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 M) and 18-glycyrrhetinic acid (18GRA; 100 M) 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 [DiBAC4(3)] was monitored ϳ200 m away. A 40-mV endothelial depolarization was accompanied by a 26.1 Ϯ 5.5-mV change in DiBAC4(3) fluorescence. DiBAC4(3) fluorescence did not change after 18GRA or when pericytes were depolarized. Similarly, propagated cytoplasmic Ca 2ϩ responses arising from mechanical perturbation of the DVR wall were attenuated by 18GRA or heptanol. Connexin (Cx) immunostaining showed predominant linear Cx40 and Cx43 in endothelia, whereas Cx37 stained smooth muscle actinpositive pericytes. We conclude that the DVR endothelium is an electrical syncytium and that gap junction coupling in DVR pericytes exists but is less pronounced.
([Ca 2ϩ ]CYT) signaling. In this study, we examined the expression of the ouabain-sensitive Na-K-ATPase ␣2 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 ␣2 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 M). 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 esterinduced contraction of DVR was slightly enhanced. Ouabain-hypertensive (OH) rats were generated by chronic ouabain treatment (30 g ⅐ kg Ϫ1 ⅐ day Ϫ1 , 5 wk). The acute endothelial [Ca 2ϩ ]CYT elevation by ouabain (100 nM) was absent in DVR endothelia of OH rats. The [Ca 2ϩ ]CYT response to 10 nM ACh was also eliminated, whereas the response to 10 M ACh was not. The endothelial [Ca 2ϩ ]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 2ϩ ]CYT responses to both ouabain-and endothelium-dependent vasodilators. kidney; medulla; microcirculation; nitric oxide; blood flow "OUABAIN-LIKE FACTORS" (OLF), synthesized by the adrenal gland and hypothalamus, inhibit Na ϩ /K ϩ exchange (19, 46) and activate signaling cascades (6, 7, 44) by binding to Na-KATPase ␣-subunits. In rodents, the ␣1 isoform of Na-KATPase that maintains Na ϩ and K ϩ gradients across cell membranes has very low affinity (K d Ͼ 10 M) for ouabain. In contrast, the ␣2 and ␣3 isoforms have high affinity for ouabain (K d Ͻ 50 nM), but are less abundantly expressed (3, 48). It has been proposed that targeting of the ␣2 isoform to cellular microdomains where ER/SR protrusions abut the plasma membrane (4, 7) may modulate intracellular Na ϩ and reduce Ca 2ϩ extrusion via Na ϩ /Ca 2ϩ exchange to enhance Ca 2ϩ sequestration into ER/SR stores. Evidence favoring that hypothesis has been accumulating. Low-dose (10 -100 nM) ouabain enhances Ca 2ϩ release in smooth muscle and endothelium (2, 35) and increases resting cytoplasmic Ca 2ϩ ([Ca 2ϩ ] CYT ) and myogenic tone (35,48).A role for OLF in hypertension is also well supported. Chronic administration of ouabain into rodents induces hypertension (26, 28), and many patients with essential hypertension have high plasma ouabain levels (43). Transgenic mice in which the ␣2 Na-K-ATPase binding site for ouabain has been mutated are resistant to both ouabain-and ACTH-induced hypertension, supporting a causal role in hypertension for both ouabain and the ␣2 ouabain receptor (12, 13).Detailed mechanisms by which ouabain induces hypertension remain to be elucidated. ...
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.
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 ([Ca²⁺]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 [Ca²⁺)]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 β-glycyrrhetinic acid (100 μM) or phosphoinositide 3 kinase inhibition with 2-morpholin-4-yl-8-phenylchromen-4-one (50 μM). [Ca²⁺]CYT responses were blocked by removal of extracellular Ca²⁺ 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 Ca²⁺ channels, and pericyte depolarization.
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.
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