Membrane current oscillations in descending vasa recta pericytes

Abstract

We investigated the origin of spontaneous transient inward current (STIC) oscillations in descending vasa recta (DVR) pericytes. In cells clamped at Ϫ80 mV, angiotensin II (ANG II; 10 nmol/l) induced oscillations with mean amplitude and frequency of Ϫ65.5 pA and 1. METHODS Isolation of DVR.Investigations involving animal use were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland. Kidneys were harvested from Sprague-Dawley rats (100 -150 g; Harlan) that had been anesthetized by an intraperitoneal injection of ketamine/ xylazine (80 mg/kg; 10 mg/kg). Tissue slices were stored at 4°C in a physiological saline solution (PSS; in mM: 155 NaCl, 5 KCl, 1 MgCl 2, 1 CaCl2, 10 HEPES, and 10 glucose, pH 7.4). Small wedges of renal medulla were dissected and transferred to Blendzyme 1 (Roche) at 0.27 mg/ml in high-glucose DMEM media (Invitrogen), incubated at 37°C for 45 min whereupon they were transferred to PSS and stored at 4°C. At intervals, DVR were isolated from the enzymedigested renal tissue and transferred to a perfusion chamber for patch-clamp recording.Whole cell patch-clamp recording. Patch pipettes were made from borosilicate glass capillaries (PG52151-4, World Precision Instruments, Sarasota, FL), using a two-stage vertical pipette puller (Narshige PP-830) and heat polished. For whole cell patch-clamp recording, the pipette solution was (in mM) 120 Kaspartate, 20 KCl, 10 NaCl, 10 HEPES, pH 7.2, with or without nystatin (100 g/ml with 0.1% DMSO) in ultrapure water. Measurements were obtained as previously described (35). Briefly, most membrane currents were measured using a CV201AU headstage and Axopatch 200 amplifier (Axon Instruments, Foster City, CA) at 10 Hz using 8-to 10-M⍀ pipettes and nystatin patches. In experiments where simultaneous recording of [Ca 2ϩ ]CYT variation and whole cell current was performed, the ruptured patch method was substituted for nystatin so that fluo-4 could diffuse into the pericyte cytoplasm.Fluorescent recording of [Ca 2ϩ ]CYT. To record [Ca 2ϩ ]CYT variations of DVR pericytes, the Ca 2ϩ -sensitive fluorescent probe, fluo-4 (Molecular Probes), was either included in the electrode buffer as the pentapotassium salt (50 M, patch-clamp recordings) or loaded into isolated DVR by incubation with the AM ester (10 mol/l, 20 min, 37°C, confocal microscopy). During patch-clamp experiments, the probe was excited at 485 nm (DeltaRam, PTI) and emissions were captured at 530 nm (B-2E/C filter cube, Nikon) using a ϫ40 CF fluor oil immersion objective, 1.3 numerical aperture. The image of the patched fluorescent pericyte was directed to a D104B photon counting photomultiplier detection assembly (PTI) set to operate in analog mode. In other experiments, to simultaneously visualize [Ca 2ϩ ]CYT transients in pericytes and endothelia and to test for their occurrence in the absence of voltage clamp, isolated, fluo-4-loaded DVR adherent to the bottom of a recording chamber were visualized by laserscanning confocal micro...

Mechanisms underlying angiotensin II-induced calcium oscillations

“…C: overall current, calculated as the sum of the currents crossing the cytosol and the microdomains, and the leak current (taken as Ϫ8 pA, assuming a 10-G⍀ seal). agonist (ANG II) stimulation (36,52,56) and KCl depolarization (36, 56) when IP 3 R and RyR stores are separated. Other authors have investigated the minimal model requirements for generation of oscillations and concluded that RyR participation is not a requisite feature (4, 16).…”

Chronic ouabain treatment induces vasa recta endothelial dysfunction in the rat

“…Accordingly, we suspected endothelial expression of a ouabain-sensitive isoform in rat vasa recta because ouabain, in nanomolar concentrations, has clear effects on DVR endothelial Ca 2ϩ content and concentration (35). In addition, basal [Ca 2ϩ ] CYT and the frequency of spontaneous transient inward currents attributable to the action of Ca 2ϩ on Ca 2ϩ -dependent chloride channels are increased by ouabain in DVR pericytes (49). Given the important roles of the ␣2 isoform in the extrarenal vasculature, we explored its expression using a subtype-specific antibody.…”

“…Tissue, whether enzymatically digested or undigested, was then transferred to microperfusion buffer (in mM): 140 NaCl, 10 NaAcetate, 5 KCl, 1.2 MgCl 2, 2 Na2HPO4/ NaH 2PO4, 1 CaCl2, 5 alanine, 5 glucose, 5 HEPES, and 0.5 g/dl albumin, pH 7.4, and stored at 4°C. At intervals, DVR were isolated and either transferred to microscope slides for fixation and immunostaining (23,24) or to the stage of an inverted microscope for microperfusion and fluorescence measurements (32,35,49).…”

“…We previously showed that nanomolar ouabain raises both pericyte (49) and endothelial (35) [Ca 2ϩ ] CYT . Given that those effects might offset one another to favor either net vasoconstriction or dilation, we tested the ability of ouabain to contract microperfused DVR from normal rats.…”

“…Acute application of ouabain to a microvessel, ex vivo, can increase [Ca 2ϩ ] CYT concentrations and induce contraction (48,49). In contrast, the chronic administration of ouabain to rodents typically requires many 1-2 wk to fully induce hypertension (19,28).…”