Adaptive responses of rat descending vasa recta to ischemia.

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.

Descending Vasa Recta Endothelial Membrane Potential Response Requires Pericyte Communication.

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.

Syncytial communication in descending vasa recta includes myoendothelial coupling.

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.

Descending vasa recta endothelial cells and pericytes form mural syncytia.

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.

Mural propagation of descending vasa recta responses to mechanical stimulation.

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 ([Ca²âº]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 [Ca²âº)]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). [Ca²âº]CYT responses were blocked by removal of extracellular Ca²âº 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 Ca²âº channels, and pericyte depolarization.

Chronic ouabain treatment induces vasa recta endothelial dysfunction in the rat.

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.

Descending vasa recta endothelia express inward rectifier potassium channels.

Descending vasa recta (DVR) are capillary-sized microvessels that supply blood flow to the renal medulla. They are composed of contractile pericytes and endothelial cells. In this study, we used the whole cell patch-clamp method to determine whether inward rectifier potassium channels (K(IR)) exist in the endothelia, affect membrane potential, and modulate intracellular Ca(2+) concentration ([Ca(2+)](cyt)). The endothelium was accessed for electrophysiology by removing abluminal pericytes from collagenase-digested vessels. K(IR) currents were recorded using symmetrical 140 mM K(+) solutions that served to maximize currents and eliminate cell-to-cell coupling by closing gap junctions. Large, inwardly rectifying currents were observed at membrane potentials below the equilibrium potential for K(+). Ba(2+) potently inhibited those currents in a voltage-dependent manner, with affinity k = 0.18, 0.33, 0.60, and 1.20 microM at -160, -120, -80, and -40 mV, respectively. Cs(+) also blocked those currents with k = 20, 48, 253, and 1,856 microM at -160, -120, -80, and -40 mV, respectively. In the presence of 1 mM ouabain, increasing extracellular K(+) concentration from 5 to 10 mM hyperpolarized endothelial membrane potential by 15 mV and raised endothelial [Ca(2+)](cyt). Both the K(+)-induced membrane hyperpolarization and the [Ca(2+)](cyt) elevation were reversed by Ba(2+). Immunochemical staining verified that both pericytes and endothelial cells of DVR express K(IR)2.1, K(IR)2.2, and K(IR)2.3 subunits. We conclude that strong, inwardly rectifying K(IR)2.x isoforms are expressed in DVR and mediate K(+)-induced hyperpolarization of the endothelium.

Descending vasa recta endothelium is an electrical syncytium.

We examined gap junction coupling of descending vasa recta (DVR). DVR endothelial cells or pericytes were depolarized to record the associated capacitance transients. Virtually all endothelia and some pericytes exhibited prolonged transients lasting 10-30 ms. Carbenoxolone (100 microM) and 18beta-glycyrrhetinic acid (18betaGRA; 100 microM) markedly shortened the endothelial transients. Carbenoxolone and heptanol (2 mM) reduced the pericyte capacitance transients when they were prolonged. Lucifer yellow (LY; 2 mM) was dialyzed into the cytoplasm of endothelial cells and pericytes. LY spread diffusely along the endothelial monolayer, whereas in most pericytes, it was confined to a single cell. In some pericytes, complex patterns of LY spreading were observed. DVR cells were depolarized by voltage clamp as fluorescence of bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)] was monitored approximately 200 microm away. A 40-mV endothelial depolarization was accompanied by a 26.1 +/- 5.5-mV change in DiBAC(4)(3) fluorescence. DiBAC(4)(3) fluorescence did not change after 18betaGRA or when pericytes were depolarized. Similarly, propagated cytoplasmic Ca(2+) responses arising from mechanical perturbation of the DVR wall were attenuated by 18betaGRA or heptanol. Connexin (Cx) immunostaining showed predominant linear Cx40 and Cx43 in endothelia, whereas Cx37 stained smooth muscle actin-positive pericytes. We conclude that the DVR endothelium is an electrical syncytium and that gap junction coupling in DVR pericytes exists but is less pronounced.