K(V)7.4 channels participate in the control of rodent renal vascular resting tone.

AIM: We tested the hypothesis that K(V)7 channels contribute to basal renal vascular tone and that they participate in agonist-induced renal vasoconstriction or vasodilation. METHODS: KV 7 channel subtypes in renal arterioles were characterized by immunofluorescence. Renal blood flow (RBF) was measured using an ultrasonic flow probe. The isometric tension of rat interlobar arteries was examined in a wire myograph. Mice afferent arteriolar diameter was assessed utilizing the perfused juxtamedullary nephron technique. RESULTS: Immunofluorescence revealed that K(V)7.4 channels were expressed in rat afferent arterioles. The K(V)7 blocker XE991 dose-dependently increased the isometric tension of rat interlobar arteries and caused a small (approx. 4.5%) RBF reduction in vivo. Nifedipine abolished these effects. Likewise, XE991 reduced mouse afferent arteriolar diameter by approx. 5%. The K(V)7.2-5 stimulator flupirtine dose-dependently relaxed isolated rat interlobar arteries and increased (approx. 5%) RBF in vivo. The RBF responses to NE or Ang II administration were not affected by pre-treatment with XE991 or flupirtine. XE991 pre-treatment caused a minor augmentation of the acetylcholine-induced increase in RBF, while flupirtine pre-treatment did not affect this response. CONCLUSION: It is concluded that K(V)7 channels, via nifedipine sensitive channels, have a role in the regulation of basal renal vascular tone. There is no indication that K(V)7 channels have an effect on agonist-induced renal vasoconstriction while there is a small effect on acetylcholine-induced vasodilation.

Endothelial nitric oxide synthase phosphorylation at Threonine 495 and mitochondrial reactive oxygen species formation in response to a high H2O2 concentration.

BACKGROUND: Hydrogen peroxide (H2O2) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr(495)) in human umbilical vein endothelial cells (HUVECs) exposed to H2O2. METHODS: HUVECs were exposed to 400 µM H2O2 for 30 min. Phosphorylation at Thr(495) was assessed by Western blotting and reactive oxygen species (ROS) monitored by flow cytometry. Protein kinase C (PKC) pathways were investigated by pretreatment with PKC-ß inhibitor ruboxistaurin or pan-PKC inhibitor GF109203X. In addition, we investigated ROCK and ERK pathways by MEKK1/2 inhibitor U0126 and ROCK inhibitor Y27632. RESULTS: H2O2 increased eNOS phosphorylation at Thr(495) (to 176% vs. control (100%), p < 0.001) along with increased mitochondrial ROS formation (from 19.7 to 45.3%, p < 0.01). This rise in phosphorylation could be prevented by U0126 and Y27632 in a dose-dependent manner, but did not result in lowered mitochondrial ROS formation. Conversely, addition of the antioxidant N-acetyl-L-cysteine only prevented mitochondrial ROS formation but did not prevent phosphorylation of eNOS Thr(495). CONCLUSION: H2O2-mediated phosphorylation of eNOS Thr(495) is mediated by ROCK and ERK activity, but not by PKC, and is uncoupled from mitochondrial ROS signaling. Furthermore, ERK inhibition increased mitochondrial ROS formation.

Mechanisms of K(+) induced renal vasodilation in normo- and hypertensive rats in vivo.

AIM: We investigated the mechanisms behind K(+) -induced renal vasodilation in vivo in normotensive Sprague-Dawley (SD) rats and spontaneously hypertensive rats (SHR). METHODS: Renal blood flow (RBF) was measured utilizing an ultrasonic Doppler flow probe. Renal vascular resistance (RVR) was calculated as the ratio of mean arterial pressure (MAP) and RBF (RVR = MAP/RBF). Test drugs were introduced directly into the renal artery. Inward rectifier K(+) (K(ir) ) channels and Na(+) ,K(+) -ATPase were blocked by Ba(2+) and ouabain (estimated plasma concentrations ∼20 and ∼7 µm) respectively. RESULTS: Confocal immunofluorescence microscopy demonstrated K(ir) 2.1 channels in pre-glomerular vessels of SD and SHR. Ba(2+) caused a transient (6-13%) increase in baseline RVR in both SD and SHR. Ouabain had a similar effect. Elevated renal plasma [K(+) ] (∼12 mm) caused a small and sustained decrease (5-13%) in RVR in both strains. This decrease was significantly larger in SHR than in SD. The K(+) -induced vasodilation was attenuated by Ba(2+) in control SD and SHR and by ouabain in SD. Nitric oxide (NO) blockade using l-NAME treatment increased MAP and decreased RBF in both rat strains, but did not affect the K(+) -induced renal vasodilation. CONCLUSION: K(+) -induced renal vasodilation is larger in SHR, mediated by K(ir) channels in SD and SHR, and in addition, by Na(+) ,K(+) -ATPase in SD. In addition, NO is not essential for K(+) -induced renal vasodilation.

Na+-independent, nifedipine-resistant rat afferent arteriolar Ca2+ responses to noradrenaline: possible role of TRPC channels.

AIM: In rat afferent arterioles we investigated the role of Na(+) entry in noradrenaline (NA)-induced depolarization and voltage-dependent Ca(2+) entry together with the importance of the transient receptor potential channel (TRPC) subfamily for non-voltage-dependent Ca(2+) entry. METHODS: R (340/380) Fura-2 fluorescence was used as an index for intracellular free Ca(2+) concentration ([Ca(2+)](i)). Immunofluorescence detected the expression of TRPC channels. RESULTS: TRPC 1, 3 and 6 were expressed in afferent arteriolar vascular smooth muscle cells. Under extracellular Na(+)-free (0 Na) conditions, the plateau response to NA was 115% of the baseline R(340/380) (control response 123%). However, as the R(340/380) baseline increased (7%) after 0 Na the plateau reached the same level as during control conditions. Similar responses were obtained after blockade of the Na(+)/Ca(2+) exchanger. The L-type blocker nifedipine reduced the plateau response to NA both under control (from 134% to 116% of baseline) and 0 Na conditions (from 112% to 103% of baseline). In the presence of nifedipine, the putative TRPC channel blockers SKF 96365 (30 µm) and Gd(3+) (100 µm) further reduced the plateau Ca(2+) responses to NA (from 117% to 102% and from 117% to 110% respectively). CONCLUSION: We found that Na(+) is not crucial for the NA-induced depolarization that mediates Ca(2+) entry via L-type channels. In addition, the results are consistent with the idea that TRPC1/3/6 Ca(2+) -permeable cation channels expressed in afferent arteriolar smooth muscle cells mediate Ca(2+) entry during NA stimulation.

SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis.

Post-transcriptional gene silencing (PTGS) provides protection in plants against virus infection and can suppress expression of transgenes. Arabidopsis plants carrying mutations at the SDE3 locus are defective in PTGS mediated by a green fluorescent protein transgene. However, PTGS mediated by tobacco rattle virus (TRV) was not affected by sde3. From these results we conclude that SDE3, like the previously described RNA polymerase encoded by SDE1, acts at a stage in the mechanism that is circumvented when PTGS is mediated by TRV. The product of SDE3 is similar to RNA helicase-like proteins including GB110 in mouse and other proteins in Drosophila and humans. These proteins are similar to, but clearly distinct from Upf1p and SMG-2, which are required for nonsense-mediated mRNA decay in yeast and Caenorhabditis elegans and, in the case of SMG-2, for PTGS.

Identification of a new promoter in the early region of the human papillomavirus type 16 genome.

Transcription of the human papillomavirus type 16 (HPV-16) genome is controlled by several promoters; the P(97) promoter is considered to be the main one. An additional promoter has been identified within the E7 ORF as well as an antisense promoter just upstream of the L2 ORF. The significance of these promoters for early and late gene expression and their activity related to cell differentiation is not known in detail. Identification of two new, previously undescribed transcription start sites at nt 542 just upstream of the E7 ORF and at nt 611 within the E7 ORF is reported. The promoter responsible for the start site at nt 542 (P(542)) was active in SiHa, HeLa and C33A cells. Very low promoter activity was found upstream of the nt 611 start site. The E7 protein has previously been shown to be synthesized from a polycistronic mRNA encoding both the E6 and E7 proteins under the control of the P(97) promoter. The data reported in the present paper suggest that promoter P(542) may control synthesis of the E7 oncoprotein from a monocistronic mRNA.