Emma J. Walsh

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.

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.

Ca2+ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries

Blood flow to our organs is maintained within a defined range to provide an adequate supply of nutrients and remove waste products by contraction and relaxation of smooth muscle cells of resistance arteries and arterioles. The ability of these cells to contract in response to an increase in intravascular pressure, and to relax following a reduction in pressure (the ‘myogenic response’), is critical for appropriate control of blood flow, but our understanding of its mechanistic basis is incomplete. Small arteries of skeletal muscles were used to test the hypothesis that myogenic constriction involves two enzymes, Rho‐associated kinase and protein kinase C, which evoke vasoconstriction by activating the contractile protein, myosin, and by reorganizing the cytoskeleton. Knowledge of the mechanisms involved in the myogenic response contributes to understanding of how blood flow is regulated and will help to identify the molecular basis of dysfunctional control of arterial diameter in disease.

Pressure‐dependent contribution of Rho kinase‐mediated calcium sensitization in serotonin‐evoked vasoconstriction of rat cerebral arteries

Our understanding of the cellular signalling mechanisms contributing to agonist‐induced constriction is almost exclusively based on the study of conduit arteries. Resistance arteries/arterioles have received less attention as standard biochemical approaches lack the necessary sensitivity to permit quantification of phosphoprotein levels in these small vessels. Here, we have employed a novel, highly sensitive Western blotting method to assess: (1) the contribution of Ca2+ sensitization mediated by phosphorylation of myosin light chain phosphatase targeting subunit 1 (MYPT1) and the 17 kDa PKC‐potentiated protein phosphatase 1 inhibitor protein (CPI‐17) to serotonin (5‐HT)‐induced constriction of rat middle cerebral arteries, and (2) whether there is any interplay between pressure‐induced myogenic and agonist‐induced mechanisms of vasoconstriction. Arterial diameter and levels of MYPT1 (T697 and T855), CPI‐17 and 20 kDa myosin light chain subunit (LC20) phosphorylation were determined following treatment with 5‐HT (1 μmol l−1) at 10 or 60 mmHg in the absence and presence of H1152 or GF109203X to suppress the activity of Rho‐associated kinase (ROK) and protein kinase C (PKC), respectively. Although H1152 and GF109203X suppressed 5‐HT‐induced constriction and reduced phospho‐LC20 content at 10 mmHg, we failed to detect any increase in MYPT1 or CPI‐17 phosphorylation. In contrast, an increase in MYPT1‐T697 and MYPT1‐T855 phosphorylation, but not phospho‐CPI‐17 content, was apparent at 60 mmHg following exposure to 5‐HT, and the phosphorylation of both MYPT1 sites was sensitive to H1152 inhibition of ROK. The involvement of MYPT1 phosphorylation in the response to 5‐HT at 60 mmHg was not dependent on force generation per se, as inhibition of cross‐bridge cycling with blebbistatin (10 μmol l−1) did not affect phosphoprotein content. Taken together, the data indicate that Ca2+ sensitization owing to ROK‐mediated phosphorylation of MYPT1 contributes to 5‐HT‐evoked vasoconstriction only in the presence of pressure‐induced myogenic activation. These findings provide novel evidence of an interplay between myogenic‐ and agonist‐induced vasoconstriction in cerebral resistance arteries.

Stromatoxin-sensitive, heteromultimeric Kv2.1/Kv9.3 channels contribute to myogenic control of cerebral arterial diameter

Cerebral vascular smooth muscle contractility plays a crucial role in controlling arterial diameter and, thereby, blood flow regulation in the brain. A number of K+ channels have been suggested to contribute to the regulation of diameter by controlling smooth muscle membrane potential (Em) and Ca2+ influx. Previous studies indicate that stromatoxin (ScTx1)‐sensitive, Kv2‐containing channels contribute to the control of cerebral arterial diameter at 80 mmHg, but their precise role and molecular composition were not determined. Here, we tested if Kv2 subunits associate with ‘silent’ subunits from the Kv5, Kv6, Kv8 or Kv9 subfamilies to form heterotetrameric channels that contribute to control of diameter of rat middle cerebral arteries (RMCAs) over a range of intraluminal pressure from 10 to 100 mmHg. The predominant mRNAs expressed by RMCAs encode Kv2.1 and Kv9.3 subunits. Co‐localization of Kv2.1 and Kv9.3 proteins at the plasma membrane of dissociated single RMCA myocytes was detected by proximity ligation assay. ScTx1‐sensitive native current of RMCA myocytes and Kv2.1/Kv9.3 currents exhibited functional identity based on the similarity of their deactivation kinetics and voltage dependence of activation that were distinct from those of homomultimeric Kv2.1 channels. ScTx1 treatment enhanced the myogenic response of pressurized RMCAs between 40 and 100 mmHg, but this toxin also caused constriction between 10 and 40 mmHg that was not previously observed following inhibition of large conductance Ca2+‐activated K+ (BKCa) and Kv1 channels. Taken together, this study defines the molecular basis of Kv2‐containing channels and contributes to our understanding of the functional significance of their expression in cerebral vasculature. Specifically, our findings provide the first evidence of heteromultimeric Kv2.1/Kv9.3 channel expression in RMCA myocytes and their distinct contribution to control of cerebral arterial diameter over a wider range of Em and transmural pressure than Kv1 or BKCa channels owing to their negative range of voltage‐dependent activation.

Cytoskeletal reorganization evoked by Rho-associated kinase- and protein kinase C-catalyzed phosphorylation of cofilin and heat shock protein 27, respectively, contributes to myogenic constriction of rat cerebral arteries.

Background: The myogenic response of cerebral arteries to intravascular pressure regulates blood flow to the brain. Results: Pressurization reduced smooth muscle G-actin and increased phospho-cofilin and -HSP27 content by a mechanism blocked by ROK or PKC inhibitors. Conclusion: ROK- and PKC-mediated control of cofilin and HSP27 contributes to actin polymerization in myogenic constriction. Significance: Knowledge of cytoskeletal dynamics is crucial for understanding myogenic control of cerebral arterial diameter. Our understanding of the molecular events contributing to myogenic control of diameter in cerebral resistance arteries in response to changes in intravascular pressure, a fundamental mechanism regulating blood flow to the brain, is incomplete. Myosin light chain kinase and phosphatase activities are known to be increased and decreased, respectively, to augment phosphorylation of the 20-kDa regulatory light chain subunits (LC20) of myosin II, which permits cross-bridge cycling and force development. Here, we assessed the contribution of dynamic reorganization of the actin cytoskeleton and thin filament regulation to the myogenic response and serotonin-evoked constriction of pressurized rat middle cerebral arteries. Arterial diameter and the levels of phosphorylated LC20, calponin, caldesmon, cofilin, and HSP27, as well as G-actin content, were determined. A decline in G-actin content was observed following pressurization from 10 mm Hg to between 40 and 120 mm Hg and in three conditions in which myogenic or agonist-evoked constriction occurred in the absence of a detectable change in LC20 phosphorylation. No changes in thin filament protein phosphorylation were evident. Pressurization reduced G-actin content and elevated the levels of cofilin and HSP27 phosphorylation. Inhibitors of Rho-associated kinase and PKC prevented the decline in G-actin; reduced cofilin and HSP27 phosphoprotein content, respectively; and blocked the myogenic response. Furthermore, phosphorylation modulators of HSP27 and cofilin induced significant changes in arterial diameter and G-actin content of myogenically active arteries. Taken together, our findings suggest that dynamic reorganization of the cytoskeleton involving increased actin polymerization in response to Rho-associated kinase and PKC signaling contributes significantly to force generation in myogenic constriction of cerebral resistance arteries.

Identification and functional characterization of protein kinase A-catalyzed phosphorylation of potassium channel Kv1.2 at serine 449.

Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro 32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.

ATP-sensitive K+ channels in pig urethral smooth muscle cells are heteromultimers of Kir6.1 and Kir6.2

The inwardly rectifying properties and molecular basis of ATP-sensitive K(+) channels (K(ATP) channels) have now been established for several cell types. However, these aspects of nonvascular smooth muscle K(ATP) channels still remain to be defined. In this study, we investigated the molecular basis of the pore of K(ATP) channels of pig urethral smooth muscle cells through a comparative study of the inwardly rectifying properties, conductance, and regulation by PKC of native and homo- and heteroconcatemeric recombinant Kir6.x channels coexpressed with sulfonylurea receptor subunit SUR2B in human embryonic kidney (HEK) 293 cells by the patch-clamp technique (conventional whole-cell and cell-attached modes). In conventional whole-cell clamp recordings, levcromakalim (> or = 1 microM) caused a concentration-dependent increase in current that demonstrated strong inward rectification at positive membrane potentials. In cell-attached mode, the unitary amplitude of levcromakalim-induced native and recombinant heteroconcatemeric Kir6.1-Kir6.2 K(ATP) channels also showed strong inward rectification at positive membrane potentials. Phorbol 12,13-dibutyrate, but not the inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate, enhanced the activity of native and heteroconcatemeric K(ATP) channels at -50 mV. The conductance of the native channels at approximately 43 pS was consistent with that of heteroconcatemeric channels with a pore-forming subunit composition of (Kir6.1)(3)-(Kir6.2). RT-PCR analysis revealed the expression of Kir6.1 and Kir6.2 transcripts in pig urethral myocytes. Our findings provide the first evidence that the predominant K(ATP) channel expressed in pig urethral smooth muscle possesses a unique, heteromeric pore structure that differs from the homomeric Kir6.1 channels of vascular myocytes and is responsible for the differences in inward rectification, conductance, and PKC regulation exhibited by the channels in these smooth muscle cell types.

α5-Integrin-mediated cellular signaling contributes to the myogenic response of cerebral resistance arteries

The myogenic response of resistance arterioles and small arteries involving constriction in response to intraluminal pressure elevation and dilation on pressure reduction is fundamental to local blood flow regulation in the microcirculation. Integrins have garnered considerable attention in the context of initiating the myogenic response, but evidence indicative of mechanotransduction by integrin adhesions, for example established changes in tyrosine phosphorylation of key adhesion proteins, has not been obtained to substantiate this interpretation. Here, we evaluated the role of integrin adhesions and associated cellular signaling in the rat cerebral arterial myogenic response using function-blocking antibodies against α5β1-integrins, pharmacological inhibitors of focal adhesion kinase (FAK) and Src family kinase (SFK), an ultra-high-sensitivity western blotting technique, site-specific phosphoprotein antibodies to quantify adhesion and contractile filament protein phosphorylation, and differential centrifugation to determine G-actin levels in rat cerebral arteries at varied intraluminal pressures. Pressure-dependent increases in the levels of phosphorylation of FAK (FAK-Y397, Y576/Y577), SFK (SFK-Y416; Y527 phosphorylation was reduced), vinculin-Y1065, paxillin-Y118 and phosphoinositide-specific phospholipase C-γ1 (PLCγ1)-Y783 were detected. Treatment with α5-integrin function-blocking antibodies, FAK inhibitor FI-14 or SFK inhibitor SU6656 suppressed the changes in adhesion protein phosphorylation, and prevented pressure-dependent phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) at T855 and 20kDa myosin regulatory light chains (LC20) at S19, as well as actin polymerization that are necessary for myogenic constriction. We conclude that mechanotransduction by integrin adhesions and subsequent cellular signaling play a fundamental role in the cerebral arterial myogenic response.