RESUMEN
Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle in vitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action.
Asunto(s)
Anoctamina-1/antagonistas & inhibidores , Hipertensión , Músculo Liso Vascular , Vasoconstricción , Animales , Presión Sanguínea/efectos de los fármacos , Canales de Cloruro , Hipertensión/tratamiento farmacológico , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Endogámicas SHRRESUMEN
Kv7 channels are voltage-gated potassium channels encoded by KCNQ genes that have a considerable physiological impact in many cell types. This reliance upon Kv7 channels for normal cellular function, as well as the existence of hereditary disorders caused by mutations to KCNQ genes, means that pharmacological targeting of these channels has broad appeal. Consequently, a plethora of chemical entities that modulate Kv7 channel activity have been developed. Moreover, Kv7 channels are influenced by many disparate intracellular mediators and trafficking processes, making upstream targeting an appealing prospect for therapeutic development. This review covers the main characteristics of these multifunctional and versatile channels with the aim of providing insight into the therapeutic value of targeting these channels.
Asunto(s)
Canales de Potasio KCNQ/genética , Canales de Potasio KCNQ/metabolismo , Animales , Transporte Biológico/genética , Humanos , Mutación/genética , Transducción de Señal/genéticaRESUMEN
OBJECTIVE: The SMIT1 (sodium:myo-inositol transporter 1) regulates myo-inositol movement into cells and responses to hypertonic stimuli. Alteration of myo-inositol levels has been associated with several diseases, including hypertension, but there is no evidence of a functional role of SMIT1 in the vasculature. Recent evidence showed that in the nervous system SMIT1 interacted and modulated the function of members of the Kv7 family of voltage-gated potassium channels, which are also expressed in the vasculature where they regulate arterial contractility. Therefore, in this study, we evaluated whether SMIT1 was functionally relevant in arterial smooth muscle. Approach and Results: Immunofluorescence and polymerase chain reaction experiments revealed that SMIT1 was expressed in rat renal and mesenteric vascular smooth muscle cells. Isometric tension recordings showed that incubation of renal arteries with raffinose and myo-inositol (which increases SMIT1 expression) reduced the contractile responses to methoxamine, an effect that was abolished by preincubation with the pan-Kv7 blocker linopirdine and by molecular knockdown of Kv7.4 and Kv7.5. Knockdown of SMIT1 increased the contraction of renal arteries induced by methoxamine, impaired the response to the Kv7.2-Kv7.5 activator ML213 but did not interfere with the relaxant responses induced by openers of other potassium channels. Proximity ligation assay showed that SMIT1 interacted with heteromeric channels formed by Kv7.4 and Kv7.5 proteins in both renal and mesenteric vascular smooth muscle cells. Patch-clamp experiments showed that incubation with raffinose plus myo-inositol increased Kv7 currents in vascular smooth muscle cells. CONCLUSIONS: SMIT1 protein is expressed in vascular smooth muscle cells where it modulates arterial contractility through an association with Kv7.4/Kv7.5 heteromers.
Asunto(s)
Canales de Potasio KCNQ/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Simportadores/metabolismo , Vasoconstricción , Animales , Células CHO , Cricetulus , Canales de Potasio KCNQ/genética , Potenciales de la Membrana , Arterias Mesentéricas/metabolismo , Unión Proteica , Ratas , Arteria Renal/metabolismo , Transducción de Señal , Simportadores/genética , Técnicas de Cultivo de TejidosRESUMEN
This study explored the mechanism by which Ca2+-activated Cl- channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl- currents (IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.
Asunto(s)
Anoctamina-1/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Regulación de la Expresión Génica , Proteínas de Neoplasias/genética , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 2/genética , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Animales , Anoctamina-1/metabolismo , Bencilaminas/farmacología , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Cantaridina/farmacología , Cloruros/metabolismo , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Células HEK293 , Humanos , Transporte Iónico/efectos de los fármacos , Ratones , Proteínas de Neoplasias/metabolismo , Ácido Ocadaico/farmacología , Técnicas de Placa-Clamp , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Sulfonamidas/farmacologíaAsunto(s)
Músculo Liso , Historia del Siglo XX , Músculo Liso/fisiología , Humanos , Fisiología/historia , Historia del Siglo XXI , AnimalesRESUMEN
Objective- In renal arteries, inhibitors of G protein ßγ subunits (Gßγ) reduce Kv7 activity and inhibit Kv7-dependent receptor-mediated vasorelaxations. However, the mechanisms underlying receptor-mediated relaxation are artery specific. Consequently, the aim of this study was to ascertain the role of Gßγ in Kv7-dependent vasorelaxations of the rat vasculature. Approach and Results- Isometric tension recording was performed in isolated rat renal, mesenteric, and cerebral arteries to study isoproterenol and calcitonin gene-related peptide relaxations. Kv7.4 was knocked down via morpholino transfection while inhibition of Gßγ was investigated with gallein and M119K. Proximity ligation assay was performed on isolated myocytes to study the association between Kv7.4 and G protein ß subunits or signaling intermediaries. Isoproterenol or calcitonin gene-related peptide-induced relaxations were attenuated by Kv7.4 knockdown in all arteries studied. Inhibition of Gßγ with gallein or M119K had no effect on isoproterenol-mediated relaxations in mesenteric artery but had a marked effect on calcitonin gene-related peptide-induced responses in mesenteric artery and cerebral artery and isoproterenol responses in renal artery. Isoproterenol increased association with Kv7.4 and Rap1a in mesenteric artery which were not sensitive to gallein, whereas in renal artery, isoproterenol increased Kv7.4-AKAP (A-kinase anchoring protein) associations in a gallein-sensitive manner. Conclusions- The Gßγ-Kv7 relationship differs between vessels and is an essential requirement for AKAP, but not Rap-mediated regulation of the channel.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/fisiología , Subunidades gamma de la Proteína de Unión al GTP/fisiología , Canales de Potasio KCNQ/fisiología , Músculo Liso Vascular/fisiología , Vasodilatación , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/farmacología , Arterias Cerebrales/efectos de los fármacos , Arterias Cerebrales/fisiología , Isoproterenol/farmacología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Ratas Wistar , Arteria Renal/efectos de los fármacos , Arteria Renal/fisiología , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , Xantenos/farmacologíaRESUMEN
Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.
Asunto(s)
Presión Sanguínea/fisiología , Caveolina 1/metabolismo , Conexinas/metabolismo , Homeostasis , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Adenosina Trifosfato/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Masculino , Ratones Noqueados , Músculo Liso Vascular/citología , Músculo Liso Vascular/inervación , Fenilefrina/farmacología , Sistema Nervioso Simpático/fisiología , Vasoconstricción/fisiologíaRESUMEN
Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein ßγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K(+) currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gßγ subunits (2-250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein-coupled receptors. Gallein, an inhibitor of Gßγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gßγ subunit inhibitors (GRK2i and a ß-subunit antibody) abolished Kv7 channel currents in the absence of either Gßγ subunit enrichment or G-protein-coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gßγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gßγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gßγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Hemodinámica/fisiología , Canales de Potasio KCNQ/metabolismo , Animales , Bovinos , Electromiografía , Células HEK293 , Humanos , Inmunoprecipitación , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Kv7.4 channels are key determinants of arterial contractility and cochlear mechanosensation that, like all Kv7 channels, have an obligatory requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). ßγ G proteins (Gßγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gßγ increased Kv7.4 open probability through an increased sensitivity to PIP2. In HEK cells stably expressing Kv7.4, PIP2 or Gßγ increased open probability in a concentration dependent manner. Depleting PIP2 prevented any Gßγ-mediated stimulation whilst an array of Gßγ inhibitors prohibited any PIP2-induced current enhancement. A combination of PIP2 and Gßγ at sub-efficacious concentrations increased channel open probability considerably. The stimulatory effects of three Kv7.2-7.5 channel activators were also lost by PIP2 depletion or Gßγ inhibitors. This study alters substantially our understanding of the fundamental processes that dictate Kv7.4 activity, revealing a more complex and subtle paradigm where the reliance on local phosphoinositide is dictated by interaction with Gßγ.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Canales de Potasio KCNQ/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Línea Celular , Células HEK293 , Humanos , Activación del Canal Iónico/fisiología , Fosfatidilinositoles/metabolismoRESUMEN
OBJECTIVE: To establish the role of Kv7 channels in EPAC (exchange protein directly activated by cAMP)-dependent relaxations of the rat vasculature and to investigate whether this contributes to ß-adrenoceptor-mediated vasorelaxations. APPROACH AND RESULTS: Isolated rat renal and mesenteric arteries (RA and MA, respectively) were used for isometric tension recording to study the relaxant effects of a specific EPAC activator and the ß-adrenoceptor agonist isoproterenol in the presence of potassium channel inhibitors and cell signaling modulators. Isolated myocytes were used in proximity ligation assay studies to detect localization of signaling intermediaries with Kv7.4 before and after cell stimulation. Our studies showed that the EPAC activator (8-pCPT-2Me-cAMP-AM) produced relaxations and enhanced currents of MA and RA that were sensitive to linopirdine (Kv7 inhibitor). Linopirdine also inhibited isoproterenol-mediated relaxations in both RA and MA. In the MA, isoproterenol relaxations were sensitive to EPAC inhibition, but not protein kinase A inhibition. In contrast, isoproterenol relaxations in RA were attenuated by protein kinase A but not by EPAC inhibition. Proximity ligation assay showed a localization of Kv7.4 with A-kinase anchoring protein in both vessels in the basal state, which increased only in the RA with isoproterenol stimulation. In the MA, but not the RA, a localization of Kv7.4 with both Rap1a and Rap2 (downstream of EPAC) increased with isoproterenol stimulation. CONCLUSIONS: EPAC-dependent vasorelaxations occur in part via activation of Kv7 channels. This contributes to the isoproterenol-mediated relaxation in mesenteric, but not renal, arteries.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Canales de Potasio KCNQ/metabolismo , Arterias Mesentéricas/metabolismo , Arteria Renal/metabolismo , Vasodilatación , Proteínas de Anclaje a la Quinasa A/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Factores de Intercambio de Guanina Nucleótido/agonistas , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Células HEK293 , Humanos , Técnicas In Vitro , Canales de Potasio KCNQ/agonistas , Canales de Potasio KCNQ/antagonistas & inhibidores , Canales de Potasio KCNQ/genética , Masculino , Potenciales de la Membrana , Arterias Mesentéricas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Ratas Wistar , Arteria Renal/efectos de los fármacos , Transducción de Señal , Transfección , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
KEY POINTS: KCNE4 alters the biophysical properties and cellular localization of voltage-gated potassium channel Kv7.4. KCNE4 is expressed in a variety of arteries and, in mesenteric arteries, co-localizes with Kv7.4, which is important in the control of vascular contractility. Knockdown of KCNE4 leads to reduced Kv7.4 membrane abundance, a depolarized membrane potential and an augmented response to vasoconstrictors. KCNE4 is a key regulator of the function and expression of Kv7.4 in vascular smooth muscle. ABSTRACT: The KCNE ancillary subunits (KCNE1-5) significantly alter the expression and function of voltage-gated potassium channels; however, their role in the vasculature has yet to be determined. The present study aimed to investigate the expression and function of the KCNE4 subunit in rat mesenteric arteries and to determine whether it has a functional impact on the regulation of arterial tone by Kv7 channels. In HEK cells expressing Kv7.4, co-expression of KCNE4 increased the membrane expression of Kv7.4 and significantly altered Kv7.4 current properties. Quantitative PCR analysis of different rat arteries found that the KCNE4 isoform predominated and proximity ligation experiments showed that KCNE4 co-localized with Kv7.4 in mesenteric artery myocytes. Morpholino-induced knockdown of KCNE4 depolarized mesenteric artery smooth muscle cells and resulted in their increased sensitivity to methoxamine being attenuated (mean ± SEM EC50 decreased from 5.7 ± 0.63 µm to 1.6 ± 0.23 µm), which coincided with impaired effects of Kv7 modulators. When KCNE4 expression was reduced, less Kv7.4 expression was found in the membrane of the mesenteric artery myocytes. These data show that KCNE4 is consistently expressed in a variety of arteries, and knockdown of the expression product leads to reduced Kv7.4 membrane abundance, a depolarized membrane potential and an augmented response to vasoconstrictors. The present study is the first to demonstrate an integral role of KCNE4 in regulating the function and expression of Kv7.4 in vascular smooth muscle.
Asunto(s)
Arterias Mesentéricas/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Vasoconstricción , Animales , Células Cultivadas , Células HEK293 , Humanos , Masculino , Potenciales de la Membrana , Arterias Mesentéricas/fisiología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Canales de Potasio con Entrada de Voltaje/genética , Ratas , Ratas WistarRESUMEN
OBJECTIVE: Middle cerebral artery (MCA) diameter is regulated by inherent myogenic activity and the effect of potent vasodilators such as calcitonin gene-related peptide (CGRP). Previous studies showed that MCAs express KCNQ1, 4, and 5 potassium channel genes, and the expression products (Kv7 channels) participate in the myogenic control of MCA diameter. The present study investigated the contribution of Kv7.4 and Kv7.5 isoforms to myogenic and CGRP regulation of MCA diameter and determined whether they were affected in hypertensive animals. APPROACH AND RESULTS: Isometric tension recordings performed on MCA from normotensive rats produced CGRP vasodilations that were inhibited by the pan-Kv7 channel blocker linopirdine (P<0.01) and after transfection of arteries with siRNA against KCNQ4 (P<0.01) but not KCNQ5. However, isobaric myography revealed that myogenic constriction in response to increases in intravascular pressure (20-80 mm Hg) was affected by both KCNQ4 and KCNQ5 siRNA. Proximity ligation assay signals were equally abundant for Kv7.4/Kv7.4 or Kv7.4/Kv7.5 antibody combinations but minimal for Kv7.5/Kv7.5 antibodies or Kv7.4/7.1 combinations. In contrast to systemic arteries, Kv7 function and Kv7.4 abundance in MCA were not altered in hypertensive rats. CONCLUSIONS: This study reveals, for the first time to our knowledge, that in cerebral arteries, Kv7.4 and Kv7.5 proteins exist predominantly as a functional heterotetramer, which regulates intrinsic myogenicity and vasodilation attributed to CGRP. Surprisingly, unlike systemic arteries, Kv7 activity in MCAs is not affected by the development of hypertension, and CGRP-mediated vasodilation is well maintained. As such, cerebrovascular Kv7 channels could be amenable for therapeutic targeting in conditions such as cerebral vasospasm.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/farmacología , Canales de Potasio KCNQ/metabolismo , Arteria Cerebral Media/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hipertensión/metabolismo , Hipertensión/fisiopatología , Canales KATP/metabolismo , Canales de Potasio KCNQ/antagonistas & inhibidores , Canales de Potasio KCNQ/genética , Masculino , Arteria Cerebral Media/metabolismo , Arteria Cerebral Media/fisiopatología , Bloqueadores de los Canales de Potasio/farmacología , Isoformas de Proteínas , Interferencia de ARN , Ratas , Ratas Endogámicas SHR , Ratas Wistar , TransfecciónRESUMEN
Ion channels play a key role in defining myometrial contractility. Modulation of ion channel populations is proposed to underpin gestational changes in uterine contractility associated with the transition from uterine quiescence to active labour. Of the myriad ion channels present in the uterus, this article will focus upon potassium channels encoded by the KCNQ genes and ether-à-go-go-related (ERG) genes. Voltage-gated potassium channels encoded by KCNQ and ERG (termed Kv7 and Kv11, respectively) are accepted as major determinants of neuronal excitability and the duration of the cardiac action potential. However, there is now growing appreciation that these ion channels have a major functional impact in vascular and non-vascular smooth muscle. Moreover, Kv7 channels may be potential therapeutic targets for the treatment of preterm labour.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/fisiología , Canales de Potasio KCNQ/fisiología , Miometrio/fisiología , Útero/fisiología , Adulto , Femenino , Humanos , Músculo Liso/fisiología , Vías Nerviosas/fisiología , Canales de Potasio/fisiología , EmbarazoRESUMEN
The KCNQ family is comprised of five genes and the expression products form voltage-gated potassium channels (Kv7.1-7.5) that have a major impact upon cellular physiology in many cell types. Each functional Kv7 channel forms as a tetramer that often associates with proteins encoded by the KCNE gene family (KCNE1-5) and is critically reliant upon binding of phosphatidylinositol bisphosphate (PIP2) and calmodulin. Other modulators like A-kinase anchoring proteins, ubiquitin ligases and Ca-calmodulin kinase II alter Kv7 channel function and trafficking in an isoform specific manner. It has now been identified that for Kv7.4, G protein ßγ subunits (Gßγ) can be added to the list of key regulators and is paramount for channel activity. This article provides an overview of this nascent field of research, highlighting themes and directions for future study.
RESUMEN
Stimulation of the calcium-sensing receptor (CaSR) regulates vascular contractility, but cellular mechanisms involved remain unclear. This study investigated the role of perivascular sensory nerves in CaSR-induced relaxations of male rat mesenteric arteries. In fluorescence studies, colocalisation between synaptophysin, a synaptic vesicle marker, and the CaSR was present in the adventitial layer of arterial segments. Using wire myography, increasing external Ca2+ concentration ([Ca2+]o) from 1 to 10 mM induced vasorelaxations, previously shown to involve the CaSR, which were inhibited by pretreatment with capsaicin. [Ca2+]o-induced vasorelaxations were partially reduced by the calcitonin gene-related peptide (CGRP) receptor blockers, CGRP 8-37 and BIBN 4096, and the neurokinin 1 (NK1) receptor blocker L733,060. The inhibitory effect of CGRP 8-37 required a functional endothelium whereas the inhibitory action of L733,060 did not. Complete inhibition of [Ca2+]o-induced vasorelaxations occurred when CGRP 8-37 and L733,060 were applied together. [Ca2+]o-induced vasorelaxations in the presence of capsaicin were abolished by the ATP-dependent K+ channel (KATP) blocker PNU 37883, but unaffected by the endothelium nitric oxide synthase (eNOS) inhibitor L-NAME. We suggest that the CaSR on perivascular sensory nerves mediate relaxations in rat mesenteric arteries via endothelium-dependent and -independent mechanisms involving CGRP and NK1 receptor-activated NO production and KATP channels, respectively.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Arterias Mesentéricas , Receptores Sensibles al Calcio , Receptores de Neuroquinina-1 , Vasodilatación , Animales , Masculino , Receptores Sensibles al Calcio/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Receptores de Neuroquinina-1/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Arterias Mesentéricas/metabolismo , Ratas , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Ratas Wistar , Antagonistas del Receptor de Neuroquinina-1/farmacología , Calcio/metabolismo , Capsaicina/farmacología , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina/farmacología , Transducción de Señal/fisiologíaRESUMEN
Stimulation of the calcium-sensing receptor (CaSR) induces both vasoconstrictions and vasorelaxations but underlying cellular processes remain unclear. This study investigates expression and effect of stimulating the CaSR by increasing external Ca2+ concentration ([Ca2+ ]o ) on contractility of rat mesenteric arteries. Immunofluorescence studies showed expression of the CaSR in perivascular nerves, vascular smooth muscle cells (VSMCs), and vascular endothelium cells. Using wire myography, increasing [Ca2+ ]o from 1 to 10 mM induced vasorelaxations which were inhibited by the calcilytic Calhex-231 and partially dependent on a functional endothelium. [Ca2+ ]o -induced vasorelaxations were reduced by endothelial NO synthase (eNOS, L-NAME) and large conductance Ca2+ -activated K+ channels (BKCa , iberiotoxin), with their inhibitory action requiring a functional endothelium. [Ca2+ ]o -induced vasorelaxations were also markedly inhibited by an ATP-dependent K+ channel (KATP ) blocker (PNU37883), which did not require a functional endothelium to produce its inhibitory action. Inhibitor studies also suggested contributory roles for inward rectifying K+ channels (Kir ), Kv7 channels, and small conductance Ca2+ -activated K+ channels (SKCa ) on [Ca2+ ]o -induced vasorelaxations. These findings indicate that stimulation of the CaSR mediates vasorelaxations involving multiple pathways, including an endothelium-dependent pathway involving NO production and activation of BKCa channels and an endothelium-independent pathway involving stimulation of KATP channels.
Asunto(s)
Receptores Sensibles al Calcio , Vasodilatación , Animales , Ratas , Adenosina Trifosfato/metabolismo , Endotelio/metabolismo , Endotelio Vascular/metabolismo , Arterias Mesentéricas/metabolismo , Receptores Sensibles al Calcio/metabolismoRESUMEN
AIMS: Sodium/glucose transporter 2 (SGLT2 or SLC5A2) inhibitors lower blood glucose and are also approved treatments for heart failure independent of raised glucose. Various studies have showed that SGLT2 inhibitors relax arteries but the underlying mechanisms are poorly understood and responses variable across arterial beds. We speculated that SGLT2 inhibitor-mediated arterial relaxation is dependent upon calcitonin gene-related peptide (CGRP) from sensory nerves independent of glucose transport. METHODS AND RESULTS: The functional effects of SGLT1 and 2 inhibitors (mizagliflozin, dapagliflozin, empagliflozin) and the sodium/hydrogen exchanger 1 (NHE1) blocker cariporide were determined on pre-contracted resistance arteries (mesenteric and cardiac septal arteries) as well as main renal conduit arteries from male Wistar rats using Wire-Myography. SGLT2, CGRP, TRPV1 and NHE1, expression was determined by Western blot and immunohistochemistry. Kv7.4/5/KCNE4 and TRPV1 currents were measured in the presence and absence of dapagliflozin and empagliflozin.All SGLT inhibitors (1µM-100µM) and cariporide (30µM) relaxed mesenteric arteries but had negligible effect on renal or septal arteries. Immunohistochemistry with TRPV1 and CGRP antibodies revealed a dense innervation of sensory nerves in mesenteric arteries that were absent in renal and septal arteries. Consistent with a greater sensory nerve component, the TRPV1 agonist capsaicin relaxed mesenteric arteries more effectively than renal or septal arteries. In mesenteric arteries, relaxations to dapagliflozin, empagliflozin and cariporide were attenuated by the CGRP receptor antagonist BIBN-4096, depletion of sensory nerves with capsaicin, and blockade of TRPV1 or Kv7 channels. Neither dapagliflozin nor empagliflozin activated heterologously expressed TRPV1 channels or Kv7 channels directly. Sensory nerves also expressed NHE1 but not SGLT2 and cariporide pre-application as well as knockdown of NHE1 by translation stop morpholinos prevented the relaxant response to SGLT2 inhibitors. CONCLUSIONS: SGLT2 inhibitors relax mesenteric arteries by promoting the release of CGRP from sensory nerves in a NHE1-dependent manner.
RESUMEN
This study investigated the molecular identity and impact of enhancing voltage-gated Na(+) (Na(V)) channels in the control of vascular tone. In rat isolated mesenteric and femoral arteries mounted for isometric tension recording, the vascular actions of the Na(V) channel activator veratridine were examined. Na(V) channel expression was probed by molecular techniques and immunocytochemistry. In mesenteric arteries, veratridine induced potent contractions (pEC(50) = 5.19 ± 0.20, E(max) = 12.0 ± 2.7 mN), which were inhibited by 1 µM TTX (a blocker of all Na(V) channel isoforms, except Na(V)1.5, Na(V)1.8, and Na(V)1.9), but not by selective blockers of Na(V)1.7 (ProTx-II, 10 nM) or Na(V)1.8 (A-80347, 1 µM) channels. The responses were insensitive to endothelium removal but were partly (~60%) reduced by chemical destruction of sympathetic nerves by 6-hydroxydopamine (2 mM) or antagonism at the α1-adrenoceptor by prazosin (1 µM). KB-R7943, a blocker of the reverse mode of the Na(+)/Ca(2+) exchanger (3 µM), inhibited veratridine contractions in the absence or presence of prazosin. T16A(inh)-A01, a Ca(2+)-activated Cl(-) channel blocker (10 µM), also inhibited the prazosin-resistant contraction to veratridine. Na(V) channel immunoreactivity was detected in freshly isolated mesenteric myocytes, with apparent colocalization with the Na(+)/Ca(2+) exchanger. Veratridine induced similar contractile effects in the femoral artery, and mRNA transcripts for Na(V)1.2 and Na(V)1.3 channels were evident in both vessel types. We conclude that, in addition to sympathetic nerves, NaV channels are expressed in vascular myocytes, where they are functionally coupled to the reverse mode of Na(+)/Ca(2+) exchanger and subsequent activation of Ca(2+)-activated Cl(-) channels, causing contraction. The TTX-sensitive Na(V)1.2 and Na(V)1.3 channels are likely involved in vascular control.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.3/metabolismo , Vasoconstricción/fisiología , Animales , Masculino , Arterias Mesentéricas/fisiología , Músculo Liso Vascular/metabolismo , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio/farmacología , Veratridina/farmacologíaRESUMEN
Sex hormones and the reproductive cycle (estrus in rodents and menstrual in humans) have a known impact on arterial function. In spite of this, sex hormones and the estrus/menstrual cycle are often neglected experimental factors in vascular basic preclinical scientific research. Recent research by our own laboratory indicates that cyclical changes in serum concentrations of sex -hormones across the rat estrus cycle, primary estradiol, have significant consequences for the subcellular trafficking and function of KV. Vascular potassium channels, including KV, are essential components of vascular reactivity. Our study represents a small part of a growing field of literature aimed at determining the role of sex hormones in regulating arterial ion channel function. This review covers key findings describing the current understanding of sex hormone regulation of vascular potassium channels, with a focus on KV channels. Further, we highlight areas of research where the estrus cycle should be considered in future studies to determine the consequences of physiological oscillations in concentrations of sex hormones on vascular potassium channel function.
Asunto(s)
Canales de Potasio , Progesterona , Femenino , Humanos , Ratas , Animales , Hormonas Esteroides Gonadales , Estradiol , Ciclo Menstrual/fisiologíaRESUMEN
In addition, to their established role in cardiac myocytes and neurons, ion channels encoded by ether-a-go-go-related genes (ERG1-3 or kcnh2,3 and 6) (kcnh2) are functionally relevant in phasic smooth muscle. The aim of the study was to determine the expression and functional impact of ERG expression products in rat urinary bladder smooth muscle using quantitative polymerase chain reaction, immunocytochemistry, whole-cell patch-clamp and isometric tension recording. kcnh2 was expressed in rat bladder, whereas kcnh6 and kcnh3 expression were negligible. Immunofluorescence for the kcnh2 expression product Kv11.1 was detected in the membrane of isolated smooth muscle cells. Potassium currents with voltage-dependent characteristics consistent with Kv11.1 channels and sensitive to the specific blocker E4031 (1 µM) were recorded from isolated detrusor smooth muscles. Disabling Kv11.1 activity with specific blockers (E4031 and dofetilide, 0.2-20 µM) augmented spontaneous contractions to a greater extent than BKCa channel blockers, enhanced carbachol-driven activity, increased nerve stimulation-mediated contractions, and impaired ß-adrenoceptor-mediated inhibitory responses. These data establish for the first time that Kv11.1 channels are key determinants of contractility in rat detrusor smooth muscle.