RESUMO
Lead optimization of piperidine amide HTS hits, based on an anilino-thiazole core, led to the identification of analogs which displayed low nanomolar blocking activity at the canonical transient receptor channels 3 and 6 (TRPC3 & 6) based on FLIPR (carbachol stimulated) and electrophysiology (OAG stimulated) assays. In addition, the anilino-thiazole amides displayed good selectivity over other TRP channels (TRPA1, TRPV1, and TRPV4), as well as against cardiac ion channels (CaV1.2, hERG, and NaV1.5). The high oxidation potential of the aliphatic piperidine and aniline groups, as well as the lability of the thiazole amide group contributed to the high clearance observed for this class of compounds. Conversion of an isoquinoline amide to a naphthyridine amide markedly reduced clearance for the bicyclic piperidines, and improved oral bioavailability for this compound series, however TRPC3 and TRPC6 blocking activity was reduced substantially. Although the most potent anilino-thiazole amides ultimately lacked oral exposure in rodents and were not suitable for chronic dosing, analogs such as 14-19, 22, and 23 are potentially valuable in vitro tool compounds for investigating the role of TRPC3 and TRPC6 in cardiovascular disease.
Assuntos
Compostos de Anilina/química , Compostos de Anilina/farmacologia , Canais de Cátion TRPC/antagonistas & inibidores , Tiazóis/química , Tiazóis/farmacologia , Diglicerídeos/metabolismo , Descoberta de Drogas , Células HEK293 , Humanos , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6RESUMO
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been recently derived and are used for basic research, cardiotoxicity assessment, and phenotypic screening. However, the hiPS-CM phenotype is dependent on their derivation, age, and culture conditions, and there is disagreement as to what constitutes a functional hiPS-CM. The aim of the present study is to characterize the temporal changes in hiPS-CM phenotype by examining five determinants of cardiomyocyte function: gene expression, ion channel functionality, calcium cycling, metabolic activity, and responsiveness to cardioactive compounds. Based on both gene expression and electrophysiological properties, at day 30 of differentiation, hiPS-CMs are immature cells that, with time in culture, progressively develop a more mature phenotype without signs of dedifferentiation. This phenotype is characterized by adult-like gene expression patterns, action potentials exhibiting ventricular atrial and nodal properties, coordinated calcium cycling and beating, suggesting the formation of a functional syncytium. Pharmacological responses to pathological (endothelin-1), physiological (IGF-1), and autonomic (isoproterenol) stimuli similar to those characteristic of isolated adult cardiac myocytes are present in maturing hiPS-CMs. In addition, thyroid hormone treatment of hiPS-CMs attenuated the fetal gene expression in favor of a more adult-like pattern. Overall, hiPS-CMs progressively acquire functionality when maintained in culture for a prolonged period of time. The description of this evolving phenotype helps to identify optimal use of hiPS-CMs for a range of research applications.
Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Canais Iônicos/fisiologia , Miócitos Cardíacos/classificação , Fenótipo , Células-Tronco Pluripotentes/classificaçãoRESUMO
Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.
Assuntos
Insuficiência Cardíaca/complicações , Moduladores de Transporte de Membrana/administração & dosagem , Moduladores de Transporte de Membrana/uso terapêutico , Edema Pulmonar/tratamento farmacológico , Edema Pulmonar/prevenção & controle , Canais de Cátion TRPV/antagonistas & inibidores , Administração Oral , Animais , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Modelos Animais de Doenças , Diuréticos/farmacologia , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Endotélio/patologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Moduladores de Transporte de Membrana/química , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Camundongos Knockout , Permeabilidade/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Edema Pulmonar/etiologia , Edema Pulmonar/patologia , Ratos , Canais de Cátion TRPV/metabolismo , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
Large conductance Ca(2+)-activated K(+) (BK) channels are known to be regulated by both intracellular Ca(2+) and voltage. Although BK channel modulators have been identified, there is a paucity of information regarding the molecular entities of this channel that govern interaction with blockers and activators. Using both whole-cell and single-channel electrophysiological studies we have characterized the possible role that a threonine residue in the pore region of the channel has on function and interaction with BK channel modulators. A threonine-to-serine substitution at position 352 (T352S) resulted in a 59-mV leftward shift in the voltage-dependent activation curve. Single-channel conductance was 236 pS for the wild-type channel and 100 pS for the T352S mutant, measured over the range -80 mV to +80 mV. In addition, there was an almost 10-fold reduction in the potency of the BK channel inhibitor 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP), the IC(50) values being 4.3 +/- 0.3 and 38.2 +/- 3.3 nM for wild-type and mutant channel, respectively. There was no significant difference between wild type and the mutant channel in response to inhibition by iberiotoxin. The IC(50) was 8.1 +/- 0.3 nM for the wild type and 7.7 +/- 0.3 nM for the mutant channel. Here, we have identified a residue in the pore region of the BK channel that alters voltage sensitivity and reduces the potency of the blocker HMIMP.
Assuntos
Cálcio/fisiologia , Indazóis/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Treonina/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Condutividade Elétrica , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Técnicas de Patch-Clamp , Homologia de Sequência de AminoácidosRESUMO
Previously we have shown that the transient receptor potential vanilloid 4 (TRPV4) channel regulates urinary bladder function, and that TRPV4 is expressed in both smooth muscle and urothelial cell types within the bladder wall.(1) Urothelial cells have also been suggested to express TRPV1 channels.(2) Therefore, we enzymatically isolated guinea-pig urothelial cells in an attempt to record TRPV4 and TRPV1-mediated currents. The identity of the isolated cells was confirmed by quantitative PCR for the urothelial marker uroplakin 1A. Whole-cell patch-clamp recordings with the TRPV4 agonist, GSK1016790A, activated urothelial currents with an EC(50) of 11 nM that were completely inhibited by the TRPV4 inhibitor ruthenium red (5 microM). Urothelial currents were also activated by challenge with hypotonic extracellular solution (220 mOsm) known to activate TRPV4 channels. However, the TRPV1 agonist capsaicin, which activated TRPV1 currents in HEK cells expressing TRPV1, was unable to evoke current in these freshly isolated guinea-pig urothelial cells. We demonstrate that TRPV4 channels are functionally expressed at the plasma membrane of freshly isolated, guinea-pig urothelial cells, further supporting the important role of TRPV4 in urinary bladder physiology.
Assuntos
Capsaicina/farmacologia , Potenciais Evocados/efeitos dos fármacos , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/metabolismo , Bexiga Urinária/fisiologia , Urotélio/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Separação Celular , Corantes/farmacologia , Relação Dose-Resposta a Droga , Potenciais Evocados/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Cobaias , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Músculo Liso/metabolismo , Rutênio Vermelho/farmacologia , Sulfonamidas/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
The large-conductance voltage-gated and calcium-dependent K(+) (BK) channels are widely distributed and play important physiological roles. Commonly used BK channel inhibitors are peptide toxins that are isolated from scorpion venoms. A high-affinity, nonpeptide, synthesized BK channel blocker with selectivity against other ion channels has not been reported. We prepared several compounds from a published patent application (Doherty et al., 2004) and identified 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP) as a potent and selective BK channel blocker. The patch-clamp technique was used for characterizing the activity of HMIMP on recombinant human BK channels (alpha subunit, alpha+beta1 and alpha+beta4 subunits). HMIMP blocked all of these channels with an IC(50) of approximately 2 nM. The inhibitory effect of HMIMP was not voltage-dependent, nor did it require opening of BK channels. HMIMP also potently blocked BK channels in freshly isolated detrusor smooth muscle cells and vagal neurons. HMIMP (10 nM) reduced the open probability significantly without affecting single BK-channel current in inside-out patches. HMIMP did not change the time constant of open states but increased the time constants of the closed states. More importantly, HMIMP was highly selective for the BK channel. HMIMP had no effect on human Na(V)1.5 (1 microM), Ca(V)3.2, L-type Ca(2+), human ether-a-go-go-related gene potassium channel, KCNQ1+minK, transient outward K(+) or voltage-dependent K(+) channels (100 nM). HMIMP did not change the action potentials of ventricular myocytes, confirming its lack of effect on cardiac ion channels. In summary, HMIMP is a highly potent and selective BK channel blocker, which can serve as an important tool in the pharmacological study of the BK channel.
Assuntos
Indazóis/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Células CHO , Canais de Cálcio/efeitos dos fármacos , Cricetinae , Cricetulus , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Cobaias , Humanos , Indóis/farmacologia , Canal de Potássio KCNQ1/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Peptídeos/farmacologia , Coelhos , Canais de Sódio/efeitos dos fármacosRESUMO
The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca2+ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4-/- null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-L-arginine methyl ester; L-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (L-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.
Assuntos
Aorta Torácica/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Leucina/análogos & derivados , Sulfonamidas/efeitos adversos , Canais de Cátion TRPV/agonistas , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Aorta Torácica/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Leucina/efeitos adversos , Leucina/farmacocinética , Masculino , Camundongos , Camundongos Knockout , Estrutura Molecular , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacocinética , Canais de Cátion TRPV/genética , Vasoconstrição/efeitos dos fármacosRESUMO
BACKGROUND: This study was designed to test the hypothesis that differences exist in the inactivation properties of voltage-gated Ca(2+) channels (Ca(V)) in hypertensive arterial smooth muscle cells (ASMCs), and that these differences contribute to enhanced Ca(V) activity. METHODS: The properties of Ca(V) were studied in freshly isolated myocytes from small mesenteric arteries (SMAs) of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs) using whole-cell patch-clamp methods. RESULTS: Peak currents (I(Ca)) were larger in SHR with either 2 mmol/l Ca(2+) or Ba(2+) as the charge carrier. In WKY and SHR, the peak current was larger with Ba(2+) than with Ca(2+) with no difference in their ratio. The voltage dependence of Ca(V) activation was shifted to the left in SHR as compared to WKY for Ca(2+) but not for Ba(2+), while availability was not different. The time course of inactivation of current could be represented by two time constants, both of which were larger in SHR than in WKY and also larger for Ba(2+) than for Ca(2+), with a greater fraction of inactivation being associated with the process slower in SHR and with Ba(2+). The time courses of availability, inactivation, and recovery from inactivation were faster in SHR than in WKY in the case of Ca(2+), but there was no difference in the case of Ba(2+). CONCLUSIONS: These results demonstrate that there are differences between WKY and SHR in the inactivation properties of SMA Ca(V), and that these differences could contribute to larger steady-state currents. The differences cannot be explained merely by the presence of a larger number of identical Ca(V) complexes, and it appears likely that differences in intrinsic compositions, primary structures, and/or regulation are involved.
Assuntos
Canais de Cálcio/metabolismo , Hipertensão/metabolismo , Artérias Mesentéricas/metabolismo , Células Musculares/metabolismo , Contração Muscular/fisiologia , Músculo Liso Vascular/metabolismo , Animais , Modelos Animais de Doenças , Hipertensão/patologia , Hipertensão/fisiopatologia , Masculino , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/patologia , Artérias Mesentéricas/fisiopatologia , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Técnicas de Patch-Clamp , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Long and short QT syndromes associated with loss and gain of human ether-a-go-go-related gene (hERG) channel activity, respectively, can cause life-threatening arrhythmias. As such, modulation of hERG channel activity is an important consideration in the development of all new therapeutic agents. In the present study, we investigated the mechanisms of action of 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD-307243), a known hERG channel activator, on hERG channels stably expressed in Chinese hamster ovary (CHO) cells using the patch-clamp technique. In the whole-cell recordings, the extracellular application of PD-307243 concentration-dependently increased the hERG current and markedly slowed hERG channel deactivation and inactivation. PD-307243 had no effect on the selectivity filter of hERG channels. The activity of PD-307243 was use-dependent. PD-307243 (3 and 10 muM) induced instantaneous hERG current with little decay at membrane potentials from -120 to -40 mV. At more positive voltages, PD-307243 induced an I(to)-like upstroke of hERG current. The actions of PD-307243 on the rapid component of delayed rectifier K(+) current (I(Kr)) in rabbit ventricular myocytes were similar to those observed in hERG channel-transfected CHO cells. Inside-out patch experiments revealed that PD-307243 increased hERG tail currents by 2.1 +/- 0.6 (n = 7) and 3.4 +/- 0.3-fold (n = 4) at 3 and 10 muM, respectively, by slowing the channel deactivation but had no effect on channel activation. During a voltage-clamp protocol using a prerecorded cardiac action potential, 3 muM PD-307243 increased the total potassium ions passed through hERG channels by 8.8 +/- 1.0-fold (n = 5). Docking studies suggest that PD-307243 interacts with residues in the S5-P region of the channel.
Assuntos
Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/fisiologia , Isoindóis/farmacologia , Niacina/análogos & derivados , Niacina/farmacologia , Ácidos Nicotínicos/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Interpretação Estatística de Dados , Relação Dose-Resposta a Droga , Condutividade Elétrica , Canais de Potássio Éter-A-Go-Go/genética , Ventrículos do Coração/citologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoindóis/química , Cinética , Masculino , Camundongos , Microeletrodos , Modelos Moleculares , Estrutura Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Niacina/química , Ácidos Nicotínicos/química , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , TransfecçãoRESUMO
Acetic acid was found to have actions on urinary bladder smooth muscle in our routine ion channel screening assays. Numerous studies have examined the mechanisms of bladder irritation by acetic acid; however, the direct effect of acetic acid on ion channels in detrusor smooth muscle cells has not been evaluated. We used whole-cell patch-clamp techniques to examine the effect of acetic acid on large-conductance Ca2+-activated K+ channels (BKCa) from guinea pig detrusor smooth muscle cells and CHO cells expressing recombinant human BKCaalphabeta1 (CHO BKCaalphabeta1) and human BKCaalpha (CHO BKCaalpha). Acetic acid activated BKCa currents in a concentration-dependent (0.01% to 0.05% v/v) manner in all the cell systems studied. Acetic acid (0.05%) increased BKCa current at +30 mV by 2764+/-918% (n=8) in guinea pig detrusor smooth muscle cells. Acetic acid (0.03%) shifted the V1/2 of conductance-voltage curve by 64+/-14 (n=5), 128+/-14 (n=5), and 126+/-12 mV (n=4) in CHO BKCaalpha, CHO BKCaalphabeta1 and detrusor smooth muscle cells, respectively. This effect of acetic acid was found to be independent of pH and was also not produced by its salt form, sodium acetate. Automated patch-clamp experiments also showed similar activation of CHO BKCaalphabeta1 by acetic acid. In conclusion, acetic acid directly activates BKCa channels in detrusor smooth muscle cells. This novel study necessitates caution while interpreting the results from acetic acid bladder irritation model.
Assuntos
Ácido Acético/farmacologia , Irritantes/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Miócitos de Músculo Liso/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Cobaias , Humanos , Técnicas In Vitro , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Técnicas de Patch-Clamp , Proteínas Recombinantes/agonistas , Transfecção , Bexiga Urinária/citologia , Bexiga Urinária/metabolismoRESUMO
Human ether-a-go-go-related gene (hERG) encodes a rapidly activating delayed rectifier potassium channel that plays important roles in cardiac action potential repolarization. Although many drugs and compounds block hERG channels, activators of the channel have only recently been described. Three structurally diverse synthetic compounds have been reported to activate hERG channels by altering deactivation or inactivation or by unidentified mechanisms. Here, we describe a novel, naturally occurring hERG channel activator, mallotoxin (MTX). The effects of MTX on hERG channels were investigated using the patch-clamp technique. MTX increased both step and tail hERG currents with EC(50) values of 0.34 and 0.52 microM, respectively. MTX leftward shifted the voltage dependence of hERG channel activation to less depolarized voltages ( approximately 24 mV at 2.5 microM). In addition, MTX increased hERG deactivation time constants. MTX did not change the half-maximal inactivation voltage of the hERG channel, but it reduced the slope of the voltage-dependent inactivation curve. All of these factors contribute to the enhanced activity of hERG channels. During a voltage-clamp protocol using prerecorded cardiac action potentials, 2.5 microM MTX increased the total potassium ions passed through hERG channels by approximately 5-fold. In conclusion, MTX activates hERG channels through distinct mechanisms and with significantly higher potency than previously reported hERG channel activators.
Assuntos
Acetofenonas/farmacologia , Benzopiranos/farmacologia , Inibidores Enzimáticos/farmacologia , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/fisiologia , Potássio/metabolismo , Proteína Quinase C/antagonistas & inibidores , CoelhosRESUMO
Hypertension is associated with a remodeling of arterial smooth muscle K(+) channels with Ca(2+)-gated K(+) channel (BK(Ca)) activity being enhanced and voltage-gated K(+) channel (K(v)) activity depressed. Because both of these channel types are modulated by intracellular Ca(2+), we tested the hypothesis that Ca(2+) had a larger effect on both BK(Ca) and K(v) channels in arterial myocytes from hypertensive animals. Myocytes were enzymatically dispersed from small mesenteric arteries (SMA) of 12-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Using whole cell patch clamp methods, BK(Ca) and K(v) current components were determined as iberiotoxin-sensitive and -insensitive currents, respectively. The effects of Ca(2+) on these K(+) current components were determined from measurements made with 0.2 and 2 mmol/L external Ca(2+). Increasing external Ca(2+) from 0.2 to 2 mmol/L Ca(2+) increased BK(Ca) currents recorded using myocytes from both WKY rats and SHR with a larger effect in SHR. Increasing external Ca(2+) decreased K(v) currents recorded using myocytes from both WKY and SHR also with a larger effect in SHR. In other experiments, currents through voltage-gated Ca(2+) channels (Ca(v)) measured at 0.2 mmol/L external Ca(2+) were 12 +/- 2% (n = 12) of those recorded at 2 mmol/L Ca(2+) with no differences in percent effect between WKY and SHR. In isolated SMA segments, isometric force development in response to 140 mmol/L KCl at 0.2 mmol/L external Ca(2+) was about 23 +/- 6% (n = 8) of that measured at 2 mmol/L external Ca(2+). These results suggest that an increase in Ca(2+) influx through Ca(v) or in intracellular Ca(2+) secondary to an increase in external Ca(2+) augments BK(Ca) currents and inhibits K(v) currents in SMA myocytes with a larger effect in SHR compared to WKY. This mechanism may contribute to the functional remodeling of K(+) currents of arterial myocytes in hypertensive animals.
Assuntos
Cálcio/metabolismo , Hipertensão/metabolismo , Artérias Mesentéricas/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Canais de Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/citologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Especificidade da EspécieRESUMO
Numerous studies have emphasized the important role of altered Ca(2+) channel function in hypertension. We previously showed that Ca(2+) currents measured in myocytes isolated from both Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) small mesenteric arteries closely correlated with systolic blood pressure (BP) during normal development. The purpose of the present experiments was to determine whether antihypertensive therapy with an angiotensin converting enzyme inhibitor normalizes Ca(2+) channel function in SHR myocytes along with BP. Ramipril (3.5 mg/kg/day) was added to the drinking water of 12-week-old male WKY and SHR for 8 weeks. Segments of small mesenteric arteries were used for isometric contraction studies, and for isolation of myocytes for measurement of Ca(2+) and K(+) currents (I(Ca) and I(K)) by patch clamp methods. Ramipril treatment decreased systolic pressure in WKY and SHR, decreased heart weight and heart weight-to-body weight ratio in SHR, and decreased body weight in WKY. Maximum contractile responses to Bay k 8644 in SMA from ramipril-treated SHR were smaller compared to untreated SHR (10% +/- 2% v 55% +/- 7% of the response to 120 mmol/L KCl). The smaller responses in WKY were not affected by ramipril treatment (11% +/- 4% v 8% +/- 3%). Contractile responses to 10 mmol/L tetraethylammonium (TEA) were not different in untreated versus ramipril-treated SHR (65% +/- 6% v 82% +/- 8%) but were increased in treated WKY (4% +/- 1% v 35% +/- 9%). Ramipril treatment decreased peak I(Ca) and equalized the voltage-dependence of I(Ca) activation between SHR and WKY. The I(K) measured from holding potentials of -60 and -20 mV were significantly smaller in treated SHR and WKY compared to their untreated counterparts, as was the component of I(K) measured in the presence of 100 nmol/L iberiotoxin. These results show that ramipril treatment decreases arterial pressure and Ca(2+) channel function in SHR as expected but unexpectedly also decreases I(K) in both WKY and SHR. These results suggest that angiotensin may have a BP independent effect on ion channel function in arterial smooth muscle.