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 Ca

Key points• 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.Abstract The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca 2+ entry via voltage-gated Ca 2+ channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC 20 ) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca 2+ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca 2+ sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC 20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC 20 was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC 20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our findings indicate that two mechanisms of Ca 2+ sensitization (ROK-mediated phosphorylation of MYPT1-T855 with augmentation of LC 20 pho...

Abstract-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 ϩ (K DR ) channels are of fundamental importance for control of arterial diameter and, thereby, peripheral vascular resistance, blood pressure, and organ-specific blood flow. Key Words: myogenic response Ⅲ delayed rectifier potassium channels Ⅲ vascular smooth muscle Ⅲ Kv1 channels T he intrinsic ability of resistance arteries to contract in response to elevations in intraluminal (or transmural) pressure, the myogenic response, was first described over 100 years ago by Bayliss. [1][2][3][4] This phenomenon is now well recognized to be an essential autoregulatory mechanism. [2][3][4] Myogenic tone development depends on L-type Ca 2ϩ channel (Cav1.2) activity within vascular myocytes. 5 The resulting rise in intracellular free Ca 2ϩ concentration via these Ca 2ϩ channels 6 activates cross-bridge cycling and contractile force development that may be enhanced and/or maintained by a Ca 2ϩ sensitization of the contractile machinery. 3,4 A current working hypothesis suggests that the activation of L-type Ca 2ϩ channels during the myogenic response is the result of low amplitude, steady-state depolarization of the vascular smooth muscle (VSM) cells attributable to increased intraluminal pressure. 2,3,6 However, very precise con...

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 (E m ) and Ca 2+ 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 Ca 2+ -activated K + (BK Ca ) 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 E m and transmural pressure than Kv1 or BK Ca channels owing to their negative range of voltage-dependent activation.

Abstract-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 [Arg 8 ]-vasopressin. A7r5 cells expressed transcripts encoding TRPC1, TRPC4␤, TRPC6, and TRPC7. TRPC4, TRPC6, and TRPC7 protein expression was confirmed by immunoblotting and association of TRPC6 with TRPC7, but not TRPC4␤, was detected by coimmunoprecipitation. The amplitude of arginine vasopressin (AVP)-induced ROC current was suppressed by dominant-negative mutant TRPC6 (TRPC6 DN ) but not TRPC5 (TRPC5 DN ) 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 Ca 2ϩ concentration ([Ca 2ϩ ] 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 [Ca 2ϩ ] o between Ϸ0.01 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. sensitization of contractile filaments. [1][2][3][4] The depolarization and influx of Ca 2ϩ evoked by GPCRs is attributable in part to the activation of receptor-operated, nonselective cation channels (ROCs) by a signaling pathway involving phospholipase and diacylglycerol (reviewed previously [3][4][5][6] ). The molecular basis of VSM ROCs is not known with certainty, but accumulating evidence suggests that transient receptor potential channel (TRPC) family subunits (TRPC1 to TRPC7) [7][8][9] are likely involved. [3][4][5][6] Moreover, ROCs owing to heterologous expression of TRPC3, TRPC6, or TRPC7 are activated by a similar mechanism involving phospholipase and diacylglycerol. 10 -12 Understanding the molecular basis of VSM ROCs is clearly warranted in light of their important role in control of VSM excitability and contractility 4 -6 and evidence of changes in TRPC expression associated with abnormal contractility and/or VSM cell proliferation. [13][14][15] Identifying the contribution of TRPC subunits to VSM ROCs has been compromised by a lack of specific/selective pharmacological blockers. For this reason, alternative strategies involving antibodies, anti-sense or small interfering RNAs (siRNAs) were used to indicate roles for: (1) TRPC1 as store-operated channels 16 ; (2) TRPC3 as ROCs 17 ; (3) TRPC6 as ROCs and/or mechanosensit...