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...

In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K(+) current (I(to)) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of I(to), even at low [Ca(2+)]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of I(to). Because channels underlying I(to) are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvalpha-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca(2+) was used in the pipette or when intracellular [Ca(2+)] ([Ca(2+)](i)) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca(2+)-dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca(2+) increase, and the association was inhibited by KN93. These results suggest that both channels underlying I(to) are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca(2+)](i) increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca(2+)-dependent modulation by this enzyme of the associated Kv4.2 channels.

Abstract-Vascular smooth muscle cells (VSMCs) perform diverse functions that can be classified into contractile and synthetic (or proliferating). All of these functions can be fulfilled by the same cell because of its capacity of phenotypic modulation in response to environmental changes. The resting membrane potential is a key determinant for both contractile and proliferating functions. Here, we have explored the expression of voltage-dependent K ϩ (Kv) channels in contractile (freshly dissociated) and proliferating (cultured) VSMCs obtained from human uterine arteries to establish their contribution to the functional properties of the cells and their possible participation in the phenotypic switch. We have studied the expression pattern (both at the mRNA and at the protein level) of Kv␣ subunits in both preparations as well as their functional contribution to the K ϩ currents of VSMCs. Our results indicate that phenotypic remodeling associates with a change in the expression and distribution of Kv channels. Whereas Kv currents in contractile VSMCs are mainly performed by Kv1 channels, Kv3.4 is the principal contributor to K ϩ currents in cultured VSMCs. Furthermore, selective blockade of Kv3.4 channels resulted in a reduced proliferation rate, suggesting a link between Kv channels expression and phenotypic remodeling. T he vascular smooth muscle cells (VSMCs) of mature animals are highly specialized cells whose main function is contraction. Although during vasculogenesis, the principal role of VSMCs is proliferation and synthesis of the matrix components of the vessel wall, differentiated VSMCs proliferate at an extremely low rate and express a unique repertoire of contractile proteins, ion channels, and signaling molecules required for contraction. However, VSMCs can undergo relatively rapid and reversible phenotypic changes in response to local environmental conditions. Accelerated proliferation of VSMCs is known to play a key role in atherosclerotic plaque formation and postangioplasty restenosis and is a common feature in hypertensive arteries. 1 VSMCs express a large repertoire of ion channels and membrane receptors that vary widely among different vascular beds and are key determinants of the electrical and contractile responses of the cells. [2][3][4][5] Although release of intracellular Ca 2ϩ is necessary for effective contraction, Ca 2ϩ influx through voltage-activated Ca 2ϩ (Cav) channels is responsible for initiating contraction, making resting membrane potential (E m ) a primary determinant of vascular smooth muscle tone. 4 Membrane depolarization opens Cav and raises [Ca 2ϩ ] i , and cytosolic-free Ca 2ϩ serves, in turn, as a critical signal transduction element in a variety of cell functions, such as contraction, migration, proliferation, and gene expression. 6 -8 There is increasing evidence showing that K ϩ channels may have an important role in dedifferentiation and proliferation of VSMCs. Modulating E m , K ϩ channels can affect not only Ca 2ϩ influx, a well-established factor influen...