coupling in the overall transduction process. In greater detail, these investigators argued that the activation of pyrimidine-sensitive P2Y receptors enhances PKC activity via Gq, phospholipase C
, and the production of diacylglycerol. By altering the phosphorylation state of key proteins, this serine/threonine kinase was subsequently proposed to inhibit outward K+ conductance and thereby initiate depolarization (18). In cerebral smooth muscle, K+ conductance is determined by four families of K+ channels including those that underlie the inwardly rectifying, the ATP-sensitive, the large-conductance Ca2+-activated (BK), and the voltage-dependent delayed rectifying (KDR) K+ currents (28). With the exception of the inwardly rectifying K+ channels, all of these K+ conductances display sensitivity to PKC and thus, in theory, could be targeted and suppressed by pyrimidine nucleotides (1, 4, 5).
This study examined whether, and by what signaling and ionic mechanisms, pyrimidine nucleotides constrict rat cerebral arteries. We report that UTP-induced constriction partly depends on the depolarization of cerebral smooth muscle cells and the activation of voltage-operated Ca2+ channels. Contrary to initial expectations, the depolarization and constriction induced by UTP did not depend on PKC but rather arose from the activation of Rho kinase. Such findings indicate that, in addition to regulating myosin light chain phosphatase, Rho kinase likely targets other downstream effectors of smooth muscle cell contraction including ion channels. Electrophysiological measurements indicate that the slowly inactivating 4-aminopyridine (4-AP)-sensitive KDR current is one of the ionic conductances suppressed by UTP through a signaling pathway that involves RhoA/Rho kinase and not PKC. In summary, our findings are the first to establish RhoA/Rho kinase as a modulator of ion channels that control smooth muscle membrane potential (Em). KDR current suppression by RhoA/Rho kinase likely plays an important role in enabling pyrimidine-sensitive P2Y receptors to depolarize and consequently constrict intact cerebral arteries.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Intact cerebral arteries. Cerebral arteries were mounted in a customized arteriograph chamber (J. B. Pierce Laboratory, New Haven, CT) and superfused with warm (37°C) physiological salt solution containing (in mM) 119 NaCl, 4.7 KCl, 20 NaHCO3, 1.7 KH2PO4, 1.2 MgSO4, 1.6 CaCl2, and 10 glucose (pH 7.4). Endothelial cells were removed from all arteries by passing air bubbles through the vessel lumen for 2 min; successful removal was confirmed by the loss of acetylcholine-induced dilations. In keeping with past studies (43, 44), cannulated arteries were pressurized to 15 mmHg. Low intravascular pressure minimizes myogenic tone and enables investigators to study the effects of pyrimidine nucleotides with and without inhibitors in isolation. Arterial diameter was monitored with an automated edge detection system (IonOptix, Milton, MA). Smooth muscle Em was assessed by inserting a glass microelectrode filled with 1 M KCl (tip resistance = 120–150 M
) carefully into the vessel wall. The criteria for successful cell impalement were 1) a sharp negative Em deflection on entry, 2) a stable recording for at least 1 min after entry, and 3) a sharp return to baseline on electrode removal. Because Em recordings were typically limited to 3–10 min, a distinct impalement was required to measure Em under each experimental condition.
Experimental protocol and intact cerebral arteries. Cerebral arteries were equilibrated for 60 min at 37°C. Before experimentation, the vessel's contractile state was determined by briefly (
10 s) exposing the tissue to a 6 x 10–2 M KCl challenge. Dose responsiveness of cerebral arteries to UTP, UDP, and U-46619 was ascertained by increasing agonist concentration every 10 min and monitoring arterial diameter. To study the effects of PKC, Rho kinase, and voltage-operated Ca2+ channel inhibition, cerebral arteries were preconstricted with agonist and then exposed to bisindolylmaleimide I (10–8–3 x 10–6 M), Y-27632 (10–7–3 x 10–5 M), and diltiazem (3 x 10–5 M), respectively. It should be noted that agonist concentration was varied slightly among experiments (UTP, 10–6–10–5 M; U-46619, 10–9–10–8 M) to set the initial constrictor response at 70–90% of maximum.
Preparation of isolated arterial smooth muscle cells. Smooth muscle cells from rat cerebral arteries were enzymatically isolated as previously described (44). In brief, arterial segments were placed in warm (37°C) cell isolation medium containing (in mM) 60 NaCl, 80 Na-glutamate, 5 KCl, 2 MgCl2, 10 glucose, and 10 HEPES with 1 mg/ml albumin (pH 7.2) for 10 min. After this equilibration step, arterial segments were initially incubated (37°C) in 0.6 mg/ml papain and 1.8 mg/ml dithioerythritol for 15 min. This was followed by a second incubation (37°C) in isolation medium containing 100 µM Ca2+, 0.7 mg/ml type F collagenase, and 0.4 mg/ml type H collagenase for 10 min. After enzyme treatment, the tissue was washed three times in ice-cold isolation medium and triturated with a fire-polished pipette to release the myocytes. Cells were stored in ice-cold isolation medium for use the same day.
Patch-clamp electrophysiology and isolated smooth muscle cells. Conventional whole cell patch clamp was used to measure KDR currents in cerebral smooth muscle cells. Recording electrodes (resistance 4–7 M) were pulled from borosilicate glass, covered in sticky wax to reduce capacitance, and backfilled with pipette solution containing (in mM) 110 K-gluconate, 30 KCl, 0.5 MgCl2, 5 HEPES, 10 EGTA, 5 Na2ATP, and 1 GTP. Recording pipettes were gently lowered onto a cerebral smooth muscle cell, and negative pressure was briefly applied to rupture the membrane and attain a gigaohm seal. Cells were voltage clamped (–60 mV) in a bath solution containing (in mM) 120 NaCl, 3 NaHCO3, 4.2 KCl, 1.2 KH2PO4, 2 MgCl2, 0.1 CaCl2, 10 glucose, and 10 HEPES. A 1 M NaCl-agar salt bridge between the bath and the Ag-AgCl reference electrode was used to minimize offset potentials. Liquid junction potentials were measured and were <2 mV. Whole cell currents, recorded on an Axopatch 200B amplifier (Axon Instruments, Union City, CA), were filtered at 1 kHz, digitized at 5 kHz, and stored on a computer for subsequent analysis with Clampfit 8.1. Cell capacitance averaged 16.4 ± 0.4 pF and was measured with the cancellation circuitry in the voltage-clamp amplifier. Cells were excluded from analysis if capacitance shifted >0.3 pF during an experiment. All electrical recordings were performed at room temperature.
Experimental procedures and isolated smooth muscle cells. Voltage-clamped cells were equilibrated for 15 min before experimentation. After equilibration, whole cell KDR currents were monitored in the presence and absence of 4-AP (5 mM), UTP (30 µM), or PMA (50 nM). To ascertain whether PKC or Rho kinase signaling was involved in the UTP-induced suppression of KDR, cerebral smooth muscle cells were preincubated for 15 min with bisindolylmaleimide I (1 x 10–7 M), calphostin C (2 x 10–7 M), Y-27632 (3 x 10–5 M), or C3 exoenzyme (10 µg/ml + 5 x 10–5 M NAD+) before the addition of UTP. Bisindolylmaleimide I, calphostin C, and Y-27632 were continuously superfused in the bath, whereas C3 exoenzyme-NAD+ was added to the pipette solution. In general, the net current-voltage relationship was determined at 5-min intervals by measuring the peak current at the end of a 300-ms pulse to voltages between –70 and +40 mV. After each 300-ms pulse, cells were voltage clamped to –40 mV (500 ms) to facilitate the monitoring of tail currents. Tail current amplitude was used to assess steady-state activation and was calculated as the difference between the peak amplitude of the tail and the sustained level of current at –40 mV. Steady-state inactivation was ascertained by measuring peak outward current at +20 mV (200-ms voltage step) after a series of prolonged test pulses (45 s) ranging from –90 to 0 mV. Data were fitted to a Boltzmann distribution function so that the voltages for half-maximal activation and inactivation could be calculated.
RT-PCR analysis and isolated smooth muscle cells. Approximately 300 smooth muscle cells were enzymatically isolated from cerebral and cerebellar arteries and placed in RNase- and DNase-free collection tubes. After total RNA extraction (RNeasy mini kit with DNase treatment; Qiagen, Valencia, CA), first-strand cDNA was synthesized with the Sensiscript RT kit (Qiagen). Subsequently, 2 µl of each first-strand cDNA reaction were used as the template in a PCR reaction containing 1.5 mM MgCl2, 0.25 µM forward and reverse primers (University of Calgary), 0.2 mM deoxynucleotide triphosphates; and 2.5 units of recombinant Taq DNA polymerase. 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 rat P2Y2, P2Y4, and P2Y6 were as follows: P2Y2 (forward) 5'-TTCCACGTCACCCGCACCCTCTTATTACT-3', P2Y2 (reverse) 5'-CGATTCCCCAACTCACACATACAAATGATTG-3'; P2Y4 (forward) 5'-CTTCTCTGCCTGGGTGTTTGGTTGGTAGTA-3', P2Y4 (reverse) 5'-TCCCCCGTGAAGAGATAGAGCACTGGA-3'; and P2Y6 (forward) 5'-GCCAGTTATGGAGCGGGACAATGG-3', P2Y6 (reverse) 5'-AGGAACAGGATGCTGCCGTGTAGGTTG-3'. The expected amplicon sizes for P2Y2, P2Y4, and P2Y6 were 539, 473, and 357 bp, respectively. DNA sequencing was used to identify each reaction product. Control experiments confirmed the absence of genomic DNA contamination by assessing where an Rho kinase-specific amplicon could be generated from samples exposed to RNase.
Chemicals, drugs and enzymes. Buffer reagents, UTP, NAD+, collagenase (types F and H), and diltiazem were obtained from Sigma (St. Louis, MO). PMA, bisindolylmaleimide I, Y-27632, C3 exoenzyme, and U-46619 were purchased from Calbiochem (La Jolla, CA). PMA, bisindolylmaleimide I, and C3 exoenzyme were dissolved in DMSO, with the final solvent concentration 
0.05%.
Statistical analysis. Data are expressed as means ± SE, and n indicates the number of vessels or cells. The EC50 of a given agonist was ascertained by fitting a four-parameter logistic function to the observed data. Paired t-tests were performed to statistically compare the effects of a given condition or treatment on arterial diameter, Em, or whole cell current. If more than two conditions or treatments were being compared, a repeated-measures ANOVA was used. When appropriate, a Tukey-Kramer pairwise comparison was used for post hoc analysis. P values 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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300 cerebral smooth muscle cells revealed product bands with the predicted sizes of 539, 473, and 357 bp for P2Y2, P2Y4, and P2Y6 receptors, respectively (Fig. 1C). DNA sequencing confirmed the identity of all PCR products.
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Cerebral smooth muscle cells depolarized from –56.8 ± 0.8 to –37.1 ± 1.6 mV in the presence of UTP (Fig. 2, A and B). Diltiazem (3 x 10–5 M), an inhibitor of voltage-operated Ca2+ channels, attenuated UTP-induced constriction by 31.3 ± 4.8%, indicating that tone development partly depended on depolarization (Fig. 2, C and D); nifedipine (1 x 10–6 M; n = 2) was also noted to attenuate constriction (data not shown). Given that P2Y receptors can activate PKC (18), subsequent experiments examined whether this kinase enabled UTP to both constrict and depolarize the cerebral vasculature. The PKC activator PMA (3 x 10–8 M) initiated a sustained constriction that was abolished by preincubation with bisindolylmaleimide I (10–7 M; Fig. 3A). Despite its effectiveness as a PKC inhibitor, UTP-constricted arteries did not dilate to the application of bisindolylmaleimide I at concentrations ranging from 10–8 to 3 x 10–7 M (Fig. 3, B and C). Preincubation of vessels with bisindolylmaleimide I (1 x 10–7 M) before the addition of UTP (5 µM) was also ineffective at attenuating constriction [diameter response in µm (n = 5): control, 77.1 ± 5.7; bisindolylmaleimide I, 74.3 ± 7.1]. Bisindolylmaleimide I did not prevent UTP from depolarizing cerebral smooth muscle cells (Fig. 3, C and D).
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Contrary to the preceding observations, the Rho kinase inhibitor Y-27632 dilated cerebral arteries preconstricted with UTP (Fig. 4, A and B). This attenuating effect was dose dependent, with 74.0 ± 5.9% (n = 7) of the agonist-induced constriction eliminated at a Y-27632 concentration of 3 x 10–5 M. In addition to its effects on agonist-induced tone, Y-27632 attenuated the ability of UTP to depolarize cerebral smooth muscle cells by 13.8 ± 2.1 mV (Fig. 4, C and D). The effects of Y-27632 on arterial diameter and Em cannot be ascribed to alterations in myogenic signaling because vessels were maintained at low intravascular pressure (15 mmHg).
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Patch-clamp electrophysiology and isolated smooth muscle cells. With conventional whole cell patch-clamp electrophysiology and pipette solutions that minimize BK channel activity, the KDR current was isolated in rat cerebral smooth muscle cells. In general, brief voltage steps positive to –30 mV activated KDR without an induction of inactivation (Fig. 5A). Steady-state activation and inactivation were ascertained by monitoring 1) tail currents (at –40 mV) after a set of 300-ms voltage pulses (range –70 to +40 mV) and 2) peak outward current (at +20 mV) after a series of prolonged test potentials (45 s, range –90 to –10 mV; Fig. 5B). Data fitted to a Boltzmann function established voltages for half-maximal activation and inactivation of +1.5 and –36.2 mV, respectively (Fig. 5C). At +40 mV, the KDR current was stable over time: a small but significant current run-up was noted at –20 mV (P 0.05; Fig. 5D).
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KDR currents in cerebral smooth muscle cells are comprised of two slowly inactivating components that vary in their sensitivity to 4-AP (33). In accordance with past studies (1, 33), 4-AP (5 x 10–3 M) partially suppressed the KDR current, thereby revealing both the 4-AP-sensitive and -insensitive components (Fig. 6, A and B). The slowly inactivating 4-AP-sensitive component of the KDR current represented 37.9 ± 3.8% of the total whole cell current at +40 mV. Superfused iberiotoxin had no significant effect on peak outward current, confirming that high pipette concentrations of EGTA minimize the activity of BK channels (Fig. 6C).
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UTP modulation of KDR current. As shown in Figs. 1 and 2, pyrimidine nucleotides depolarize and constrict cerebral arteries. Consistent with the view that this depolarization partly depends on the modulation of outward K+ conductance, UTP (3 x 10–5 M) was observed to inhibit KDR by 35.0 ± 3.4% at +40 mV (Fig. 7, A and B). Characteristically, KDR current suppression was observable 5–10 min after the addition of UTP, with peak inhibition occurring at 20 min. In the presence of 4-AP (5 x 10–3 M), UTP had no effect on the remaining whole cell KDR current (Fig. 7C). Such observations are consistent with pyrimidine nucleotides modulating the 4-AP-sensitive component of the KDR current.
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Subsequent experiments defined whether PKC enables UTP to suppress the 4-AP-sensitive component of the KDR current. PMA initiated a sustained constriction in intact cerebral arteries that was abolished by the PKC inhibitors calphostin C and bisindolylmaleimide I [diameter response in µm: PMA (3 x 10–8 M), 75.7 ± 8.8; calphostin C (2 x 10–7 M) + PMA, 1.2 ± 1.5; see Fig. 2A for bisindolylmaleimide I]. Despite the effectiveness of PMA as a PKC activator, this agent (5 x 10–8 M) had no discernible effect on the KDR current (Fig. 8). The ability of UTP to suppress the KDR current was not blocked by preincubating cerebral smooth muscle cells with calphostin C (2 x 10–7 M; Fig. 9, A and C) or bisindolylmaleimide I (1 x 10–7 M; Fig. 9, B and C).
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In light of recent observations showing that the UTP-sensitive P2Y receptor can activate RhoA and Rho kinase (37), subsequent experiments examined whether these signaling proteins enable UTP to suppress the 4-AP-sensitive component of the KDR current. Under resting conditions, the application of Y-27632 (3 x 10–5 M) elicited a small but statistically insignificant increase in whole cell KDR current (Fig. 10). Although the effects of Y-27632 were small under basal conditions, this Rho kinase inhibitor abolished the ability of UTP to suppress KDR. Inhibiting RhoA activation with C3 exoenzyme (10 µg/ml) also eliminated the UTP-induced suppression of KDR (Fig. 11, A and C). Control experiments confirmed that NAD+ alone could not prevent UTP from suppressing the KDR current (Fig. 11, B and D).
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U-46619 and intact cerebral arteries. Further experiments tested whether the thromboxane mimetic U-46619 constricts and depolarizes cerebral arteries through a transduction process that also involves Rho kinase rather than PKC. As shown in Fig. 12A, U-46619 elicited a dose-dependent constriction in cerebral arteries stripped of endothelium and pressurized to 15 mmHg to minimize myogenic tone (EC50 = 1.7 x 10–9 M). This constriction was not blocked by bisindolylmaleimide I [diameter in µm (n = 6): control, 226.5 ± 8.3; U-46619, 137.2 ± 7.4; U-46619 + bisindolylmaleimide I, 133.3 ± 8.2] but was attenuated in a dose-dependent manner by Y-27632 (Fig. 12, B and C). U-46619 initiated a depolarization in cerebral smooth muscle cells that was unaffected by bisindolylmaleimide I (Fig. 12D). In contrast, when bisindolylmaleimide I and Y-27632 were applied together, the depolarization induced by U-46619 was diminished by 9.4 ± 0.6 mV.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pyrimidine nucleotides and cerebral vasculature. Pyrimidine nucleotides such as UTP and UDP are important signaling compounds released from cells during mechanical stress and cellular injury (23). When secreted in close proximity to cerebral smooth muscle cells, these agents elicit a sustained constrictor response (12, 19, 27). This maintained constriction was readily observed in the present study and is the result of P2Y receptor activation. Molecular studies have identified at least five subtypes of P2Y receptors that are coupled to trimeric G proteins (3, 31). Pyrimidine nucleotides potently activate three of these subtypes (i.e., P2Y2, P2Y4, and P2Y6), all of which are expressed at the mRNA level in isolated cerebral smooth muscle cells. To elicit constriction, pyrimidine-sensitive P2Y receptors must activate a signaling pathway that enhances the Ca2+ sensitivity of the myofilament (39) or increases cytosolic Ca2+ through mechanisms that are either dependent or independent of resting membrane potential (28). Two key findings support the view that electromechanical coupling plays an important role in enabling UTP to constrict cerebral arteries. First, the bath application of UTP depolarized cerebral smooth muscle cells by 19.6 ± 1.6 mV. Second, inhibiting depolarization-induced increases in Ca2+ channel activity (with diltiazem) attenuated constriction by >30%. At present, there is little consensus as to which signaling mechanisms enable pyrimidine-sensitive P2Y receptors to depolarize and constrict intact cerebral arteries. Of the few studies conducted in this area, Jaggar and Nelson (18) proposed a prominent role for PKC, a serine/threonine kinase coupled to P2Y receptors through Gq, phospholipase C, and the production of diacylglycerol. This hypothesis was predicated on patch-clamp observations of spontaneous transient outward currents but did not involve a direct examination of intact cerebral arteries (18). Surprisingly, when this study applied a confirmed PKC inhibitor to cerebral arteries preconstricted with UTP, there was no measurable effect on either constriction or depolarization.
Although PKC activation is not a key factor in enabling UTP-sensitive P2Y receptors to depolarize or constrict cerebral arteries, these findings should not be interpreted to suggest that arterial constriction is independent of Gq signaling. Indeed, pyrimidine nucleotides have been observed in cerebral arteries to induce Ca2+ waves, transitory Ca2+ events driven by phospholipase C activation and the production of inositol (1,4,5)-trisphosphate (18). Although Ca2+ waves have been associated with arterial constriction (10, 35), studies have yet to resolve how a periodic event can elicit sustained constriction. We propose that a sustained constrictor response could be achieved if Ca2+ wave activation coincided with an inhibition of myosin light chain phosphatase. Indeed, it could be the combination of Ca2+ waves and phosphatase inhibition (via Rho kinase) that underlies a significant part of the UTP-mediated constriction insensitive to Ca2+ channel blockade. The periodic release of Ca2+ may also stimulate store-operated channels that in theory could increase cytosolic Ca2+ and elicit a constriction independent of membrane potential.
Recent findings in the coronary circulation have shown that pyrimidine-induced contraction can be effectively abolished by the Rho kinase inhibitor Y-27632 (37). From these observations, it was hypothesized that UTP-sensitive P2Y receptors mediate arterial constriction by activating G12/13 and its associated signaling pathway including p115 Rho guanine nucleotide exchange factor (GEF), RhoA, and Rho kinase (37). Observations from the current study indicate that Rho kinase signaling is also important in the regulation of cerebral vascular tone. More specifically, we observed that cerebral arteries preconstricted with UTP dilated in a dose-dependent manner to Y-27632. The vascular literature characteristically assumes that if Rho kinase inhibition impairs arterial constriction, such an alteration must exclusively reflect a change in the Ca2+ sensitivity of the myofilament (37, 40). This assumption arises from the known ability of Rho kinase to target and regulate myosin light chain phosphatase (40, 42). Although an important regulator of pharmacomechanical coupling, Rho kinase could conceivably target other downstream effectors of smooth muscle cell contraction including ion channels involved in membrane potential regulation. Consistent with this view, Y-27632 was observed to reverse the depolarization initiated by UTP. These physiological findings appear to be the first in the resistance vasculature to support a role for Rho kinase in the regulation of ion channel activity and electromechanical coupling.
To depolarize cerebral smooth muscle cells, P2Y receptors along with Rho kinase must enhance depolarizing inward currents and/or inhibit hyperpolarizing K+ conductances. Although voltage-operated Ca2+ (34), nonselective cation (44), and four distinct K+ (28) currents have been isolated in cerebral smooth muscle cells, studies have never ascertained whether any of these conductances are sensitive to Rho kinase modulation. Despite this lack of evidence in native tissue, findings in expression systems have shown that voltage-dependent KDR channels may be one of the primary targets (6). This view is based on the work of Cachero et al. (6), who observed the ability of G protein-coupled receptors to suppress Kv1.2 by activating RhoA, the upstream regulator of Rho kinase. Intriguingly, Kv1.2 is one of the key pore-forming subunits of the slowly inactivating 4-AP-sensitive KDR channel in vascular smooth muscle (20, 43). This KDR channel contributes to resting Em, and its inhibition by vasoconstrictors is known to induce arterial depolarization (7, 16). In light of these observations, a supplemental examination was undertaken to examine the relationship between pyrimidine nucleotides and KDR channel regulation.
KDR current in cerebral smooth muscle cells. Conventional whole cell patch-clamp electrophysiology was used to isolate and examine the KDR current in cerebral smooth muscle cells. Generally speaking, whole cell KDR current was characterized by 1) measurable activation at voltages positive to –30 mV, 2) slow time-dependent inactivation, and 3) voltages of half-maximal activation and inactivation of +1.5 and –36.1 mV, respectively. These documented KDR properties are similar to those in rat mesenteric arteries (16) and indicate that steady-state KDR current will be small at physiological Em (–30 to –60 mV). Despite their limited magnitude, the high input resistance of vascular smooth muscle cells (5–20 G) dictates that such currents will play an important role in controlling membrane potential (28). Like KDR, outward current through inwardly rectifying K+ channels and inward current through nonselective cation channels are equally small and difficult to monitor with patch-clamp electrophysiology (30, 32, 44). Nevertheless, both currents have been shown to alter the resting Em of intact cerebral arteries by 10–15 mV (22, 44). Quantitative three-dimensional electrical models of the resistance vasculature also indicate that small currents are functionally relevant and important to dynamic vessel function (45).
The KDR current in vascular smooth muscle is divisible into distinct components that include 1) a slowly inactivating 4-AP-sensitive current, 2) a slowly inactivating 4-AP-insensitive current, and 3) a rapidly inactivating 4-AP-sensitive A-type current (KTO; Refs. 1 and 20). Unlike portal vein, KTO current could not be discerned in cerebral smooth muscle cells. The slowly inactivating currents were, however, readily observable, with the 4-AP-sensitive component representing 37.9 ± 3.7% of the overall current. Interestingly, this proportion is sizably smaller than that previously reported for portal vein (7) and pulmonary (8) arteries. Although work is limited in the resistance vasculature, observations in portal vein have implicated Kv1.2 and Kv1.5 as the key pore-forming subunits of 4-AP-sensitive channels (20). In contrast, the pore-forming subunits of 4-AP-insensitive channels are thought to include members of the Kv7 family (29).
UTP-induced regulation of KDR current. Intact vessel measurements demonstrated that pyrimidine nucleotides constrict intact cerebral arteries through a mechanism that partly depends on smooth muscle cell depolarization and Ca2+ influx through voltage-operated Ca2+ channels (18). Consistent with depolarization arising from a reduction in outward K+ conductance, UTP was observed to potently suppress the KDR current. This suppression of KDR was particularly evident at positive potentials where the current is large and less susceptible to subtle time-dependent changes in current amplitude and seal resistance. The onset of current suppression typically began 5–10 min after agonist application and peaked by 20 min. The slow nature of agonist-induced current suppression was not entirely unexpected in light of past findings (7) and given that the current experiments were performed on enzymatically isolated cells maintained at room temperature and under conditions that result in significant cellular dialysis. The two slowly inactivating components of KDR current were differentially affected by this pyrimidine nucleotide. More specifically, UTP elicited a near-complete inhibition of the 4-AP-sensitive current whereas the 4-AP-insensitive component remained unaffected. Although this study is the first to demonstrate that pyrimidine-sensitive P2Y receptors target specific KDR channels, this pattern of preferential regulation is not unique. For example, vasoconstrictors such as angiotensin II have been reported to selectively suppress 4-AP-sensitive KDR currents in portal vein (7). Correspondingly, activators of PKA and PKC have been shown to preferentially target the same KDR channels (1, 9). Such observations support the view that 4-AP-sensitive channels are more tightly controlled by phosphorylation events than KDR channels insensitive to this K+ channel blocker (7).
To inhibit the slowly inactivating 4-AP-sensitive component of the KDR current, UTP-sensitive P2Y receptors must activate a trimeric G protein along with its associated signaling cascade. Traditionally, it has been asserted that this receptor class is coupled to Gq and its downstream effectors including phospholipase C and PKC (24, 41). PKC is a serine/threonine kinase whose activity is known to suppress 4-AP-sensitive KDR channels in portal vein and mesenteric artery (1, 7, 16). Although UTP-sensitive P2Y receptors can in theory stimulate PKC, two key findings indicate that this kinase is not responsible for KDR current suppression in cerebral artery myocytes. First, the PKC activator PMA failed to inhibit KDR when applied at concentrations known to constrict intact cerebral arteries. Second, preincubation of cerebral smooth muscle cells with PKC inhibitors (calphostin C or bisindolylmaleimide I) did not prevent UTP from suppressing the KDR current. These electrophysiological findings nicely parallel functional observations that demonstrated that PKC inhibition did not impair the depolarization of intact cerebral arteries by UTP. Although Rho kinase is conventionally viewed as a regulator of Ca2+ sensitivity, it is conceivable for this phosphotransferase protein along with its upstream regulator RhoA to mediate KDR current suppression by UTP (6). Supporting a role for Rho kinase signaling, Y-27632 was observed in this study to prevent UTP from inhibiting KDR in isolated cerebral myocytes. Likewise, the RhoA inhibitor C3 exoenzyme was also effective at preventing pyrimidine nucleotides from regulating KDR.
Physiological implications. In summary, this study demonstrated that pyrimidine nucleotides constrict intact cerebral arteries through a mechanism that partly involves the depolarization of vascular smooth muscle cells and the activation of voltage-operated Ca2+ channels. We report for the first time in cerebral arterial smooth muscle that depolarization is dependent on the activation of Rho kinase and not PKC. One of the ionic conductances inhibited by UTP via Rho kinase is the 4-AP-sensitive KDR current. We hypothesize that pyrimidine-sensitive P2Y receptors activate Rho kinase through a signal transduction cascade that involves G12/13, p115 Rho GEF, and RhoA (Fig. 13). It is conceivable that Rho kinase regulation of ion channels might be narrowly limited to the cerebral vasculature or to the preceding receptor class. This view, however, is challenged by recent mesenteric observations showing that Rho kinase inhibition partially attenuates norepinephrine-induced depolarization (13) as well as by the findings shown in Fig. 12, which indicate that the thromboxane mimetic U-46619 also depolarizes and constricts cerebral arteries by activating Rho kinase and not PKC. Further investigation is required to determine whether other ion channels are modulated by Rho kinase and thus contribute to the depolarizations induced by UTP and other constrictor agonists. Indeed, the recent observations by Ghisdal et al. (13) have indirectly implied that this serine/threonine kinase could activate a depolarizing nonselective cation current. In closing, we suggest that in the cerebral circulation Rho kinase regulation may be a common mechanism by which G protein-coupled receptors control both the electrical and the contractile state of vascular smooth muscle.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by the Canadian Institute for Health Research and the Alberta Heritage Foundation for Medical Research (AHFMR). D. G. Welsh was supported by a Heart and Stroke Foundation New Investigator Scholarship Award and an AHFMR Scholar Award.
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FOOTNOTES |
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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.
* K. D. Luykenaar and S. E. Brett contributed equally to this work. 
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 L. Stirling, M. R. Williams, and A. D. Morielli Dual Roles for RHOA/RHO-Kinase In the Regulated Trafficking of a Voltage-sensitive Potassium Channel Mol. Biol. Cell, June 15, 2009; 20(12): 2991 - 3002. [Abstract] [Full Text] [PDF]
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 R. P. Johnson, A. F. El-Yazbi, M. F. Hughes, D. C. Schriemer, E. J. Walsh, M. P. Walsh, and W. C. Cole Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449 J. Biol. Chem., June 12, 2009; 284(24): 16562 - 16574. [Abstract] [Full Text] [PDF]
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 R. P. Johnson, A. F. El-Yazbi, K. Takeya, E. J. Walsh, M. P. Walsh, and W. C. Cole Ca2+ sensitization via phosphorylation of myosin phosphatase targeting subunit at threonine-855 by Rho kinase contributes to the arterial myogenic response J. Physiol., June 1, 2009; 587(11): 2537 - 2553. [Abstract] [Full Text] [PDF]
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 K. D. Luykenaar, R. A. El-Rahman, M. P. Walsh, and D. G. Welsh Rho-kinase-mediated suppression of KDR current in cerebral arteries requires an intact actin cytoskeleton Am J Physiol Heart Circ Physiol, April 1, 2009; 296(4): H917 - H926. [Abstract] [Full Text] [PDF]
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L.-M. Jin Rock 'n' Rho: regulation of ion channels Am J Physiol Heart Circ Physiol, April 1, 2009; 296(4): H908 - H909. [Full Text] [PDF]
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P. D. Smith, S. E. Brett, K. D. Luykenaar, S. L. Sandow, S. P. Marrelli, E. J. Vigmond, and D. G. Welsh KIR channels function as electrical amplifiers in rat vascular smooth muscle J. Physiol., February 15, 2008; 586(4): 1147 - 1160. [Abstract] [Full Text] [PDF]
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V. Telezhkin, T. Goecks, A. D. Bonev, G. Osol, and N. I. Gokina Decreased function of voltage-gated potassium channels contributes to augmented myogenic tone of uterine arteries in late pregnancy Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H272 - H284. [Abstract] [Full Text] [PDF]
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R. L. Corteling, S. E. Brett, H. Yin, X.-L. Zheng, M. P. Walsh, and D. G. Welsh The functional consequence of RhoA knockdown by RNA interference in rat cerebral arteries Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H440 - H447. [Abstract] [Full Text] [PDF]
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K. D. Luykenaar and D. G. Welsh Activators of the PKA and PKG pathways attenuate RhoA-mediated suppression of the KDR current in cerebral arteries Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2654 - H2663. [Abstract] [Full Text] [PDF]
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M. C. Jantzi, S. E. Brett, W. F. Jackson, R. Corteling, E. J. Vigmond, and D. G. Welsh Inward rectifying potassium channels facilitate cell-to-cell communication in hamster retractor muscle feed arteries Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1319 - H1328. [Abstract] [Full Text] [PDF]
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 S. Chrissobolis and C. G. Sobey Recent Evidence for an Involvement of Rho-Kinase in Cerebral Vascular Disease Stroke, August 1, 2006; 37(8): 2174 - 2180. [Abstract] [Full Text] [PDF]
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 N. I. Gokina, K. M. Park, K. McElroy-Yaggy, and G. Osol Effects of Rho kinase inhibition on cerebral artery myogenic tone and reactivity J Appl Physiol, May 1, 2005; 98(5): 1940 - 1948. [Abstract] [Full Text] [PDF]
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S. A. Reading, S. Earley, B. J. Waldron, D. G. Welsh, and J. E. Brayden TRPC3 mediates pyrimidine receptor-induced depolarization of cerebral arteries Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2055 - H2061. [Abstract] [Full Text] [PDF]
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 T. Nagaoka, K. A. Fagan, S. A. Gebb, K. G. Morris, T. Suzuki, H. Shimokawa, I. F. McMurtry, and M. Oka Inhaled Rho Kinase Inhibitors Are Potent and Selective Vasodilators in Rat Pulmonary Hypertension Am. J. Respir. Crit. Care Med., March 1, 2005; 171(5): 494 - 499. [Abstract] [Full Text] [PDF]
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 F. Plane, R. Johnson, P. Kerr, W. Wiehler, K. Thorneloe, K. Ishii, T. Chen, and W. Cole Heteromultimeric Kv1 Channels Contribute to Myogenic Control of Arterial Diameter Circ. Res., February 4, 2005; 96(2): 216 - 224. [Abstract] [Full Text] [PDF]
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S Shabir, L Borisova, S. Wray, and T Burdyga Rho-kinase inhibition and electromechanical coupling in rat and guinea-pig ureter smooth muscle: Ca2+-dependent and -independent mechanisms J. Physiol., November 1, 2004; 560(3): 839 - 855. [Abstract] [Full Text] [PDF]
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J. T. Sylvester The tone of pulmonary smooth muscle: ROK and Rho music? Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L624 - L630. [Full Text] [PDF]
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