Alejandro Moreno-Domínguez

Characterization of the Kv channels of mouse carotid body chemoreceptor cells and their role in oxygen sensing

As there are wide interspecies variations in the molecular nature of the O2‐sensitive Kv channels in arterial chemoreceptors, we have characterized the expression of these channels and their hypoxic sensitivity in the mouse carotid body (CB). CB chemoreceptor cells were obtained from a transgenic mouse expressing green fluorescent protein (GFP) under the control of tyrosine hydroxylase (TH) promoter. Immunocytochemical identification of TH in CB cell cultures reveals a good match with GFP‐positive cells. Furthermore, these cells show an increase in [Ca2+]i in response to low PO2, demonstrating their ability to engender a physiological response. Whole‐cell experiments demonstrated slow‐inactivating K+ currents with activation threshold around −30 mV and a bi‐exponential kinetic of deactivation (τ of 6.24 ± 0.52 and 32.85 ± 4.14 ms). TEA sensitivity of the currents identified also two different components (IC50 of 17.8 ± 2.8 and 940.0 ± 14.7 μm). Current amplitude decreased reversibly in response to hypoxia, which selectively affected the fast deactivating component. Hypoxic inhibition was also abolished in the presence of low (10–50 μm) concentrations of TEA, suggesting that O2 interacts with the component of the current most sensitive to TEA. The kinetic and pharmacological profile of the currents suggested the presence of Kv2 and Kv3 channels as their molecular correlates, and we have identified several members of these two subfamilies by single‐cell PCR and immunocytochemistry. This report represents the first functional and molecular characterization of Kv channels in mouse CB chemoreceptor cells, and strongly suggests that O2‐sensitive Kv channels in this preparation belong to the Kv3 subfamily.

Contribution of Kv Channels to Phenotypic Remodeling of Human Uterine Artery Smooth Muscle Cells

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+ (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&agr; subunits in both preparations as well as their functional contribution to the K+ 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+ 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.

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.

Characterization of Ion Channels Involved in the Proliferative Response of Femoral Artery Smooth Muscle Cells

Objective—Vascular smooth muscle cells (VSMCs) contribute significantly to occlusive vascular diseases by virtue of their ability to switch to a noncontractile, migratory, and proliferating phenotype. Although the participation of ion channels in this phenotypic modulation (PM) has been described previously, changes in their expression are poorly defined because of their large molecular diversity. We obtained a global portrait of ion channel expression in contractile versus proliferating mouse femoral artery VSMCs, and explored the functional contribution to the PM of the most relevant changes that we observed. Methods and Results—High-throughput real-time polymerase chain reaction of 87 ion channel genes was performed in 2 experimental paradigms: an in vivo model of endoluminal lesion and an in vitro model of cultured VSMCs obtained from explants. mRNA expression changes showed a good correlation between the 2 proliferative models, with only 2 genes, Kv1.3 and Kv&bgr;2, increasing their expression on proliferation. The functional characterization demonstrates that Kv1.3 currents increased in proliferating VSMC and that their selective blockade inhibits migration and proliferation. Conclusion—These findings establish the involvement of Kv1.3 channels in the PM of VSMCs, providing a new therapeutical target for the treatment of intimal hyperplasia.

De novo expression of Kv6.3 contributes to changes in vascular smooth muscle cell excitability in a hypertensive mice strain

Essential hypertension involves a gradual and sustained increase in total peripheral resistance, reflecting an increased vascular tone. This change associates with a depolarization of vascular myocytes, and relies on a change in the expression profile of voltage‐dependent ion channels (mainly Ca2+ and K+ channels) that promotes arterial contraction. However, changes in expression and/or modulation of voltage‐dependent K+ channels (Kv channels) are poorly defined, due to their large molecular diversity and their vascular bed‐specific expression. Here we endeavor to characterize the molecular and functional expression of Kv channels in vascular smooth muscle cells (VSMCs) and their regulation in essential hypertension, by using VSMCs from resistance (mesenteric) or conduit (aortic) arteries obtained from a hypertensive inbred mice strain, BPH, and the corresponding normotensive strain, BPN. Real‐time PCR reveals a differential distribution of Kv channel subunits in the different vascular beds as well as arterial bed‐specific changes under hypertension. In mesenteric arteries, the most conspicuous change was the de novo expression of Kv6.3 (Kcng3) mRNA in hypertensive animals. The functional relevance of this change was studied by using patch‐clamp techniques. VSMCs from BPH arteries were more depolarized than BPN ones, and showed significantly larger capacitance values. Moreover, Kv current density in BPH VSMCs is decreased mainly due to the diminished contribution of the Kv2 component. The kinetic and pharmacological profile of Kv2 currents suggests that the expression of Kv6.3 could contribute to the natural development of hypertension.

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.

High blood pressure associates with the remodelling of inward rectifier K+ channels in mice mesenteric vascular smooth muscle cells

•  Essential hypertension involves an electrical remodelling of vascular smooth muscle cells (VSMCs), particularly relevant for K+ channels, as key determinants of resting membrane potential (VM) and excitability. •  We explored mRNA expression levels of inward rectifier K+ channel genes in five different vascular beds from normotensive (BPN) and hypertensive (BPH) mice. In mesenteric VSMCs, their functional contribution to cell excitability and vascular reactivity was investigated. •  BPH mesenteric VSMCs show a decreased functional expression of both classical inward rectifiers (KIR, Kir2 and Kir4 channels) and ATP‐sensitive K+ channels (KATP, Kir6 channels). However, only the changes in the functional expression of KATP channels seem to contribute to the increased vascular reactivity of BPH arteries. •  BPN and BPH mice are a useful model to provide integrated information of the impact in the pathophysiology of essential hypertension of the changes in ion channel functional expression.

PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization

Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca(2+)-dependent PKC, and in the absence of extracellular Ca(2+). Biochemical evidence indicates that either + or - extracellular Ca(2+), PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries.

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.