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Human coronaviruses (HCoVs) modify host proteins to evade the antiviral defense and sustain viral expansion. Here, we report tonicity-responsive enhancer (TonE) binding protein (TonEBP) as a cellular target of HCoVs. TonEBP was cleaved into N-terminal and C-terminal fragments (TonEBP NT and TonEBP CT, respectively) by NSP5 from all the HCoVs tested. This cleavage resulted in the loss of TonEBP's ability to stimulate the TonE-driven transcription. On the other hand, TonEBP NT promoted viral expansion in association with the suppression of IFN-β expression. TonEBP NT competed away NF-κB binding to the PRD II domain on the IFN-β promoter. A TonEBP mutant resistant to the cleavage by NSP5 did not promote the viral expansion nor suppress the IFN-β expression. These results demonstrate that HCoVs use a common strategy of targeting TonEBP to suppress the host immune defense. [ABSTRACT FROM AUTHOR]

The phenotypic and functional plasticity of adipose tissue macrophages (ATMs) during obesity plays a crucial role in orchestration of adipose and systemic inflammation. Tonicity-responsive enhancer binding protein (TonEBP) (also called NFAT5) is a stress protein that mediates cellular responses to a range of metabolic insults. Here, we show that myeloid cell-specific TonEBP depletion reduced inflammation and insulin resistance in mice with high-fat diet-induced obesity but did not affect adiposity. This phenotype was associated with a reduced accumulation and a reduced proinflammatory phenotype of metabolically activated macrophages, decreased expression of inflammatory factors related to insulin resistance, and enhanced insulin sensitivity. TonEBP expression was elevated in the ATMs of obese mice, and Sp1 was identified as a central regulator of TonEBP induction. TonEBP depletion in macrophages decreased induction of insulin resistance-related genes and promoted induction of insulin sensitivity-related genes under obesity-mimicking conditions and thereby improved insulin signaling and glucose uptake in adipocytes. mRNA expression of TonEBP in peripheral blood mononuclear cells was positively correlated with blood glucose levels in mice and humans. These findings suggest that TonEBP in macrophages promotes obesity-associated systemic insulin resistance and inflammation, and downregulation of TonEBP may induce a healthy metabolic state during obesity. [ABSTRACT FROM AUTHOR]

Descending vasa recta (DVR) are 15-[micro]m vessels that perfuse the renal medulla. Ouabain has been shown to augment DVR endothelial cytoplasmic [Ca.sup.2+] ([[[Ca.sup.2+]].sub.CYT]) signaling. In this study, we examined the expression of the ouabain-sensitive Na-K-ATPase [alpha]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 [alpha]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 [micro]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 ester-induced contraction of DVR was slightly enhanced. Ouabain-hypertensive (OH) rats were generated by chronic ouabain treatment (30 [micro]g x [kg.sup.-1] x [day.sup.-1], 5 wk). The acute endothelial [[Ca.sup.2+]].sub.CYT] elevation by ouabain (100 nM) was absent in DVR endothelia of OH rats. The [[Ca.sup.2+]].sub.CYT] response to 10 nM ACh was also eliminated, whereas the response to 10 [micro]M ACh was not. The endothelial [[Ca.sup.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.sup.2+]].sub.CYT] responses to both ouabain- and endothelium-dependent vasodilators. kidney; medulla; microcirculation; nitric oxide; blood flow

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 (Kin) exist in the endothelia, affect membrane potential, and modulate intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.cyt]). The endothelium was accessed for electrophysiology by removing abluminal pericytes from collagenase-digested vessels. [K.sub.IR] currents were recorded using symmetrical 140 mM [K.sup.+] 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.sup.+]. [Ba.sup.2+] potently inhibited those currents in a voltage- dependent manner, with affinity k = 0.18, 0.33, 0.60, and 1.20 [micro]M at -160, -120, -80, and -40 mV, respectively. [Cs.sup.+] also blocked those currents with k = 20, 48, 253, and 1,856 [micro]M at -160, -120, -80, and -40 mV, respectively. In the presence of 1 mM ouabain, increasing extracellular [K.sup.+] concentration from 5 to 10 mM hyperpolarized endothelial membrane potential by 15 mV and raised endothelial [[[Ca.sup.2+]].sub.cyt]. Both the [K.sup.+]-induced membrane hyperpolarization and the [[[Ca.sup.2+]].sub.cyt] elevation were reversed by [Ba.sup.2+]. Immunochemical staining verified that both pericytes and endothelial cells of DVR express [K.sub.IR]2.1, [K.sub.IR]2.2, and [K.sub.IR]2.3 subunits. We conclude that strong, inwardly rectifying [K.sub.IR]2.x isoforms are expressed in DVR and mediate [K.sup.+]-induced hyperpolarization of the endothelium. kidney; medulla; microcirculation; electrophysiology; potassium channel; endothelium

Rat descending vasa recta (DVR) express a tetrodotoxin (TTX)-sensitive voltage-operated [Na.sup.+]([Na.sub.v]) conductance. We examined expression of [Na.sub.v] isoforms in DVR and tested for regulation of [Na.sub.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.sub.v] 1.3. Immunoblot of outer medullary homogenate verified [Na.sub.v] 1.3 expression, and fluorescent immunochemistry showed [Na.sub.v] 1.3 expression in isolated vessels. Immunochemistry in outer medullary serial sections confirmed that [Na.sub.v] 1.3 is confined to [alpha]-smooth muscle actin-positive vascular bundles. [Na.sub.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.sub.v] 1.3 or a GST-[Na.sub.v] 1.3 COOH-terminal fusion protein. In patch-clamp experiments, [Na.sub.v] currents were suppressed by calmodulin inhibitory peptide (CIP; 100 nM) or the CaM inhibitor N-(6-aminohexyl)-5-chloro-l-naphthalene-sulphonamide hydrochloride (W7). Neither CIP nor W7 altered the voltage dependence of pericyte [Na.sub.v] currents; however, raising electrode free C[A.sub.2]+ from 20 to -2,000 nM produced a depolarizing shift of activation. In vitro binding of CaM to GST-[Na.sub.v] 1.3C was not affected by C[A.sub.2]+ concentration. We conclude that [Na.sub.v] 1.3 is expressed by DVR, binds to CaM, and is regulated by CaM and [Ca.sup.2+]. Inhibition of CaM binding suppresses pericyte [Na.sub.v] currents. kidney; medulla; microcirculation; patch clamp; channel

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 [micro]M) and 18[beta]-glycyrrhetinic acid (18[beta]GRA; 100 [micro]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 [DiBA[C.sub.4](3)] was monitored ~200 [micro]m away. A 40-mV endothelial depolarization was accompanied by a 26.1 [+ or -] 5.5-mV change in DiBA[C.sub.4](3) fluorescence. DiBA[C.sub.4](3) fluorescence did not change after 18[beta]GRA or when pericytes were depolarized. Similarly, propagated cytoplasmic [Ca.sup.2+] responses arising from mechanical perturbation of the DVR wall were attenuated by 18[beta]GRA 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. rat; kidney; medulla; microcirculation; electrophysiology; blood flow

Strong inward rectifier potassium channels are expressed by some vascular smooth muscle cells and facilitate [K.sup.+]-induced hyperpolarization. Using whole cell patch clamp of isolated descending vasa recta (DVR), we tested whether strong inward rectifier [K.sup.+] currents are present in smooth muscle and pericytes. Increasing extracellular [K.sup.+] from 5 to 50 and 140 mmol/l induced inward rectifying currents. Those currents were [Ba.sup.2+] sensitive and reversed at the [K.sup.+] equilibrium potential imposed by the electrode and extracellular buffers. [Ba.sup.2+] binding constants in symmetrical [K.sup.+] varied between 0.24 and 24 [micro]mol/l at -150 and -20 mV, respectively. [Ba.sup.2+] blockade was time and voltage dependent. Extracellular [Cs.sup.+] also blocked the inward currents with binding constants between 268 and 4,938 [micro]mol/l at -150 and -50 mV, respectively. [Ba.sup.2+] (30 [micro]mol/l) and ouabain (1 mmol/l) depolarized pericytes by an average of 11 and 24 mV, respectively. Elevation of extracellular [K.sup.+] from 5 to 10 mmol/l hyperpolarized pericytes by 6 mV. That hyperpolarization was reversed by [Ba.sup.2+] (30 [micro] mol/l). We conclude that strong inward rectifier [K.sup.+] channels and [Na.sup.+]-[K.sup.+]-ATPase contribute to resting potential and that [K.sub.IR] channels can mediate [K.sup.+]-induced hyperpolarization of DVR pericytes. kidney; medulla; microcirculation; electrophysiology; potassium channel

Using nystatin-perforated patch-clamp and whole cell recording, we tested the hypothesis that KATP channels contribute to resting conductance of rat descending vasa recta (DVR) pericytes and are modulated by vasoconstrictors. The KATP blocker glybenclamide (Glb; 10 [micro]M) depolarized pericytes and inhibited outward currents of cells held at -40 mV. KATP openers pinacidil (Pnc; 10 [micro]M) and P-1075 (1 [micro]M) 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.sup.+] were markedly augmented by Pnc and fully reversed by Glb. Ramp depolarizations in symmetrical [K.sup.+], performed in Pnc and Pnc + Glb, yielded a Pnc-induced, Glb-sensitive KATP difference current that lacked rectification and reversed at 0 mV. Immunostaining identified both KIR6.1, KIR6.2 inward rectifier subunits and sulfonurea receptor subtype 2B. ANG II (1 and 10 nM) and endothe1in-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 KATP channels that make a significant contribution to basal [K.sup.+] conductance and are inhibited by ANG II and endothelin-1. kidney; medulla; microcirculation; electrophysiology; vasoconstriction

The PDZ domain adaptor protein [Na.sup.+]/[H.sup.+] 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 [Ca.sup.2+], 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 [Ca.sup.2+] signaling. PDZ proteins; calcium channels; medulla; pericytes; endothelium; microcirculation

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 x [kg.sup.-1] x [min.sup.-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.sup.[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.sup.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. medulla; kidney; microcirculation; calcium; oxidative stress

The intestinal brush border (BB) [Na.sup.+]/[H.sup.+] exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) by the cholinergic agonist carbachol and [Ca.sup.2+] ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [[[Ca.sup.2+]].sub.i] with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in [Ca.sup.2+] regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the [Ca.sup.2+]-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Go-6976 and a specific PKC[alpha] pseudosubstrate-derived inhibitor peptide. [[[Ca.sup.2+]].sub.i] elevation caused translocation of PKC[alpha] from cytosol to membrane. PKC[alpha] bound to the PDZ1 domain of GST-E3KARP in vitro in a [Ca.sup.2+]-dependent manner. PKC[alpha] and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [[[Ca.sup.2+]].sub.i] and was increased with ionomycin exposure. Biotinylation studies demonstrated that [[[Ca.sup.2+]].sub.i] elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKC[alpha] is not necessary for the [Ca.sup.2+]-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis. Na absorption; PDZ domains; signal complex

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. NHE3; Npt2; ezrin; PDZ domains; immunolocalization

Research has been conducted on the cytoplasmic domain of the insulin receptor (IR) beta-subunit. The ability of the cross-linking reagent 1,6-bismaleimidohexane to link covalently IR with interacting proteins is examined.

A study was conducted to examine the significance of various insulin receptor cytoplasmic domains in the antiapoptotic capacity of insulin, as well as the signal transduction pathways involved in this action of insulin. Findings indicated that the antiapoptotic action of insulin is dependent upon intact insulin receptor kinase activity. It was also shown that treatment with orthovanadate mimicked the ability of insulin to exert antiapoptotic function while triggering activation of the p38 monoamine phosphate kinase cascade.