Biomedical subjects

W H Wang

Publications and source records attributed to W H Wang.

At least 19 recordsLinked to original sources

PubMed2001

Optimal procedure for extracting RNA from human ocular tissues and expression profiling of the congenital glaucoma gene FOXC1 using quantitative RT-PCR.

PURPOSE: To develop methods for obtaining high quality RNA from human donor eyes and to determine the expression profile of the congenital glaucoma gene FOXC1 in human ocular tissues. METHODS: To obtain high quality RNA from donor eyes, several different preservation methods were tested including storing eyes on ice, freezing eyes, and placing eyes in the commercial fixative RNAlaterTM prior to dissection and RNA extraction. Nine different ocular tissues from human donors were dissected and examined. Pigment-free total RNA was isolated and used for quantitative real-time RT-PCR using FOXC1 and GAPDH (internal standard) primers to assess the quality and expression of FOXC1. RESULTS: An expression profile of FOXC1 in human ocular tissues was determined using quantitative PCR of RNA isolated using a simple and effective procedure for ocular tissue preservation and pigment-free RNA isolation. Higher quality RNA was obtained from human donor eyes preserved in RNAlaterTM compared to RNA extracted from eyes stored on ice or frozen at -80 degrees C. RNA extraction techniques that removed interfering pigment from ocular tissues produced RNA that could be easily amplified by PCR. In the adult human eye, expression of FOXC1 was greatest in the trabecular meshwork (TM) followed by the optic nerve head, choroid/RPE, ciliary body, cornea, and iris. FOXC1 expression levels were much lower in other non-ocular human tissues, such as liver, muscle, lung, heart, and kidney. CONCLUSIONS: Using an optimized donor eye preservation method and tissue RNA isolation procedure, we show that the FOXC1 transcription factor gene, which is known to be associated with developmental glaucoma, also may have an important role in the adult eye.

Aged↗

PubMed2001

The ameliorating effect of the water layer of Fructus Schisandrae on cycloheximide-induced amnesia in rats: interaction with drugs acting at neurotransmitter receptors.

Our previous study indicated that the water layer present in Fructus Schisandra(FS(w)) at 10 and 25 mg kg(-1)significantly counteracted cycloheximide (CXM)-induced amnesia. Therefore, the mechanism of action of the ameliorating effect of FS(w)on CXM-induced amnesia in the passive avoidance task was investigated in rats. The ameliorating effect of FS(w)on CXM-induced amnesia was depressed by scopolamine. The serotonin releaser, p -chloroamphetamine significantly antagonized the ameliorating effect of FS(w)on CXM-induced amnesia. Furthermore, the ameliorating effect was also inhibited by the 5-HT(1A)receptor agonist 8-OH-DPAT, but potentiated by the 5-HT(2)receptor antagonist ritanserin. Finally, the GABA(A)receptor antagonist bicuculline blocked the ameliorating effect of FS(w). These results suggest that the beneficial effect of FS(w)on CXM-induced amnesia is amplified by treatment with serotonergic 5-HT(2)receptor antagonists, but reduced by serotonergic 5-HT(1A)receptor agonists as well as GABA(A)and cholinergic receptor antagonists.

Administration, Oral↗

PubMed2001

The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes.

OBJECTIVE: To image spindles in living human oocytes and to examine the relation between spindles and fertilization after ICSI. DESIGN: The LC polscope was used to examine spindles in an observational study of living oocytes. SETTING: Academic IVF clinic. PATIENT(S): Women being treated for infertility. INTERVENTION(S): Oocytes retrieved from patients for infertility treatment were examined before ICSI. Aged, unfertilized oocytes after IVF or ICSI were examined with polscope and confocal microscopes to compare the two methods. MAIN OUTCOME MEASURE(S): Spindle structure in living oocytes and fertilization after ICSI. RESULT(S): Spindles could be imaged in 61.4% of oocytes. More oocytes with spindles than oocytes without spindles fertilized normally after ICSI (61.8% vs. 44.2%). Spindles in most aged oocytes were partially or completely disassembled, and only a few microtubules around the chromosomes or dispersed microtubules in the cytoplasm were observed. Confocal images of immunostained spindles were almost identical to polscope images of spindle birefringence. CONCLUSION(S): Spindles in living human oocytes can be imaged by using the polscope. A birefringent spindle in human oocytes may clinically predict the quality and age of oocytes. This method also can be used to monitor spindle position during ICSI.

Cellular Senescence↗

PubMed2001

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.

Animals↗

PubMed2001

The regulation of surface charged residues on the properties of cytochrome b5.

To understand the roles of negatively surface charged residues, the cytochrome b5 (Cyt b5) E48A/D60A mutant was constructed. UV-visible and CD spectra confirmed that the mutation did not cause overall structural changes of the protein. The mutant presents an unexpected high stability toward the thermal and denaturant compared with the wild type Cyt b5, which shows that these surface charged residues can influence the interactions between the heme b group and the polypeptide chain. Functional properties were clarified through the electron transfer reactions between Cyt b5 and Cyt c. The driving force of the electron transfer reactions is conservative. Although the association constant of Cyt b5 E48A/D60A with Cyt c is much lower than that of the wild type Cyt b5, their electron transfer rate constants do not differ significantly. The results show that these surface charged residues play important roles in regulating both the stability and functional properties of Cyt b5.

Base Sequence↗

PubMed2001

One-week ranitidine bismuth citrate-based triple therapy for the eradication of Helicobacter pylori in Hong Kong with high prevalence of metronidazole resistance.

AIM: To compare 1-week ranitidine bismuth citrate-based (RBC) triple therapy vs. omeprazole-based (O) triple therapy for the eradication of Helicobacter pylori infection in Hong Kong with high prevalence of metronidazole resistance. METHODS: Patients with non-ulcer dyspepsia and H. pylori infection were randomized to receive either: (i) RBCCM: ranitidine bismuth citrate (pylorid) 400 mg, clarithromycin 250 mg and metronidazole 400 mg; or (ii) OCM: omeprazole 20 mg, clarithromycin 250 mg and metronidazole 400 mg, each given twice daily for 1 week. Endoscopy (CLO test, histology and culture) and 13C-urea breath test were performed before randomization and 6 weeks after drug treatment. RESULTS: A total of 180 patients were randomized. H. pylori eradication rates (intention-to-treat, n=180/per protocol, n=166) were 83%/92% for RBCCM and 66%/70% for OCM (P=0.01, intention-to-treat and P=0.001, per protocol, respectively). RBCCM treatment was unaffected by metronidazole susceptibility and achieved a significantly higher eradication rate in metronidazole-resistant cases (89%) than the OCM group (45%, P=0.0064). CONCLUSION: One-week ranitidine bismuth citrate-based triple therapy is significantly better than omeprazole-based triple therapy for the eradication of H. pylori infection, especially in metronidazole-resistant cases. It is an effective regimen for the eradication of H. pylori infection in regions with a high prevalence of metronidazole resistance.

Adult↗

PubMed2001

An evaluation of invasive and non-invasive tests for the diagnosis of Helicobacter pylori infection in Chinese.

BACKGROUND: Different tests are available for diagnosing Helicobacter pylori infection. AIM: To compare the most commonly used tests either alone or in combination in Chinese patients with respect to routine clinical use or research purpose. METHODS: A total of 294 consecutive dyspeptic patients without previous H. pylori treatment were recruited. During upper endoscopy, biopsies were taken from the antrum and corpus, for a commercially available CLO-test, an in-house rapid urease test, culture, polymerase chain reaction and histological examination. Patients then received a 13C-urea breath test. The H. pylori status of each patient was determined by a concordance of test results. RESULTS: For routine clinical use, histology (antral plus corpus biopsies) had an accuracy of 100%, whilst the rapid urease test had an accuracy of 99.7%. The 13C-urea breath test was equally reliable, with an accuracy of 94.5%. Combinations of two tests did not confer additional advantage over the most accurate single test. For research purposes, the accuracy of using the criteria of two positives out of three diagnostic tests was 100% and equivocal results were not found. CONCLUSION: Histology with or without a rapid urease test was highly accurate for routine clinical use. Alternatively, the 13C-urea breath test was an equally reliable non-invasive test. The two positives out of three tests approach was highly reliable in predicting H. pylori status of untreated Chinese patients in a research setting.

Biopsy↗

PubMed2001

High prevalence of mixed infections by Helicobacter pylori in Hong Kong: metronidazole sensitivity and overall genotype.

BACKGROUND: Diversity in metronidazole susceptibility and genotypes of Helicobacter pylori have been reported with varying results in different areas. AIMS: To investigate the prevalence of multiple strain infection in a symptomatic Chinese population and to determine the metronidazole susceptibility pattern and genotypic characteristics of these infecting strains. METHODS: Gastric biopsies from antrum, body and cardia were taken during upper endoscopy in symptomatic patients referred to our department. Pooled cultures and single colony isolates were obtained and tested for metronidazole susceptibility and random amplified polymorphic DNA (RAPD) fingerprint patterns. RESULTS: A total of 461 isolates were successfully cultured from 46 patients. Fifty-seven per cent of subjects had metronidazole-resistant strains. Among them, 77% carried a mixture of sensitive and resistant strains, non-uniformly distributed in the gastric mucosa. Mixed genotypes were found by RAPD typing in 24% of subjects. These did not correlate with the metronidazole susceptibility/resistance pattern. CONCLUSION: H. pylori infections with mixed metronidazole sensitive/resistant strains and mixed genotypes are common in Hong Kong. This makes it prudent to use bacterial strains from several biopsy sites when testing for traits such as drug resistance or virulence in relation to disease.

Adult↗

PubMed2001

Three-day lansoprazole quadruple therapy for Helicobacter pylori-positive duodenal ulcers: a randomized controlled study.

AIM: To compare the efficacy and tolerability of a 3-day quadruple therapy with a standard 7-day triple therapy in eradicating Helicobacter pylori infection and healing duodenal ulcers. METHODS: Patients with H. pylori-positive duodenal ulcers were randomized to receive either lansoprazole 30 mg, clarithromycin 500 mg, and metronidazole 400 mg twice daily for 7 days (LCM-7) or lansoprazole 30 mg, clarithromycin 500 mg, metronidazole 400 mg, and bismuth subcitrate 240 mg twice daily for 3 days (LCMB-3). No pre- or post-treatment acid suppression was used. Follow-up endoscopy was performed at week 6. RESULTS: A total of 118 patients were recruited. Sixty patients in the LCM-7 group and 53 patients in the LCMB-3 group returned for endoscopy. Intention-to-treat eradication rates were 87% and 86% (P=0.94) and per protocol eradication rates were 87% and 94% (P=0.29) in the LCM-7 and LCMB-3 groups, respectively. Per protocol and intention-to-treat ulcer healing rates were 98% and 98% in LCM-7 and 100% and 91% in LCMB-3, respectively. There were no significant differences in efficacy in relation to the initial metronidazole and clarithromycin susceptibility. Significant reduction in the duration of side-effects was found in the LCMB-3 group. CONCLUSION: The 3-day quadruple therapy is highly effective, better tolerated and can be considered as a first-line therapy in duodenal ulcer management.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed2001

Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy.

BACKGROUND: Spindles are formed from microtubules and are exquisitely sensitive to changes in temperature. An orientation-independent polarized light microscope, the Polscope, can be used to image spindles in living oocytes allowing analysis of spindle kinetics in the living state. This study examined the effects of cooling on spindle disassembly in living human oocytes and spindle recovery after rewarming. METHODS: Oocytes were imaged continuously with the Polscope during cooling and rewarming. The quantity of microtubules in the spindles was measured by its birefringence using the Polscope. RESULTS: Spindles had completely disassembled by 5 min after cooling and recovered by 20 min after rewarming to 37 degrees C if rewarming started soon after the oocyte's temperature dropped to room temperature. However, when oocytes were cooled and kept at 33, 28 or 25 degrees C for 10 min and then warmed, it was found that warming allowed 5/5, 2/5 and 0/5 oocytes of the spindles to recover respectively. CONCLUSIONS: These results indicate that human meiotic spindles are exquisitely sensitive to alterations in temperature. The maintenance of temperature at 37 degrees C during in-vitro manipulation is important for spindle integrity and, therefore, is likely to be important for normal fertilization and subsequent embryo development.

Cold Temperature↗

PubMed2001

Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination.

BACKGROUND: Birefringent spindles imaged with the Polscope can predict fertilization rates after intracytoplasmic sperm injection (ICSI). The present study examined the development of human oocytes with or without birefringent spindles, imaged with the Polscope before ICSI. METHODS: Oocytes were obtained from stimulated ovaries of consenting patients undergoing oocyte retrieval for ICSI. Spindles were imaged with the Polscope combined with a computerized image analysis system. After imaging and ICSI, oocytes with or without spindles were cultured separately for examination of fertilization and embryo development. A total of 1544 oocytes from 136 cycles were examined with the Polscope and inseminated by ICSI. RESULTS: Spindles were imaged in 82% of oocytes. After ICSI, more oocytes (P < 0.05) with spindles (69.4%) fertilized normally, forming 2 pronuclei, than oocytes without spindles (62.9%). At day 3, more oocytes (P < 0.01) with spindles (66.3%) developed to 4-11 cell stages than oocytes without spindles (55.4%). Significantly more (P < 0.001) oocytes with spindles developed to morula and blastocyst by day 5 (51.1 versus 30.3%) and day 6 (53.2 versus 29.3%) compared with oocytes without spindles. CONCLUSIONS: The results indicate that the presence of a birefringent spindle in human oocytes can predict not only higher fertilization rate, but also higher embryo developmental competence.

Adult↗

PubMed2001

Nonaspiration puncture biopsy for suspected thoracic cavum tumor: experience of 2,010 clinical cases.

In our hospital from October 1980 to February 2000, 2,010 patients with suspected thoracic cavum cancer were diagnosed by nonaspiration puncture with multiple-hole type and surface trough-type biopsy needles. The positive rate of cytology or pathology examination for lesions in pleura, mediastinum, and lung tissue were 88.6% (148/167), 83% (297/358), and 80.2% (1,191/1,485), respectively. The mean positive rate in all cases was 81.4% (1,636/2,010). Complications rates for pneumothorax or hemoptysis were 1.3% and 0.8%, respectively, in this group. Such revised puncture needles have been used by us in various organs, such as brain tumors, metastatic lymph nodes of head and neck cancers, malignant disease of breast, liver, kidney, prostate, rectum and bone, etc. The use of such nonaspiration puncture needles is very simple, safe, and effective; even for beginners with this technique, the rate of successful diagnoses is fairly high.

Biopsy, Needle↗

PubMed2001

Ca2+ mediates the effect of inhibition of Na+-K+-ATPase on the basolateral K+ channels in the rat CCD.

We investigated the effect of inhibiting Na+-K+-ATPase on the basolateral 18-pS K+ channel in the cortical collecting duct (CCD) of the rat kidney. Inhibiting Na+-K+-ATPase with strophanthidin decreased the activity of the 18-pS K+ channel and increased the intracellular Ca2+ to 420 nM. Removal of extracellular Ca2+ abolished the effect of strophanthidin. When intracellular Ca2+ was raised with 5 microM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the 18-pS K+ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism of Ca2+-induced inhibition, the effect of 400 nM Ca2+ on channel activity was studied in the presence of calphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62, inhibitors of calmodulin-dependent kinase II. Addition of calphostin C or KN-93 or KN-62 failed to block the inhibitory effect of high concentrations of Ca2+ . This suggested that the inhibitory effect of high concentrations of Ca2+ was not mediated by protein kinase C or calmodulin-dependent kinase II pathways. To examine the possibility that the inhibitory effect of high concentrations of Ca2+ was mediated by the interaction of nitric oxide with superoxide, we investigated the effect of 400 nM Ca2+ on channel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) or N(omega)-nitro-L-arginine methyl ester. Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonic acid or N(omega)-nitro-L-arginine methyl ester completely abolished the inhibitory effect of 400 nM Ca2+ on channel activity. Moreover, application of 4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effect of strophanthidin. We conclude that the effect of inhibiting Na+-K+-ATPase is mediated by intracellular Ca2+ and the inhibitory effect of high concentrations of Ca2+ is the result of interaction of nitric oxide with superoxide.

Animals↗

PubMed2001

Effects of protein tyrosine kinase and protein tyrosine phosphatase on apical K(+) channels in the TAL.

We have previously demonstrated that the protein level of c-Src, a nonreceptor type of protein tyrosine kinase (PTK), was higher in the renal medulla from rats on a K-deficient (KD) diet than that in rats on a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000). We have now used the patch-clamp technique to investigate the role of PTK in regulating the apical K channels in the medullary thick ascending limb (mTAL) of the rat kidney. Inhibition of PTK with herbimycin A increased NP(o), a product of channel number (N) and open probability (P(o)), of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on a KD diet but had no significant effect in tubules from animals on a HK diet. In contrast, herbimycin A did not affect the activity of the 30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition of N-methylsulfonyl-12,12-dibromododec-11-enamide, an agent that inhibits the cytochrome P-450-dependent production of 20-hydroxyeicosatetraenoic acid, further increased NP(o) of the 70-pS K channel in the presence of herbimycin A. Furthermore, Western blot detected the presence of PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in the renal outer medulla. Inhibition of PTP with phenylarsine oxide (PAO) decreased NP(o) of the 70-pS K channel in the mTAL from rats on a HK diet. However, PAO did not inhibit the activity of the 30-pS K channel in the mTAL. The effect of PAO on the 70-pS K channel was due to indirectly stimulating PTK because pretreatment of the mTAL with herbimycin A abolished the inhibitory effect of PAO. Finally, addition of exogenous c-Src reversibly blocked the activity of the 70-pS K channel in inside-out patches. We conclude that PTK and PTP have no effect on the low-conductance K channels in the mTAL and that PTK-induced tyrosine phosphorylation inhibits, whereas PTP-induced tyrosine dephosphorylation stimulates, the apical 70-pS K channel in the mTAL.

Amides↗

PubMed2001

Effect of dietary K intake on apical small-conductance K channel in CCD: role of protein tyrosine kinase.

We have used Western blot to examine the expression of cSrc protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP)-1D in the renal cortex, and the patch-clamp technique to determine the role of PTK in mediating the effect of dietary K intake on the small-conductance K (SK) channel in the cortical collecting duct (CCD). When rats were on a K-deficient (KD) diet for 1, 3, 5, and 7 days, the expression of cSrc increased by 40, 90, 140, and 135%, respectively. In contrast, the expression of cSrc in the renal cortex from rats on a high-K (HK) diet for 1, 2, and 3 days decreased by 40, 60, and 75%, respectively. However, the protein level of PTP-1D was not significantly changed by dietary K intake. The addition of 1 microM herbimycin A increased NP(o), a product of channel number (N) and open probability (P(o)) in the CCD from rats on a normal diet or on a KD diet. The increase in NP(o) was 0.30 (normal), 0.45 (1-day KD), 0.65 (3-day KD), 1.55 (5-day KD), and 1.85 (7-day KD), respectively. Treatment of the CCD with herbimycin A from rats on a KD diet increased NP(o) per patch from the control value (0.7) to 1.4 (1-day KD), 1.6 (3-day KD), 2.6 (5-day KD), and 3.5 (7-day KD), respectively. In contrast, HK intake for as short as 1 day abolished the effect of herbimycin A. Furthermore, the expression of ROMK channels in the renal cortex was the same between rats on a KD diet or on a HK diet. Moreover, treatment with herbimycin A did not further increase NP(o) in the CCDs from rats on a HK diet. We conclude that dietary K intake plays a key role in regulating the activity of the SK channels and that PTK is involved in mediating the effect of the K intake on channel activity in the CCD.

Animals↗

PubMed2001

CaR-mediated COX-2 expression in primary cultured mTAL cells.

Primary cultures of medullary thick ascending limb (mTAL) cells retain the capacity to express calcium-sensing receptor (CaR) mRNA and protein. Increases in cyclooxygenase-2 (COX-2) mRNA accumulation, protein expression, and PGE(2) synthesis were observed in a dose- and time-dependent manner after exposure of these cells to extracellular calcium (Ca(o)(2+)). Moreover, transfection of mTAL cells with a CaR overexpression vector significantly enhanced COX-2 expression and PGE(2) production in response to calcium compared with cells transfected with an empty vector. Challenge with the CaR-selective agonist poly-L-arginine (PLA) also increased COX-2 mRNA accumulation, protein expression, and PGE(2) synthesis. Furthermore, Ca(o)(2+)- and PLA-mediated PGE(2) production was abolished in the presence of NS-398 or nimesulide, two different COX-2-selective inhibitors. These data suggest that intracellular signaling mechanisms initiated via activation of CaR contribute to COX-2-dependent PGE(2) synthesis in the mTAL. Because Ca(o)(2+) concentration varies along Henle's loop, calcium may contribute to salt and water balance via a COX-2- and CaR-dependent mechanism. Thus novel calcimimetics might be useful in conditions such as hypertension in which manipulation of extracellular fluid volume provides beneficial effects.

Animals↗

PubMed2001

Carbon monoxide of vascular origin attenuates the sensitivity of renal arterial vessels to vasoconstrictors.

Rat renal interlobar arteries express heme oxygenase 2 (HO-2) and manufacture carbon monoxide (CO), which is released into the headspace gas. CO release falls to 30% and 54% of control, respectively, after inhibition of HO activity with chromium mesoporphyrin (CrMP) or of HO-2 expression with antisense oligodeoxynucleotides (HO-2 AS-ODN). Patch-clamp studies revealed that CrMP decreases the open probability of a tetraethylammonium-sensitive (TEA-sensitive) 105 pS K channel in interlobar artery smooth muscle cells, and that this effect of CrMP is reversed by CO. Assessment of phenylephrine-induced tension development revealed reduction of the EC(50) in vessels treated with HO-2 AS-ODN, CrMP, or TEA. Exogenous CO greatly minimized the sensitizing effect on agonist-induced contractions of agents that decrease vascular CO production, but not the sensitizing effect of K channel blockade with TEA. Collectively, these data suggest that vascular CO serves as an inhibitory modulator of vascular reactivity to vasoconstrictors via a mechanism that involves a TEA-sensitive K channel.

Animals↗

PubMed2000

Regulation of ROMK1 channels by protein-tyrosine kinase and -tyrosine phosphatase.

We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K(+) current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K(+) current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K(+) current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 microg/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels.

Alanine↗