RESUMO
tested whether rat descending vasa recta (DVR) undergo regulatory adaptations after the kidney is exposed to ischemia. Left kidneys (LK) were subjected to 30-min renal artery cross clamp. After 48 h, the postischemic LK and contralateral right kidney (RK) were harvested for study. When compared with DVR isolated from either sham-operated LK or the contralateral RK, postischemic LK DVR markedly increased their NO generation. The selective inducible NOS (iNOS) inhibitor 1400W blocked the NO response. Immunoblots from outer medullary homogenates showed a parallel 2.6-fold increase in iNOS expression ( P = 0.01). Microperfused postischemic LK DVR exposed to angiotensin II (ANG II, 10 nM), constricted less than those from the contralateral RK, and constricted more when exposed to 1400W (10 µM). Resting membrane potentials of pericytes from postischemic LK DVR pericytes were hyperpolarized relative to contralateral RK pericytes (62.0 ± 1.6 vs. 51.8 ± 2.2 mV, respectively, P < 0.05) or those from sham-operated LK (54.9 ± 2.1 mV, P < 0.05). Blockade of NO generation with 1400W did not repolarize postischemic pericytes (62.5 ± 1.4 vs. 61.1 ± 3.4 mV); however, control pericytes were hyperpolarized by exposure to NO donation from S-nitroso- N-acetyl- dl-penicillamine (51.5 ± 2.9 to 62.1 ± 1.4 mV, P < 0.05). We conclude that postischemic adaptations intrinsic to the DVR wall occur after ischemia. A rise in 1400W sensitive NO generation and iNOS expression occurs that is associated with diminished contractile responses to ANG II. Pericyte hyperpolarization occurs that is not explained by the rise in ambient NO generation within the DVR wall.
Assuntos
Arteríolas/fisiopatologia , Isquemia/fisiopatologia , Rim/irrigação sanguínea , Artéria Renal/fisiopatologia , Circulação Renal , Vasoconstrição , Adaptação Fisiológica , Amidinas/farmacologia , Angiotensina II/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/metabolismo , Benzilaminas/farmacologia , Constrição , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Isquemia/metabolismo , Isquemia/patologia , Rim/patologia , Potenciais da Membrana , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Pericitos/metabolismo , Ratos Sprague-Dawley , Artéria Renal/cirurgia , Circulação Renal/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
Patients with chronic kidney disease (CKD) may have nonlinear serum creatinine concentration (SC) trajectories, especially as CKD progresses. Variability in SC is associated with renal failure and death. However, present methods for measuring SC variability are unsatisfactory because they blend information about SC slope and variance. We propose an improved method for defining and calculating a patient's SC slope and variance so that they are mathematically distinct, and we test these methods in a large sample of US veterans, examining the correlation of SC slope and SC nonlinearity (SCNL) and the association of SCNL with time to stage 4 CKD (CKD4) and death. We found a strong correlation between SCNL and rate of CKD progression, time to CKD4, and time to death, even in patients with normal renal function. We therefore argue that SCNL may be a measure of renal autoregulatory dysfunction that provides an early warning sign for CKD progression.
Assuntos
Creatinina/sangue , Testes de Função Renal , Insuficiência Renal Crônica/sangue , Animais , Humanos , Dinâmica não Linear , Insuficiência Renal Crônica/diagnósticoRESUMO
Using dual cell patch-clamp recording, we examined pericyte, endothelial, and myoendothelial cell-to-cell communication in descending vasa recta. Graded current injections into pericytes or endothelia yielded input resistances of 220 ± 21 and 128 ± 20 MΩ, respectively (P < 0.05). Injection of positive or negative current into an endothelial cell depolarized and hyperpolarized adjacent endothelial cells, respectively. Similarly, current injection into a pericyte depolarized and hyperpolarized adjacent pericytes. During myoendothelial studies, current injection into a pericyte or an endothelial cell yielded small, variable, but significant change of membrane potential in heterologous cells. Membrane potentials of paired pericytes or paired endothelia were highly correlated and identical. Paired measurements of resting potentials in heterologous cells were also correlated, but with slight hyperpolarization of the endothelium relative to the pericyte, -55.2 ± 1.8 vs. -52.9 ± 2.2 mV (P < 0.05). During dual recordings, angiotensin II or bradykinin stimulated temporally identical variations of pericyte and endothelial membrane potential. Similarly, voltage clamp depolarization of pericytes or endothelial cells induced parallel changes of membrane potential in the heterologous cell type. We conclude that the descending vasa recta endothelial syncytium is of lower resistance than the pericyte syncytium and that high-resistance myoendothelial coupling also exists. The myoendothelial communication between pericytes and endothelium maintains near identity of membrane potentials at rest and during agonist stimulation. Finally, endothelia membrane potential lies slightly below pericyte membrane potential, suggesting a tonic role for the former to hyperpolarize the latter and provide a brake on vasoconstriction.
Assuntos
Angiotensina II/farmacologia , Comunicação Celular/fisiologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Animais , Bradicinina/farmacologia , Comunicação Celular/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Medula Renal/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Pericitos , Ratos , Ratos Sprague-DawleyRESUMO
Using patch clamp, we induced depolarization of descending vasa recta (DVR) pericytes or endothelia and tested whether it was conducted to distant cells. Membrane potential was measured with the fluorescent voltage dye di-8-ANEPPS or with a second patch-clamp electrode. Depolarization of an endothelial cell induced responses in other endothelia within a millisecond and was slowed by gap junction blockade with heptanol. Endothelial response to pericyte depolarization was poor, implying high-resistance myo-endothelial coupling. In contrast, dual patch clamp of neighboring pericytes revealed syncytial coupling. At high sampling rate, the spread of depolarization between pericytes and endothelia occurred in 9 ± 2 or 12 ± 2 µs, respectively. Heptanol (2 mM) increased the overall input resistance of the pericyte layer to current flow and prevented transmission of depolarization between neighboring cells. The fluorescent tracer Lucifer yellow (LY), when introduced through ruptured patches, spread between neighboring endothelia in 1 to 7 s, depending on location of the flanking cell. LY diffused to endothelial cells on the ipsilateral but not contralateral side of the DVR wall and minimally between pericytes. We conclude that both DVR pericytes and endothelia are part of individual syncytia. The rate of conduction of membrane potential exceeds that for diffusion of hydrophilic molecules by orders of magnitude. Gap junction coupling of adjacent endothelial cells may be spatially oriented to favor longitudinal transmission along the DVR axis.
Assuntos
Comunicação Celular , Células Endoteliais/fisiologia , Junções Comunicantes/fisiologia , Células Gigantes/fisiologia , Rim/irrigação sanguínea , Pericitos/fisiologia , Animais , Difusão , Impedância Elétrica , Células Endoteliais/metabolismo , Corantes Fluorescentes/metabolismo , Isoquinolinas/metabolismo , Potenciais da Membrana , Técnicas de Patch-Clamp/métodos , Pericitos/metabolismo , Compostos de Piridínio/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
To investigate the responses of descending vasa recta (DVR) to deformation of the abluminal surface, we devised an automated method that controls duration and frequency of stimulation by utilizing a stream of buffer from a micropipette. During stimulation at one end of the vessel, fluorescent responses from fluo4 or bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [DiBAC4(3)], indicating cytoplasmic calcium ([Ca²âº]CYT) or membrane potential, respectively, were recorded from distant cells. Alternately, membrane potential was recorded from DVR pericytes by nystatin whole cell patch-clamp. Mechanical stimulation elicited reversible [Ca²âº)]CYT responses that increased with frequency. Individual pericyte responses along the vessel were initiated within a fraction of a second of one another. Those responses were inhibited by gap junction blockade with 18 ß-glycyrrhetinic acid (100 µM) or phosphoinositide 3 kinase inhibition with 2-morpholin-4-yl-8-phenylchromen-4-one (50 µM). [Ca²âº]CYT responses were blocked by removal of extracellular Ca²âº or L-type voltage-gated channel blockade with nifedipine (10 µM). At concentrations selective for the T-type channel blockade, mibefradil (100 nM) was ineffective. During mechanostimulation, pericytes rapidly depolarized, as documented with either DiBAC4(3) fluorescence or patch-clamp recording. Single stimuli yielded depolarizations of 22.5 ± 2.2 mV while repetitive stimuli at 0.1 Hz depolarized pericytes by 44.2 ± 4.0 mV. We conclude that DVR are mechanosensitive and that rapid transmission of signals along the vessel axis requires participation of gap junctions, L-type Ca²âº channels, and pericyte depolarization.
Assuntos
Neovascularização Fisiológica/fisiologia , Circulação Renal/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Cromonas/farmacologia , Citoplasma/metabolismo , Corantes Fluorescentes , Junções Comunicantes/fisiologia , Técnicas In Vitro , Microscopia de Fluorescência , Morfolinas/farmacologia , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Pericitos/efeitos dos fármacos , Estimulação Física , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular resistance, and thereby raises blood pressure (BP), are poorly understood. Here we summarize recent evidence that defines specific molecular links between Na(+) and the elevated vascular resistance that directly produces high BP. In this new paradigm, high dietary salt raises cerebrospinal fluid [Na(+)]. This leads, via the Na(+)-sensing circumventricular organs of the brain, to increased sympathetic nerve activity (SNA), a major trigger of vasoconstriction. Plasma levels of endogenous ouabain (EO), the Na(+) pump ligand, also become elevated. Remarkably, high cerebrospinal fluid [Na(+)]-evoked, locally secreted (hypothalamic) EO participates in a pathway that mediates the sustained increase in SNA. This hypothalamic signaling chain includes aldosterone, epithelial Na(+) channels, EO, ouabain-sensitive α(2) Na(+) pumps, and angiotensin II (ANG II). The EO increases (e.g.) hypothalamic ANG-II type-1 receptor and NADPH oxidase and decreases neuronal nitric oxide synthase protein expression. The aldosterone-epithelial Na(+) channel-EO-α(2) Na(+) pump-ANG-II pathway modulates the activity of brain cardiovascular control centers that regulate the BP set point and induce sustained changes in SNA. In the periphery, the EO secreted by the adrenal cortex directly enhances vasoconstriction via an EO-α(2) Na(+) pump-Na(+)/Ca(2+) exchanger-Ca(2+) signaling pathway. Circulating EO also activates an EO-α(2) Na(+) pump-Src kinase signaling cascade. This increases the expression of the Na(+)/Ca(2+) exchanger-transient receptor potential cation channel Ca(2+) signaling pathway in arterial smooth muscle but decreases the expression of endothelial vasodilator mechanisms. Additionally, EO is a growth factor and may directly participate in the arterial structural remodeling and lumen narrowing that is frequently observed in established hypertension. These several central and peripheral mechanisms are coordinated, in part by EO, to effect and maintain the salt-induced elevation of BP.
Assuntos
Hipertensão/induzido quimicamente , Cloreto de Sódio na Dieta/efeitos adversos , Animais , Cardiotônicos/farmacologia , Feminino , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/fisiopatologia , Masculino , Camundongos , Ouabaína/sangue , Ouabaína/farmacologia , Gravidez , Ratos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiopatologiaRESUMO
It has been observed that vasoactivity of explanted descending vasa recta (DVR) is modulated by intrinsic nitric oxide (NO) and superoxide (O(2)(-)) production (Cao C, Edwards A, Sendeski M, Lee-Kwon W, Cui L, Cai CY, Patzak A, Pallone TL. Am J Physiol Renal Physiol 299: F1056-F1064, 2010). To elucidate the cellular mechanisms by which NO, O(2)(-) and hydrogen peroxide (H(2)O(2)) modulate DVR pericyte cytosolic Ca(2+) concentration ([Ca](cyt)) and vasoactivity, we expanded our mathematical model of Ca(2+) signaling in pericytes. We incorporated simulations of the pathways that translate an increase in [Ca](cyt) to the activation of myosin light chain (MLC) kinase and cell contraction, as well as the kinetics of NO and reactive oxygen species formation and their effects on [Ca](cyt) and MLC phosphorylation. The model reproduced experimentally observed trends of DVR vasoactivity that accompany exposure to N(ω)-nitro-L-arginine methyl ester, 8-Br-cGMP, Tempol, and H(2)O(2). Our results suggest that under resting conditions, NO-induced activation of cGMP maintains low levels of [Ca](cyt) and MLC phosphorylation to minimize basal tone. This results from stimulation of Ca(2+) uptake from the cytosol into the SR via SERCA pumps, Ca(2+) efflux into the extracellular space via plasma membrane Ca(2+) pumps, and MLC phosphatase (MLCP) activity. We predict that basal concentrations of O(2)(-) and H(2)O(2) have negligible effects on Ca(2+) signaling and MLC phosphorylation. At concentrations above 1 nM, O(2)(-) is predicted to modulate [Ca(cyt)] and MCLP activity mostly by reducing NO bioavailability. The DVR vasoconstriction that is induced by high concentrations of H(2)O(2) can be explained by H(2)O(2)-mediated downregulation of MLCP and SERCA activity. We conclude that intrinsic generation of NO by the DVR wall may be sufficient to inhibit vasoconstriction by maintaining suppression of MLC phosphorylation.
Assuntos
Medula Renal/irrigação sanguínea , Óxido Nítrico/fisiologia , Pericitos/fisiologia , Artéria Renal/fisiologia , Vasodilatação/fisiologia , Animais , Cálcio/análise , Cálcio/metabolismo , Simulação por Computador , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Óxidos N-Cíclicos/farmacologia , Peróxido de Hidrogênio/metabolismo , Modelos Biológicos , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Pericitos/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Artéria Renal/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Marcadores de Spin , Tionucleotídeos/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
Multiple voltage-gated Ca(2+) channel (Ca(V)) subtypes have been reported to participate in control of the juxtamedullary glomerular arterioles of the kidney. Using the patch-clamp technique, we examined whole cell Ca(V) currents of pericytes that contract descending vasa recta (DVR). The dihydropyridine Ca(V) agonist FPL64176 (FPL) stimulated inward Ca(2+) and Ba(2+) currents that activated with threshold depolarizations to -40 mV and maximized between -20 and -10 mV. These currents were blocked by nifedipine (1 µM) and Ni(2+) (100 and 1,000 µM), exhibited slow inactivation, and conducted Ba(2+) > Ca(2+) at a ratio of 2.3:1, consistent with "long-lasting" L-type Ca(V). In FPL, with 1 mM Ca(2+) as charge carrier, Boltzmann fits yielded half-maximal activation potential (V(1/2)) and slope factors of -57.9 mV and 11.0 for inactivation and -33.3 mV and 4.4 for activation. In the absence of FPL stimulation, higher concentrations of divalent charge carriers were needed to measure basal currents. In 10 mM Ba(2+), pericyte Ca(V) currents activated with threshold depolarizations to -30 mV, were blocked by nifedipine, exhibited voltage-dependent block by diltiazem (10 µM), and conducted Ba(2+) > Ca(2+) at a ratio of â¼2:1. In Ca(2+), Boltzmann fits to the data yielded V(1/2) and slope factors of -39.6 mV and 10.0 for inactivation and 2.8 mV and 7.7 for activation. In Ba(2+), V(1/2) and slope factors were -29.2 mV and 9.2 for inactivation and -5.6 mV and 6.1 for activation. Neither calciseptine (10 nM), mibefradil (1 µM), nor ω-agatoxin IVA (20 and 100 nM) blocked basal Ba(2+) currents. Calciseptine (10 nM) and mibefradil (1 µM) also failed to reverse ANG II-induced DVR vasoconstriction, although raising mibefradil concentration to 10 µM was partially effective. We conclude that DVR pericytes predominantly express voltage-gated divalent currents that are carried by L-type channels.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Medula Renal/metabolismo , Pericitos/metabolismo , Animais , Bário/metabolismo , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Medula Renal/citologia , Mibefradil/farmacologia , Modelos Animais , Níquel/farmacologia , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Pericitos/citologia , Pericitos/efeitos dos fármacos , Pirróis/farmacologia , Ratos , ômega-Agatoxina IVA/farmacologiaRESUMO
Descending vasa recta (DVR) are 12- to 15-µm microvessels that supply the renal medulla with blood flow. We examined the ability of intrinsic nitric oxide (NO) and reactive oxygen species (ROS) generation to regulate their vasoactivity. Nitric oxide synthase (NOS) inhibition with N(ω)-nitro-l-arginine methyl ester (l-NAME; 100 µmol/l), or asymmetric N(G),N(G)-dimethyl-l-arginine (ADMA; 100 µmol/l), constricted isolated microperfused DVR by 48.82 ± 4.34 and 27.91 ± 2.91%, respectively. Restoring NO with sodium nitroprusside (SNP; 1 mmol/l) or application of 8-Br-cGMP (100 µmol/l) reversed DVR vasoconstriction by l-NAME. The superoxide dismutase mimetic Tempol (1 mmol/l) and the NAD(P)H inhibitor apocynin (100, 1,000 µmol/l) also blunted ADMA- or l-NAME-induced vasoconstriction, implicating a role for concomitant generation of ROS. A role for ROS generation was also supported by an l-NAME-associated rise in oxidation of dihydroethidium that was prevented by Tempol or apocynin. To test whether H(2)O(2) might play a role, we examined its direct effects. From 1 to 100 µmol/l, H(2)O(2) contracted DVR whereas at 1 mmol/l it was vasodilatory. The H(2)O(2) scavenger polyethylene glycol-catalase reversed H(2)O(2) (10 µmol/l)-induced vasoconstriction; however, it did not affect l-NAME-induced contraction. Finally, the previously known rise in DVR permeability to (22)Na and [(3)H]raffinose that occurs with luminal perfusion was not prevented by NOS blockade. We conclude that intrinsic production of NO and ROS can modulate DVR vasoactivity and that l-NAME-induced vasoconstriction occurs, in part, by modulating superoxide concentration and not through H(2)O(2) generation. Intrinsic NO production does not affect DVR permeability to hydrophilic solutes.
Assuntos
Óxido Nítrico/biossíntese , Óxido Nítrico/fisiologia , Circulação Renal/fisiologia , Superóxidos/metabolismo , Vasoconstrição/fisiologia , Animais , Inibidores Enzimáticos/farmacologia , Etídio/análogos & derivados , Corantes Fluorescentes , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Indicadores e Reagentes , Tono Muscular/fisiologia , Músculo Liso Vascular/fisiologia , NADPH Oxidases/antagonistas & inibidores , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Oxidantes/farmacologia , Oxirredução , Permeabilidade , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Circulação Renal/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacosRESUMO
We hypothesized that in salt-dependent forms of hypertension, endogenous ouabain acts on arterial smooth muscle to cause enhanced vasoconstriction. Here, we tested for the involvement of the arterial endothelium and perivascular sympathetic nerve terminals in ouabain-induced vasoconstriction. Segments of rat mesenteric or renal interlobar arteries were pressurized to 70 mmHg at 37 degrees C and exposed to ouabain (10(-11)-10(-7) M). Removal of the endothelium enhanced ouabain-induced vasoconstriction by as much as twofold (at an ouabain concentration of 10(-9) M). A component of the ouabain-induced vasoconstriction is due to the enhanced spontaneous release of norepinephrine (NE) from nerve terminals in the arterial wall. The alpha(1)-adrenoceptor blocker prazosin (10(-6) M) decreased ouabain-induced vasoconstrictions by as much as 50%. However, neither the contraction induced by sympathetic nerve activity (SNA) nor the NE release evoked by SNA (measured directly by carbon fiber amperometry) was increased by ouabain (<10(-7) M). Nevertheless, the converse case was true: after brief bursts of SNA, vasoconstrictor responses to ouabain were transiently increased (1.75-fold). This effect may be mediated by neuropeptide Y and Y(1) receptors on smooth muscle. In arteries lacking the endothelium and exposed to prazosin, ouabain (10(-11) M and greater) caused vasoconstriction, indicating a direct effect of very "low" concentrations of ouabain on arterial smooth muscle. In conclusion, in intact arteries, the endothelium opposes ouabain (10(-11)-10(-7)M)-induced vasoconstriction, which is caused by both enhanced spontaneous NE release and direct effects on smooth muscle. Ouabain (<10(-7)M) does not enhance SNA-mediated contractions, but SNA enhances ouabain-induced contractions. The effects of endogenous ouabain may be accentuated in forms of hypertension that involve sympathetic nerve hyperactivity and/or endothelial dysfunction.
Assuntos
Endotélio Vascular/fisiologia , Artérias Mesentéricas/fisiologia , Ouabaína/farmacologia , Artéria Renal/fisiologia , Sistema Nervoso Simpático/fisiologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Relação Dose-Resposta a Droga , Fenômenos Eletrofisiológicos , Endotélio Vascular/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Modelos Animais , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Neuropeptídeo Y/metabolismo , Neurotransmissores/metabolismo , Norepinefrina/metabolismo , Ratos , Ratos Sprague-Dawley , Artéria Renal/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
We used the whole cell patch-clamp technique to investigate the regulation of descending vasa recta (DVR) pericyte Ca(2+)-dependent Cl(-) currents (CaCC) by cytoplasmic Ca(2+) concentration ([Ca](CYT)), voltage, and kinase activity. Murine CaCC increased with voltage and electrode Ca(2+) concentration. The current saturated at [Ca](CYT) of â¼1,000 nM and exhibited an EC(50) for Ca(2+) of â¼500 nM, independent of depolarization potential. Activation time constants were between 100 and 200 ms, independent of electrode Ca(2+). Repolarization-related tail currents elicited by stepping from +100 mV to varying test potentials exhibited deactivation time constants of 50-200 ms that increased with voltage when electrode [Ca](CYT) was 1,000 nM. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, 30 µM) blocked CaCC. The myosin light chain kinase blockers 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7, 1-50 µM) and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9, 10 µM) were similarly effective. Resting pericytes were hyperpolarized by ML-7. Pericytes exposed to ANG II (10 nM) depolarized from a baseline of -50 ± 6 to -29 ± 3 mV and were repolarized to -63 ± 7 mV by exposure to 50 µM ML-7. The Ca(2+)/calmodulin-dependent kinase inhibitor KN-93 reduced pericyte CaCC only when it was present in the electrode and extracellular buffer from the time of membrane break-in. We conclude that murine DVR pericytes are modulated by [Ca](CYT), membrane potential, and phosphorylation events, suggesting that Ca(2+)-dependent Cl(-) conductance may be a target for regulation of vasoactivity and medullary blood flow in vivo.
Assuntos
Cálcio/metabolismo , Cloretos/metabolismo , Sistema Justaglomerular/irrigação sanguínea , Microvasos/enzimologia , Pericitos/enzimologia , Proteínas Quinases/metabolismo , Angiotensina II/metabolismo , Animais , Azepinas/farmacologia , Benzilaminas/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Transporte de Íons , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/metabolismo , Naftalenos/farmacologia , Técnicas de Patch-Clamp , Pericitos/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
PURPOSE: To determine whether a type of contrast medium (CM), iodixanol, modifies outer medullary descending vasa recta (DVR) vasoreactivity and nitric oxide (NO) production in isolated microperfused DVR. MATERIALS AND METHODS: Animal handling conformed to the Animal Care Committee Guidelines of all participating institutions. Single specimens of DVR were isolated from rats and perfused with a buffered solution containing iodixanol. A concentration of 23 mg of iodine per milliliter was chosen to mimic that expected to be used in usual examinations in humans. Luminal diameter was determined by using video microscopy, and NO was measured by using fluorescent techniques. RESULTS: Iodixanol led to 52% reduction of DVR luminal diameter, a narrowing that might interfere with passage of erythrocytes in vivo. Vasoconstriction induced by angiotensin II was enhanced by iodixanol. Moreover, iodixanol decreased NO bioavailability by more than 82%. Use of 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (a superoxide dismutase mimetic) prevented both vasoconstriction with iodixanol alone and increased constriction with angiotensin II caused by CM. CONCLUSION: Iodixanol in doses typically used for coronary interventions constricts DVR, intensifies angiotensin II-induced constriction, and reduces bioavailability of NO. CM-induced nephropathy may be related to these events and scavenging of reactive oxygen species might exert a therapeutic benefit by preventing the adverse effects that a CM has on medullary perfusion.
Assuntos
Meios de Contraste/farmacologia , Meios de Contraste/toxicidade , Nefropatias/induzido quimicamente , Medula Renal/irrigação sanguínea , Medula Renal/efeitos dos fármacos , Circulação Renal/efeitos dos fármacos , Ácidos Tri-Iodobenzoicos/farmacologia , Ácidos Tri-Iodobenzoicos/toxicidade , Vasoconstrição/efeitos dos fármacos , Análise de Variância , Angiotensina II/farmacologia , Animais , Endotélio Vascular/efeitos dos fármacos , Masculino , Óxido Nítrico/biossíntese , Ratos , Ratos Sprague-Dawley , RiscoRESUMO
Using dual-cell electrophysiological recording, we examined the routes for equilibration of membrane potential between the pericytes and endothelia that comprise the descending vasa recta (DVR) wall. We measured equilibration between pericytes in intact vessels, between pericytes and endothelium in intact vessels and between pericytes physically separated from the endothelium. Dual pericyte recording on the abluminal surface of DVR showed that both resting potential and subsequent time-dependent voltage fluctuations after vasoconstrictor stimulation remained closely equilibrated, regardless of the agonist employed (angiotensin II, vasopressin or endothelin 1). When pericytes where removed from the vessel wall but retained physical contact with one another, membrane potential responses were also highly coordinated. In contrast, responses of pericytes varied independently when they were isolated from both the endothelium and from contact with one another. When pericytes and endothelium were in contact, their resting potentials were similar and their temporal responses to stimulation were highly coordinated. After completely isolating pericytes from the endothelium, their mean resting potentials became discordant. Finally, complete endothelial isolation eliminated all membrane potential responses to angiotensin II. We conclude that cell-to-cell transmission through the endothelium is not needed for pericytes to equilibrate their membrane potentials. AngII dependent responses of DVR endothelia may originate from gap junction coupling to pericytes rather than via receptor dependent signaling in the endothelium, per se.
Assuntos
Comunicação Celular , Endotélio Vascular/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Pericitos/citologia , Angiotensina II/farmacologia , Animais , Comunicação Celular/efeitos dos fármacos , Separação Celular , Endotélio Vascular/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Modelos Lineares , Técnicas de Patch-Clamp , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacosRESUMO
The renal medullary microcirculation is a distinctive arrangement of blood vessels that serve multiple functions in the renal medulla. This article begins with a description of the unique anatomy of this vascular bed and the role it plays in transport and countercurrent exchange in the medulla. A segment of the review is then devoted to the important role mathematical modeling has played in the understanding of this vascular bed's function. Succeeding sections focus upon the hematocrit in the vasa recta capillaries and methods utilized to assess blood flow in the renal medulla. An extensive portion of the article is then devoted to the regulation of the medullary circulation, from ion channel architecture to neurohormonal signaling. Finally, we discuss the importance of the renal medullary circulation in the regulation of fluid and electrolyte homeostasis and arterial blood pressure regulation.
Assuntos
Medula Renal/irrigação sanguínea , Modelos Cardiovasculares , Animais , Pressão Arterial/fisiologia , Transporte Biológico/fisiologia , Técnicas de Diagnóstico Urológico , Hematócrito , Homeostase/fisiologia , Humanos , Canais Iônicos/fisiologia , Medula Renal/fisiologia , Medula Renal/ultraestrutura , Microcirculação/fisiologia , Microscopia Eletrônica , Natriurese/fisiologia , Circulação Renal/fisiologiaRESUMO
BACKGROUND: Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined. METHODOLOGY/PRINCIPAL FINDINGS: Fourth-order mesenteric arteries (170-250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1-100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K(+) channel blockade with glibenclamide (K(ATP)), 4-aminopyradine (K(V)), BaCl(2) (K(IR)) or tetraethylammonium (TEA, K(Ca)) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca(2+) fluorescence (â¼50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca(2+) (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca(2+) responses (â¼50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction. CONCLUSION: In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca(2+) responses, independent of muscarinic receptor, NO or K(+) channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs.
Assuntos
Ácido Glicodesoxicólico/farmacologia , Resistência Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Animais , Ácidos e Sais Biliares/farmacologia , Músculo Liso Vascular , Ratos , Ratos Sprague-DawleyRESUMO
Research over the last decade has uncovered roles for bile acids (BAs) that extend beyond their traditional functions in regulating lipid digestion and cholesterol metabolism. BAs are now recognized as signaling molecules that interact with both plasma membrane and nuclear receptors. Emerging evidence indicates that by interacting with these receptors, BAs regulate their own synthesis, glucose and energy homeostasis, and other important physiological events. Herein, we provide a comprehensive review of the actions of BAs on cardiovascular function. In the heart and the systemic circulation, BAs interact with plasma membrane G-protein-coupled receptors, for example, TGR5 and muscarinic receptors, and nuclear receptors, for example, the farnesoid (FXR) and pregnane (PXR) xenobiotic receptors. BA receptors are expressed in cardiovascular tissue, however, the mechanisms underlying BA-mediated regulation of cardiovascular function remain poorly understood. BAs reduce heart rate by regulating channel conductance and calcium dynamics in sino-atrial and ventricular cardiomyocytes and regulate vascular tone via both endothelium-dependent and -independent mechanisms. End-stage liver disease, obstructive jaundice, and intrahepatic cholestasis of pregnancy are prominent conditions in which elevated serum BAs alter vascular dynamics. This review focuses on BAs as newly recognized signaling molecules that modulate cardiovascular function.
Assuntos
Ácidos e Sais Biliares/metabolismo , Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Animais , Membrana Celular/metabolismo , Frequência Cardíaca , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisAssuntos
Rim/irrigação sanguínea , Animais , Arteríolas/anatomia & histologia , Arteríolas/fisiologia , Transporte Biológico Ativo , Permeabilidade Capilar , Dissecação/métodos , Corantes Fluorescentes , Técnicas In Vitro , Microscopia de Vídeo , Perfusão/instrumentação , Perfusão/métodos , Vasoconstrição , Água/metabolismoRESUMO
Descending vasa recta (DVR) are 15-microm vessels that perfuse the renal medulla. Ouabain has been shown to augment DVR endothelial cytoplasmic Ca(2+) ([Ca(2+)](CYT)) signaling. In this study, we examined the expression of the ouabain-sensitive Na-K-ATPase alpha2 subunit in the rat renal vasculature and tested effects of acute ouabain exposure and chronic ouabain treatment on DVR. Immunostaining with antibodies directed against the alpha2 subunit verified its expression in both DVR pericytes and endothelium. Acute application of ouabain (100 or 500 nM) augmented the DVR nitric oxide generation stimulated by acetylcholine (ACh; 10 microM). At a concentration of 1 mM, ouabain constricted microperfused DVR, whereas at 100 nM, it was without effect. Acute ouabain (100 nM) did not augment constriction by angiotensin II (0.5 or 10 nM), whereas l-nitroarginine methyl ester-induced contraction of DVR was slightly enhanced. Ouabain-hypertensive (OH) rats were generated by chronic ouabain treatment (30 microg.kg(-1).day(-1), 5 wk). The acute endothelial [Ca(2+)](CYT) elevation by ouabain (100 nM) was absent in DVR endothelia of OH rats. The [Ca(2+)](CYT) response to 10 nM ACh was also eliminated, whereas the response to 10 microM ACh was not. The endothelial [Ca(2+)](CYT) response to bradykinin (100 nM) was significantly attenuated. We conclude that endothelial responses may offset the ability of acute ouabain exposure to enhance DVR vasoconstriction. Chronic exposure to ouabain, in vivo, leads to hypertension and DVR endothelial dysfunction, manifested as reduced [Ca(2+)](CYT) responses to both ouabain- and endothelium-dependent vasodilators.