William C. Cole

Transient Receptor Potential Channel 6–Mediated, Localized Cytosolic [Na+] Transients Drive Na+/Ca2+ Exchanger–Mediated Ca2+ Entry in Purinergically Stimulated Aorta Smooth Muscle Cells

The Na+/Ca2+ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca2+ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca2+ influx by the NCX (1) is the primary mechanism of Ca2+ entry in purinergically stimulated rat aorta smooth muscle cells and (2) requires functional coupling with transient receptor potential channel 6 nonselective cation channels. Using the Na+ indicator CoroNa Green, we now directly observed and characterized the localized cytosolic [Na+] ([Na+]i) elevations that have long been hypothesized to underlie physiological NCX reversal but that have never been directly shown. Stimulation of rat aorta smooth muscle cells caused both global and monotonic [Na+]i elevations and localized [Na+]i transients (LNats) at the cell periphery. Inhibition of nonselective cation channels with SKF-96365 (50 &mgr;mol/L) and 2-amino-4-phosphonobutyrate (75 &mgr;mol/L) reduced both global and localized [Na+]i elevations in response to ATP (1 mmol/L). This effect was mimicked by expression of a dominant negative construct of transient receptor potential channel 6. Selective inhibition of NCX-mediated Ca2+ entry with KB-R7943 (10 &mgr;mol/L) enhanced the LNats, whereas the global cytosolic [Na+] signal was unaffected. Inhibition of mitochondrial Na+ uptake with CGP-37157 (10 &mgr;mol/L) increased both LNats and global cytosolic [Na+] elevations. These findings directly demonstrate NCX regulation by LNats, which are restricted to subsarcolemmal, cytoplasmic microdomains. Analysis of the LNats, which facilitate Ca2+ entry via NCX, suggests that mitochondria limit the cytosolic diffusion of LNats generated by agonist-mediated activation of transient receptor potential channel 6–containing channels.

Heteromultimeric Kv1 Channels Contribute to Myogenic Control of Arterial Diameter

Inhibition of vascular smooth muscle (VSM) delayed rectifier K+ channels (KDR) by 4-aminopyridine (4-AP; 200 &mgr;mol/L) or correolide (1 &mgr;mol/L), a selective inhibitor of Kv1 channels, enhanced myogenic contraction of rat mesenteric arteries (RMAs) in response to increases in intraluminal pressure. The molecular identity of KDR of RMA myocytes was characterized using RT-PCR, real-time PCR, and immunocytochemistry. Transcripts encoding the pore-forming Kv&agr; subunits, Kv1.2, Kv1.4, Kv1.5, and Kv1.6, were identified and confirmed at the protein level with subunit-specific antibodies. Kv&bgr; transcript (&bgr;1.1, &bgr;1.2, &bgr;1.3, and &bgr;2.1) expression was also identified. Kv1.5 message was ≈2-fold more abundant than that for Kv1.2 and Kv1.6. Transcripts encoding these three Kv1&agr; subunits were ≈2-fold more abundant in 1st/2nd order conduit compared with 4th order resistance RMAs, and Kv&bgr;1 was 8-fold higher than Kv&bgr;2 message. RMA KDR activated positive to −50 mV, exhibited incomplete inactivation, and were inhibited by 4-AP and correolide. However, neither &agr;-dendrotoxin or &kgr;-dendrotoxin affected RMA KDR, implicating the presence of Kv1.5 in all channels and the absence of Kv1.1, respectively. Currents mediated by channels because of coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv&bgr;1.2 in human embryonic kidney 293 cells had biophysical and pharmacological properties similar to those of RMA KDR. It is concluded that KDR channels composed of heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter.

Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes.

1. The effect of angiotension II (Ang) on delayed rectifier K+ current (IK(V)) was studied in isolated rabbit portal vein smooth muscle cells using standard whole‐cell voltage clamp technique. The effect of 100 nM Ang on macroscopic, whole‐cell IK(V) was assessed in myocytes dialysed with 10 mM BAPTA, 5 mM ATP and 1 mM GTP either at room temperature or at 30 degrees C. 2. Application of Ang caused a decline in IK(V) which was reversed upon washout of the drug. Tail current recorded after 250 ms pulses to +30 mV and repolarization to ‐40 mV was reduced from 3.9 +/‐ 0.7 to 2.5 +/‐ 0.5 pA pF‐1 at 20 degrees C (n = 6) and from 4.5 +/‐ 0.5 to 3.13 +/‐ 0.4 pA pF‐1 at 30 degrees C(n = 17). 3. Ang had no effect on outward current in the presence of an AT1 selective antagonist, losartan (1 microM), which alone had no direct effect on the amplitude of IK(V). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 microM intracellular BAPTA did not affect the suppression of IK(V) by Ang. 4. Ang induced a decrease in time constant for the rapid phase of inactivation of the macroscopic current (tau 1 reduced from 377 +/‐ 32 to 245 +/‐ 11 ms; tau 2 unchanged, n = 17). Neither the voltage dependence of activation nor inactivation were affected by Ang. 5. The inhibition of IK(V) by Ang was abolished by intracellular dialysis with the selective PKC inhibitors, calphostin C (1 microM) and chelerythrine (50 microM). These data provide strong evidence that the decline in IK(V) due to Ang treatment is due to PKC activation. 6. The pattern of expression of PKC isoforms was examined in rabbit portal vein using isoenzyme‐specific antibodies: alpha, epsilon and zeta isoenzymes were detected, but beta, gamma, delta and eta isoenzymes were not. 7. The lack of requirement for Ca2+, as well as the sensitivity of the Ang response to chelerythrine, suggest the involvement of the Ca(2+)‐independent PKC isoenzyme epsilon in the signal transduction pathway responsible for IK(V) inhibition by Ang.

Participation of KCNQ (Kv7) potassium channels in myogenic control of cerebral arterial diameter

KCNQ gene expression was previously shown in various rodent blood vessels, where the products of KCNQ4 and KCNQ5, Kv7.4 and Kv7.5 potassium channel subunits, respectively, have an influence on vascular reactivity. The aim of this study was to determine if small cerebral resistance arteries of the rat express KCNQ genes and whether Kv7 channels participate in the regulation of myogenic control of diameter. Quantitative reverse transcription polymerase chain reaction (QPCR) was undertaken using RNA isolated from rat middle cerebral arteries (RMCAs) and immunocytochemistry was performed using Kv7 subunit‐specific antibodies and freshly isolated RMCA myocytes. KCNQ4 message was more abundant than KCNQ5=KCNQ1, but KCNQ2 and KCNQ3 message levels were negligible. Kv7.1, Kv7.4 and Kv7.5 immunoreactivity was present at the sarcolemma of freshly isolated RMCA myocytes. Linopirdine (1 μm) partially depressed, whereas the Kv7 activator S‐1 (3 and/or 20 μm) enhanced whole‐cell Kv7.4 (in HEK 293 cells), as well as native RMCA myocyte Kv current amplitude. The effects of S‐1 were voltage‐dependent, with progressive loss of stimulation at potentials of >−15 mV. At the concentrations employed linopirdine and S‐1 did not alter currents due to recombinant Kv1.2/Kv1.5 or Kv2.1/Kv9.3 channels (in HEK 293 cells) that are also expressed by RMCA myocytes. In contrast, another widely used Kv7 blocker, XE991 (10 μm), significantly attenuated native Kv current and also reduced Kv1.2/Kv1.5 and Kv2.1/Kv9.3 currents. Pressurized arterial myography was performed using RMCAs exposed to intravascular pressures of 10–100 mmHg. Linopirdine (1 μm) enhanced the myogenic response at ≥20 mmHg, whereas the activation of Kv7 channels with S‐1 (20 μm) inhibited myogenic constriction at >20 mmHg and reversed the increased myogenic response produced by suppression of Kv2‐containing channels with 30 nm stromatoxin (ScTx1). These data reveal a novel contribution of KCNQ gene products to the regulation of myogenic control of cerebral arterial diameter and suggest that Kv7 channel activating drugs may be appropriate candidates for the development of an effective therapy to ameliorate cerebral vasospasm.

Ca2+ sensitization via phosphorylation of myosin phosphatase targeting subunit at threonine-855 by Rho kinase contributes to the arterial myogenic response

Ca2+ sensitization has been postulated to contribute to the myogenic contraction of resistance arteries evoked by elevation of transmural pressure. However, the biochemical evidence of pressure‐induced increases in phosphorylated myosin light chain phosphatase (MLCP) targeting subunit 1 (MYPT1) and/or 17 kDa protein kinase C (PKC)‐potentiated protein phosphatase 1 inhibitor protein (CPI‐17) required to sustain this view is not currently available. Here, we determined whether Ca2+ sensitization pathways involving Rho kinase (ROK)‐ and PKC‐dependent phosphorylation of MYPT1 and CPI‐17, respectively, contribute to the myogenic response of rat middle cerebral arteries. ROK inhibitors (Y27632, 0.03–10 μmol l−1; H1152, 0.001–0.3 μmol l−1) and PKC inhibitors (GF109203X, 3 μmol l−1; Gö6976; 10 μmol l−1) suppressed myogenic vasoconstriction between 40 and 120 mmHg. An improved, highly sensitive 3‐step Western blot method was developed for detection and quantification of MYPT1 and CPI‐17 phosphorylation. Increasing pressure from 10 to 60 or 100 mmHg significantly increased phosphorylation of MYPT1 at threonine‐855 (T855) and myosin light chain (LC20). Phosphorylation of MYPT1 at threonine‐697 (T697) and CPI‐17 were not affected by pressure. Pressure‐evoked elevations in MYPT1‐T855 and LC20 phosphorylation were reduced by H1152, but MYPT1‐T697 phosphorylation was unaffected. Inhibition of PKC with GF109203X did not affect MYPT1 or LC20 phosphorylation at 100 mmHg. Our findings provide the first direct, biochemical evidence that a Ca2+ sensitization pathway involving ROK‐dependent phosphorylation of MYPT1 at T855 (but not T697) and subsequent augmentation of LC20 phosphorylation contributes to myogenic control of arterial diameter in the cerebral vasculature. In contrast, suppression of the myogenic response by PKC inhibitors cannot be attributed to block of Ca2+ sensitization mediated by CPI‐17 or MYPT1 phosphorylation.

Heteromultimeric TRPC6-TRPC7 Channels Contribute to Arginine Vasopressin-Induced Cation Current of A7r5 Vascular Smooth Muscle Cells

The molecular identity of receptor-operated, nonselective cation channels (ROCs) of vascular smooth muscle (VSM) cells is not known for certain. Mammalian homologues of the Drosophila canonical transient receptor potential channels (TRPCs) are possible candidates. This study tested the hypothesis that heteromultimeric TRPC channels contribute to ROC current of A7r5 VSM cells activated by [Arg8]-vasopressin. A7r5 cells expressed transcripts encoding TRPC1, TRPC4&bgr;, TRPC6, and TRPC7. TRPC4, TRPC6, and TRPC7 protein expression was confirmed by immunoblotting and association of TRPC6 with TRPC7, but not TRPC4&bgr;, was detected by coimmunoprecipitation. The amplitude of arginine vasopressin (AVP)-induced ROC current was suppressed by dominant-negative mutant TRPC6 (TRPC6DN) but not TRPC5 (TRPC5DN) mutant subunit expression. These data indicate a role for TRPC6- and/or TRPC7-containing channels and rule a more complex subunit composition including TRPC1 and TRPC4. Increasing extracellular Ca2+ concentration ([Ca2+]o) from 0.05 to 1 mmol/L suppressed currents owing to native, TRPC7, and heteromultimeric TRPC6-TRPC7 channels, but not TRPC6 current, which was slightly enhanced. The relative changes in native and heteromultimeric TRPC6-TRPC7 current amplitudes for [Ca2+]o between ≈0.01 and 1 mmol/L were identical, but the changes in homomultimeric TRPC6 and TRPC7 currents were significantly less and greater, respectively, compared with the native channels. Taken together, the data provide biochemical and functional evidence supporting the view that heteromultimeric TRPC6-TRPC7 channels contribute to receptor-activated, nonselective cation channels of A7r5 VSM cells.

The role of actin filament dynamics in the myogenic response of cerebral resistance arteries

The myogenic response has a critical role in regulation of blood flow to the brain. Increased intraluminal pressure elicits vasoconstriction, whereas decreased intraluminal pressure induces vasodilatation, thereby maintaining flow constant over the normal physiologic blood pressure range. Improved understanding of the molecular mechanisms underlying the myogenic response is crucial to identify deficiencies with pathologic consequences, such as cerebral vasospasm, hypertension, and stroke, and to identify potential therapeutic targets. Three mechanisms have been suggested to be involved in the myogenic response: (1) membrane depolarization, which induces Ca2+ entry, activation of myosin light chain kinase, phosphorylation of the myosin regulatory light chains (LC20), increased actomyosin MgATPase activity, cross-bridge cycling, and vasoconstriction; (2) activation of the RhoA/Rho-associated kinase (ROCK) pathway, leading to inhibition of myosin light chain phosphatase by phosphorylation of MYPT1, the myosin targeting regulatory subunit of the phosphatase, and increased LC20 phosphorylation; and (3) activation of the ROCK and protein kinase C pathways, leading to actin polymerization and the formation of enhanced connections between the actin cytoskeleton, plasma membrane, and extracellular matrix to augment force transmission. This review describes these three mechanisms, emphasizing recent developments regarding the importance of dynamic actin polymerization in the myogenic response of the cerebral vasculature.

Key role of Kv1 channels in vasoregulation.

Small arteries play an essential role in the regulation of blood pressure and organ-specific blood flow by contracting in response to increased intraluminal pressure, ie, the myogenic response. The molecular basis of the myogenic response remains to be defined. To achieve incremental changes in arterial diameter, as well as blood pressure or organ-specific blood flow, the depolarizing influence of intravascular pressure on vascular smooth muscle membrane potential that elicits myogenic contraction must be precisely controlled by an opposing hyperpolarizing influence. Here we use a dominant-negative molecular strategy and pressure myography to determine the role of voltage-dependent Kv1 potassium channels in vasoregulation, specifically, whether they act as a negative-feedback control mechanism of the myogenic response. Functional Kv1 channel expression was altered by transfection of endothelium-denuded rat middle cerebral arteries with cDNAs encoding c-myc epitope-tagged, dominant-negative mutant or wild-type rabbit Kv1.5 subunits. Expression of mutant Kv1.5 dramatically enhanced, whereas wild-type subunit expression markedly suppressed, the myogenic response over a wide range of intraluminal pressures. These effects on arterial diameter were associated with enhanced and reduced myogenic depolarization by mutant and wild-type Kv1.5 subunit expression, respectively. Expression of myc-tagged mutant and wild-type Kv1.5 subunit message and protein in transfected but not control arteries was confirmed, and isolated myocytes of transfected but not control arteries exhibited anti-c-myc immunofluorescence. No changes in message encoding other known, non-Kv1 elements of the myogenic response were apparent. These findings provide the first molecular evidence that Kv1-containing delayed rectifier K+ (KDR) channels are of fundamental importance for control of arterial diameter and, thereby, peripheral vascular resistance, blood pressure, and organ-specific blood flow.

NO/PGI2-independent vasorelaxation and the cytochrome P450 pathway in rabbit carotid artery

1 The nature and cellular mechanisms that are responsible for endothelium‐dependent relaxations resistant to indomethacin and NG‐nitro‐L‐arginine methyl ester (l‐NAME) were investigated in phenylephrine (PE) precontracted isolated carotid arteries from the rabbit. 2 In the presence of the cyclo‐oxygenase inhibitor, indomethacin (10 μm), acetylcholine (ACh) induced a concentration‐and endothelium‐dependent relaxation of PE‐induced tone which was more potent than the calcium ionophore A23187 with pD2 values of 7.03 ± 0.12 (n = 8) and 6.37 ± 0.12 (n = 6), respectively. The ACh‐induced response was abolished by removal of the endothelium, but was not altered when indomethacin was omitted (pD2 value 7.00 ± 0.10 and maximal relaxation 99 ± 3%, n = 6). Bradykinin and histamine (0.01–100 μm) had no effect either upon resting or PE‐induced tone (n = 5). 3 In the presence of indomethacin plus the NO synthase inhibitor, L‐NAME (30 μm), the response to A23187 was abolished. However, the response to ACh was not abolished, although it was significantly inhibited with the pD2 value and the maximal relaxation decreasing to 6.48 ± 0.10 and 67 ± 3%, respectively (for both P < 0.01, n = 8). The L‐NAME/indomethacin insensitive vasorelaxation to ACh was completely abolished by preconstriction of the tissues with potassium chloride (40 mM, n = 8). 4 The Ca2+‐activated K+ (KCa) channel blockers, tetrabutylammonium (TBA, 1 mM, n =5) and charybdotoxin (CTX, 0.1 μM, n =5), completely inhibited the nitric oxide (NO) and prostacyclin (PGI2)‐independent relaxation response to ACh. However, iberiotoxin (ITX, 0.1 μm, n = 8) or apamin (1–3 μm, n = 6) only partially inhibited the relaxation. 5 Inhibitors of the cytochrome P450 mono‐oxygenase, SKF‐525A (1–10 μm, n = 6), clotrimazole (1 μM, n = 5) and 17‐octadecynoic acid (17‐ODYA, 3 μm, n = 7) also reduced the NO/PGI2‐independent relaxation response to ACh. 6 In endothelium‐denuded rings of rabbit carotid arteries, the relaxation response to exogenous NO was not altered by either KCa channel blockade with apamin (1 μm, n = 5) or CTX (0.1 μM, n = 5), or by the cytochrome P450 mono‐oxygenase blockers SKF‐525A (10 μm, n = 4) and clotrimazole (10 μm, n = 5). However, the NO‐induced response was shifted to the right by LY83583 (10 μm, n = 4), a guanylyl cyclase inhibitor, with the pD2 value decreasing from 6.95 ± 0.14 to 6.04 ± 0.09 (P <0.01). 7 ACh (0.01–100 μm) induced a concentration‐dependent relaxation of PE‐induced tone in endothelium‐denuded arterial segments sandwiched with endothelium‐intact donor segments. This relaxation to ACh was largely unaffected by indomathacin (10 μm) plus L‐NAME (30 μM), but abolished by the combination of indomethacin, L‐NAME and TBA (1 mM, n = 5). 8 These data suggest that in the rabbit carotid artery: (a) ACh can induce the release of both NO and EDHF, whereas A23187 only evokes the release of NO from the endothelium, (b) the diffusible EDHF released by ACh may be a cytochrome P450‐derived arachidonic acid metabolite, and (c) EDHF‐induced relaxation involves the opening of at least two types of KCa channels, whereas NO mediates vasorelaxation via a guanosine 3′: 5′‐cyclic monophosphate (cyclic GMP)‐mediated pathway, in which a cytochrome P450 pathway and KCa channels do not seem to be involved.

Role of myosin light chain kinase and myosin light chain phosphatase in the resistance arterial myogenic response to intravascular pressure.

The intrinsic ability of vascular smooth muscle cells (VSMCs) within arterial resistance vessels to respectively contract and relax in response to elevation and reduction of intravascular pressure is essential for appropriate blood flow autoregulation. This fundamental mechanism, referred to as the myogenic response, is dependent on apposite control of myosin regulatory light chain (LC(20)) phosphorylation, a prerequisite for force generation, through the coordinated activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Here, we highlight the molecular basis of the smooth muscle contractile mechanism and review the regulatory pathways demonstrated to participate in the control of LC(20) phosphorylation in the myogenic response, with a focus on the Ca(2+)-dependent and Rho-associated kinase (ROK)-mediated regulation of MLCK and MLCP, respectively.