Control of renal Na+ excretion by heme oxygenase.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
Thomas L Pallone
Publications and source records attributed to Thomas L Pallone.
Vasa recta voltage-gated Na+ channel Nav1.3 is regulated by calmodulin.
Rat descending vasa recta (DVR) express a tetrodotoxin (TTX)-sensitive voltage-operated Na(+) (Na(V)) conductance. We examined expression of Na(V) isoforms in DVR and tested for regulation of Na(V) currents by calmodulin (CaM). RT-PCR in isolated permeabilized DVR using degenerate primers targeted to TTX-sensitive isoforms amplified a product whose sequence identified only Na(V)1.3. Immunoblot of outer medullary homogenate verified Na(V)1.3 expression, and fluorescent immunochemistry showed Na(V)1.3 expression in isolated vessels. Immunochemistry in outer medullary serial sections confirmed that Na(V)1.3 is confined to alpha-smooth muscle actin-positive vascular bundles. Na(V)1.3 possesses a COOH-terminal CaM binding motifs. Using pull-down assays and immunoprecipitation experiments, we verified that CaM binds to either full-length Na(V)1.3 or a GST-Na(V)1.3 COOH-terminal fusion protein. In patch-clamp experiments, Na(V) currents were suppressed by calmodulin inhibitory peptide (CIP; 100 nM) or the CaM inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide hydrochloride (W7). Neither CIP nor W7 altered the voltage dependence of pericyte Na(V) currents; however, raising electrode free Ca(2+) from 20 to approximately 2,000 nM produced a depolarizing shift of activation. In vitro binding of CaM to GST-Na(V)1.3C was not affected by Ca(2+) concentration. We conclude that Na(V)1.3 is expressed by DVR, binds to CaM, and is regulated by CaM and Ca(2+). Inhibition of CaM binding suppresses pericyte Na(V) currents.
Animals↗
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.
Animals↗
Ouabain modulation of endothelial calcium signaling in descending vasa recta.
Using fura 2-loaded vessels, we tested whether ouabain modulates endothelial cytoplasmic calcium concentration ([Ca(2+)](CYT)) in rat descending vasa recta (DVR). Over a broad range between 10(-10) and 10(-4) M, ouabain elicited biphasic peak and plateau [Ca(2+)](CYT) elevations. Blockade of voltage-gated Ca(2+) entry with nifedipine did not affect the response to ouabain mitigating against a role for myo-endothelial gap junctions. Reduction of extracellular Na(+) concentration ([Na(+)](o)) or Na(+)/Ca(2+) exchanger (NCX) inhibition with SEA-0400 (10(-6) M) elevated [Ca(2+)](CYT), supporting a role for NCX in the setting of basal [Ca(2+)](CYT). SEA-0400 abolished the [Ca(2+)](CYT) response to ouabain implicating NCX as a mediator. The transient peak phase of [Ca(2+)](CYT) elevation that followed either ouabain or reduction of [Na(+)](o) was abolished by 2-aminoethoxydiphenyl borate (5 x 10(-5) M). Cation channel blockade with La(3+) (10 muM) or SKF-96365 (10 muM) also attenuated the ouabain-induced [Ca(2+)](CYT) response. Ouabain pretreatment increased the [Ca(2+)](CYT) elevation elicited by bradykinin (10(-7) M). We conclude that inhibition of ouabain-sensitive Na(+)-K(+)-ATPase enhances DVR endothelial Ca(2+) store loading and modulates [Ca(2+)](CYT) signaling through mechanisms that involve NCX, Ca(2+) release, and cation channel activation.
Animals↗
Vasa recta pericytes express a strong inward rectifier K+ conductance.
Strong inward rectifier potassium channels are expressed by some vascular smooth muscle cells and facilitate K+-induced hyperpolarization. Using whole cell patch clamp of isolated descending vasa recta (DVR), we tested whether strong inward rectifier K+ currents are present in smooth muscle and pericytes. Increasing extracellular K+ from 5 to 50 and 140 mmol/l induced inward rectifying currents. Those currents were Ba2+ sensitive and reversed at the K+ equilibrium potential imposed by the electrode and extracellular buffers. Ba2+ binding constants in symmetrical K+ varied between 0.24 and 24 micromol/l at -150 and -20 mV, respectively. Ba2+ blockade was time and voltage dependent. Extracellular Cs+ also blocked the inward currents with binding constants between 268 and 4,938 micromol/l at -150 and -50 mV, respectively. Ba2+ (30 micromol/l) and ouabain (1 mmol/l) depolarized pericytes by an average of 11 and 24 mV, respectively. Elevation of extracellular K+ from 5 to 10 mmol/l hyperpolarized pericytes by 6 mV. That hyperpolarization was reversed by Ba2+ (30 micromol/l). We conclude that strong inward rectifier K+ channels and Na+-K+-ATPase contribute to resting potential and that KIR channels can mediate K+-induced hyperpolarization of DVR pericytes.
Animals↗
Is oxidative stress differentially regulated in the renal cortex and medulla?
Explore the source record for details and available documents.
Animals↗
KATP channel conductance of descending vasa recta pericytes.
Using nystatin-perforated patch-clamp and whole cell recording, we tested the hypothesis that K(ATP) channels contribute to resting conductance of rat descending vasa recta (DVR) pericytes and are modulated by vasoconstrictors. The K(ATP) blocker glybenclamide (Glb; 10 microM) depolarized pericytes and inhibited outward currents of cells held at -40 mV. K(ATP) openers pinacidil (Pnc; 10 microM) and P-1075 (1 microM) hyperpolarized pericytes and transiently augmented outward currents. All effects of Pnc and P-1075 were fully reversed by Glb. Inward currents of pericytes held at -60 mV in symmetrical 140 mM K(+) were markedly augmented by Pnc and fully reversed by Glb. Ramp depolarizations in symmetrical K(+), performed in Pnc and Pnc + Glb, yielded a Pnc-induced, Glb-sensitive K(ATP) difference current that lacked rectification and reversed at 0 mV. Immunostaining identified both K(IR)6.1, K(IR)6.2 inward rectifier subunits and sulfonurea receptor subtype 2B. ANG II (1 and 10 nM) and endothelin-1 (10 nM) but not vasopressin (100 nM) significantly lowered holding current at -40 mV and abolished Pnc-stimulated outward currents. We conclude that DVR pericytes express K(ATP) channels that make a significant contribution to basal K(+) conductance and are inhibited by ANG II and endothelin-1.
ATP-Binding Cassette Transporters↗
Descending vasa recta pericytes express voltage operated Na+ conductance in the rat.
We studied the properties of a voltage-operated Na+ conductance in descending vasa recta (DVR) pericytes isolated from the renal outer medulla. Whole-cell patch-clamp recordings revealed a depolarization-induced, rapidly activating and rapidly inactivating inward current that was abolished by removal of Na+ but not Ca+ from the extracellular buffer. The Na+ current (I(Na)) is highly sensitive to tetrodotoxin (TTX, Kd = 2.2 nM). At high concentrations, mibefradil (10 microM) and Ni+ (1 mM) blocked I(Na). I(Na) was insensitive to nifedipine (10 microM). The L-type Ca+ channel activator FPL-64176 induced a slowly activating/inactivating inward current that was abolished by nifedipine. Depolarization to membrane potentials between 0 and 30 mV induced inactivation with a time constant of approximately 1 ms. Repolarization to membrane potentials between -90 and -120 mV induced recovery from inactivation with a time constant of approximately 11 ms. Half-maximal activation and inactivation occurred at -23.9 and -66.1 mV, respectively, with slope factors of 4.8 and 9.5 mV, respectively. The Na+ channel activator, veratridine (100 microM), reduced peak inward I(Na) and prevented inactivation. We conclude that a TTX-sensitive voltage-operated Na+ conductance, with properties similar to that in other smooth muscle cells, is expressed by DVR pericytes.
Animals↗
Microvascular effects of aldosterone and angiotensin type 2 receptors.
Explore the source record for details and available documents.
Aldosterone↗
Expression of TRPC4 channel protein that interacts with NHERF-2 in rat descending vasa recta.
The PDZ domain adaptor protein Na+/H+ exchanger regulatory factor (NHERF)-2 is expressed in renal medullary descending vasa recta (DVR), although its function has not been defined. Transient receptor potential channels (TRPC) TRPC4 and TRPC5, nonselective cation channels that transport Ca2+, were recently demonstrated to complex with the NHERF proteins. We investigated whether TRPC4 and/or TRPC5 are associated with NHERF-2 in DVR. RT-PCR revealed mRNA for TRPC4 and NHERF-2, but not for TRPC5 or NHERF-1, in microdissected DVR. Immunohistochemical studies demonstrated expression of TRPC4 and NHERF-2 proteins in both the endothelial cells and pericytes. These proteins colocalized in some cells of the DVR. TRPC4 coimmunoprecipitated with NHERF-2 from renal medullary lysates, and NHERF-2 coimmunoprecipitated with TRPC4. TRPC5 was not detected in DVR with the use of immunohistochemistry or in NHERF-2 immunoprecipitates. We conclude that DVR pericytes and endothelia coexpress TRPC4 and NHERF-2 mRNA and protein and that these proteins colocalize and coimmunoprecipitate, indicating a possible physical association. These findings suggest that TRPC4 and NHERF-2 may play a role in interactions related to Ca2+ signaling.
Animals↗
Chronic ANG II infusion increases NO generation by rat descending vasa recta.
We tested whether chronic ANG II infusion into rats affects descending vasa recta (DVR) contractility, synthesis of superoxide, or synthesis of nitric oxide (NO). Rats were infused with ANG II at 250 ng.kg(-1).min(-1) for 11-13 days. DVR were loaded with dihydroethidium (DHE) to measure superoxide and 3-amino-4-aminomethyl-2',7'-difluorofluorescein (DAFFM) to measure NO. Acute constriction of DVR by ANG II (0.1, 1, and 10 nM) was diminished, and NO generation rate was raised by chronic ANG II infusion. DHE oxidation by DVR from ANG II-infused rats was similar to controls and was significantly higher when NO synthesis was prevented with N(omega)-nitro-L-arginine methyl ester (L-NAME). The superoxide dismutase mimetic Tempol (1 mM) increased NO generation compared with controls. The increased synthesis of NO by chronic ANG II-treated vessels persisted in the presence of Tempol. DVR endothelial cytoplasmic Ca(2+) response to ACh was diminished by chronic ANG II treatment, but the capacity of ACh to increase NO generation was unaltered. We conclude that DVR generation of superoxide is not affected by chronic ANG II exposure but that basal NO synthesis is increased. DVR superoxide is unlikely to be an important mediator of chronic ANG II slow pressor hypertension in rats.
Acetylcholine↗
Inhibition of K+ conductance in descending vasa recta pericytes by ANG II.
We tested whether K(+) channel inhibition accompanies ANG II-induced depolarization of descending vasa recta (DVR) pericytes. An increase in extracellular K(+) concentration ([K(+)](o)) from 5 to 100 mM depolarized resting pericytes but had no effect after prolonged (10 nM, 20 min) ANG II exposure. In contrast, reduction of extracellular Cl(-) concentration ([Cl(-)](o)) from 154 to 34 mM had a minor effect on resting membrane potential but strongly depolarized pericytes treated with ANG II. The K(+) channel blockers BaCl(2) (0.1, 1 mM) and tetraethylammonium (TEA; 30 mM) depolarized resting pericytes but did not affect membrane potential of ANG II-treated pericytes. Pericyte whole cell currents were reduced by ANG II and nearly eliminated by combined ANG II exposure and the Cl(-) channel blocker niflumic acid (100 muM). Augmentation of inward current induced by raising [K(+)](O) from 5 to 50 mM was eliminated by preexposure to ANG II. TEA- and BaCl(2)-sensitive outward currents, generated by depolarizing pericytes from -80 to -40 mV, were eliminated by ANG II. We conclude that ANG II depolarizes DVR pericytes by a combination of Cl(-) channel activation and K(+) channel inhibition.
Angiotensin II↗
Response of descending vasa recta to luminal pressure.
We tested whether luminal perfusion and pressurization induce an endothelial cytoplasmic Ca(2+) ([Ca(2+)](CYT)) response in descending vasa recta (DVR). DVR isolated from the rat outer medulla were cannulated and subjected to free-flow microperfusion (5 nl/min); the onset of which increased [Ca(2+)](CYT) from a baseline of 76 +/- 13 to 221 +/- 65 nM. A graded increase in luminal pressure from 0 to 45 mmHg in stopped-flow experiments induced a parallel increase in [Ca(2+)](CYT) from a baseline of 74 +/- 24 to 194 +/- 33 nM at 45 mmHg, with a tendency for [Ca(2+)](CYT) to plateau at pressures >25 mmHg. The removal of extracellular Ca(2+) and blockade by either La(3+) (10 microM) or SKF-96365 (100 microM) eliminated the response. Luminal pressurization to 25 mmHg increased nitric oxide (NO) generation, a response blocked by NO synthase inhibition or removal of extracellular Ca(2+). The NO generation was not affected by the superoxide dismutase mimetic tempol. We conclude that DVR endothelia are mechanosensitive and respond to luminal pressure by elevating [Ca(2+)](CYT) and generating NO. That response might augment medullary perfusion and saliuresis.
Animals↗
ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species.
ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.
Angiotensin II↗
Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse.
In expression systems and in yeast, Na/H exchanger regulatory factor (NHERF)-1 and NHERF-2 have been demonstrated to interact with the renal brush border membrane proteins NHE3 and Npt2. In renal tissue of mice, however, NHERF-1 is required for cAMP regulation of NHE3 and for the apical targeting of Npt2 despite the presence of NHERF-2, suggesting another order of specificity. The present studies examine the subcellular location of NHERF-1 and NHERF-2 and their interactions with target proteins including NHE3, Npt2, and ezrin. The wild-type mouse proximal tubule expresses both NHERF-1 and NHERF-2 in a distinct pattern. NHERF-1 is strongly expressed in microvilli in association with NHE3, Npt2, and ezrin. Although NHERF-2 can be detected weakly in the microvilli, it is expressed predominantly at the base of the microvilli in the vesicle-rich domain. NHERF-2 appears to associate directly with ezrin and NHE3 but not Npt2. NHERF-1 is involved in the apical expression of Npt2 and the presence of other Npt2-binding proteins does not compensate totally for the absence of NHERF-1 in NHERF-1-null mice. Although NHERF-1 links NHE3 to the actin cytoskeleton through ezrin, the absence of NHERF-1 does not result in a generalized disruption of the architecture of the cell. Thus the mistargeting of Npt2 seen in NHERF-1-null mice likely represents a specific disruption of pathways mediated by NHERF-1 to achieve targeting of Npt2. These findings suggest that the organized subcellular distribution of the NHERF isoforms may play a role in the specific interactions mediating physiological control of transporter function.
Animals↗
Countercurrent exchange in the renal medulla.
The microcirculation of the renal medulla traps NaCl and urea deposited to the interstitium by the loops of Henle and collecting ducts. Theories have predicted that countercurrent exchanger efficiency is favored by high permeability to solute. In contrast to the conceptualization of vasa recta as simple "U-tube" diffusive exchangers, many findings have revealed surprising complexity. Tubular-vascular relationships in the outer and inner medulla differ markedly. The wall structure and transport properties of descending vasa recta (DVR) and ascending vasa recta (AVR) are very different. The recent discoveries of aquaporin-1 (AQP1) water channels and the facilitated urea carrier UTB in DVR endothelia show that transcellular as well as paracellular pathways are involved in equilibration of DVR plasma with the interstitium. Efflux of water across AQP1 excludes NaCl and urea, leading to the conclusion that both water abstraction and diffusion contribute to transmural equilibration. Recent theory predicts that loss of water from DVR to the interstitium favors optimization of urinary concentration by shunting water to AVR, secondarily lowering blood flow to the inner medulla. Finally, DVR are vasoactive, arteriolar microvessels that are anatomically positioned to regulate total and regional blood flow to the outer and inner medulla. In this review, we provide historical perspective, describe the current state of knowledge, and suggest areas that are in need of further exploration.
Animals↗
Physiology of the renal medullary microcirculation.
Perfusion of the renal medulla plays an important role in salt and water balance. Pericytes are smooth muscle-like cells that impart contractile function to descending vasa recta (DVR), the arteriolar segments that supply the medulla with blood flow. DVR contraction by ANG II is mediated by depolarization resulting from an increase in plasma membrane Cl(-) conductance that secondarily gates voltage-activated Ca(2+) entry. In this respect, DVR may differ from other parts of the efferent microcirculation of the kidney. Elevation of extracellular K(+) constricts DVR to a lesser degree than ANG II or endothelin-1, implying that other events, in addition to membrane depolarization, are needed to maximize vasoconstriction. DVR endothelial cytoplasmic Ca(2+) is increased by bradykinin, a response that is inhibited by ANG II. ANG II inhibition of endothelial Ca(2+) signaling might serve to regulate the site of origin of vasodilatory paracrine agents generated in the vicinity of outer medullary vascular bundles. In the hydropenic kidney, DVR plasma equilibrates with the interstitium both by diffusion and through water efflux across aquaporin-1. That process is predicted to optimize urinary concentration by lowering blood flow to the inner medulla. To optimize urea trapping, DVR endothelia express the UT-B facilitated urea transporter. These and other features show that vasa recta have physiological mechanisms specific to their role in the renal medulla.
Animals↗
Microdissected perfused vessels.
Explore the source record for details and available documents.
Animals↗