Biomedical subjects

Hiroyuki Watanabe

Publications and source records attributed to Hiroyuki Watanabe.

At least 109 records · Page 6Linked to original sources

PubMed2003

Abnormalities of tumor suppressor gene p16 in pancreatic carcinoma: immunohistochemical and genetic findings compared with clinicopathological parameters.

BACKGROUND: Abnormalities of the tumor suppressor gene p16 have been reported in a variety of human tumors but are rare in pancreatic carcinoma except for cancer cell lines and xenografts. Their clinicopathological significance remains unknown. The purpose of this study was to examine immunohistochemical and genetic alterations of p16 in primary pancreatic carcinoma tissues and to investigate the relation between abnormalities of p16 and clinicopathological parameters to elucidate their clinicopathological significance. METHODS: We investigated p16 expression in 60 pancreatic carcinoma cases by immunohistochemistry using a monoclonal antibody clone G175-405. In addition, we analyzed genetic alterations of the p16 gene using DNA extracted from microdissected tissue of pancreatic carcinoma, by polymerase chain reaction, nonradioisotopic single-strand conformation polymorphism (non-RI-SSCP), DNA sequencing, and hypermethylation analyses using restriction enzymes. We compared the abnormalities of p16 alterations with clinicopathological parameters to elucidate their significance. RESULTS: On immunohistochemical study, staining for p16 protein was strongly positive in 22 (37%) of 60 pancreatic carcinoma cases, weakly positive in 24 (40%), and negative in 14 (23%). In contrast, p16 mutations were recognized in 9 (15%) of the 60 pancreatic carcinoma cases. The incidence of p16 mutations was 2 (9%) in 22 cases of pancreatic carcinoma with strongly positive staining, 4 (17%) in 24 with weakly positive staining, and 3 (21%) in 14 with negative staining. Hypermethylation of p16 was detected in the two pancreatic carcinoma cases with weakly positive staining, although homozygous deletions were not found in any case. There was no significant correlation between the expression of p16 protein and any of the clinicopathological parameters. However, there was a tendency for the tumor to be larger in patients with decreased expression of p16 protein than in those with normal expression levels. In contrast, the tumor was significantly larger and the survival period significantly shorter for patients with pancreatic carcinoma with p16 mutation or hypermethylation than for those with pancreatic carcinoma with an intact p16 gene (P < 0.05). CONCLUSIONS: These findings suggest that p16 alterations may participate in the aggressiveness of pancreatic carcinoma.

Adult↗

PubMed2003

Intrathecal histamine induces spinally mediated behavioral responses through tachykinin NK1 receptors.

Intrathecal injection of histamine elicited a behavioral response consisting of scratching, biting and licking in conscious mice. Here, we have examined the involvement of substance P (SP) by using intrathecal injection of tachykinin neurokinin (NK)(1) receptor antagonists and SP antiserum. Histamine-induced behavioral response was evoked significantly 5-10 min after intrathecal injection and reached a maximum at 10-15 min. Dose-dependency of the induced response showed a bell-shaped pattern from 200 to 3200 pmol, and maximum effect was observed at 800-1000 pmol. The H(1) receptor antagonist, d-chlorpheniramine and pyrilamine but not the H(2) receptor antagonists, ranitidine and zolantidine, inhibited histamine-induced behavioral response. The NK(1) receptor antagonists, CP-99,994, RP-67580 and sendide, inhibited histamine-induced behavioral response in a dose-dependent manner. A significant antagonistic effect of [D-Phe(7), D-His(9)]SP (6-11), a selective antagonist for SP receptors, was observed against histamine-induced response. The NK(2) receptor antagonist, MEN-10376, had no effect on the response elicited by histamine. Pretreatment with SP antiserum resulted in a significant reduction of the response to histamine. No significant reduction of histamine-induced response was detected in mice pretreated with NK A antiserum. The present results suggest that elicitation of scratching, biting and licking behavior induced by intrathecal injection of histamine may be largely mediated by NK(1) receptors via H(1) receptors in the spinal cord.

Animals↗

PubMed2003

Reduction of ST elevation in repeated coronary occlusion model depends on both altered metabolic response and conduction property.

The aim of this study was to elucidate the mechanisms of altered electrical response to ischemia in repeated coronary occlusion model. To test its dependence on metabolic response, extracellular K+ concentration (eKC), myocardial pH and PCO2 were simultaneously measured with epicardial ECG during three consecutive 4 min of left anterior descending coronary artery (LAD) occlusion separated by 15 min of reperfusion in canine hearts. ECG changes induced by infusion of high K+-buffer (10 mM) into the coronary arterial bed via carotid artery-LAD bypass (referred to as high K+-challenges: HKC) were also tested prior to (the first HKC), and during each reperfusion period (the second to the fourth HKC). ST elevation was significantly reduced in subsequent occlusions (3.14 +/- 0.48 and 2.98 +/- 0.47 mV in the second and third occlusion, both P<0.05, compared to 4.91 +/- 0.78 mV in the first). This was accompanied by significant attenuation of the changes in eKC, tissue pH and PCO2. ST elevation induced by HKC also significantly reduced after repeated occlusion (4.09 +/- 0.79 mV in the fourth HKC vs. 5.64 +/- 0.68 mV in the first, P<0.05) in spite of the identical changes in eKC during HKC. This progressive decrease in ST changes by HKC was rather consistent with augmented conduction delay (86.4 +/- 7.1% increase in activation time in the fourth vs. 54.3 +/- 3.4% in the first, P<0.01). These findings indicate that repeated ischemia induces altered electrical response to subsequent ischemia based on both attenuated metabolic response and altered conduction property.

Animals↗

PubMed2003

Binding site of novel 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides in the D1 protein of Photosystem II.

A series of replacement experiments of [(14)C]-triazines, [(14)C]-atrazine and [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine, bound to thylakoids isolated from wild-type and atrazine-resistant Chenopodium album (lambsquarters) were conducted. Replacement experiments of [(14)C]-triazines bound to wild-type Chenopodium thylakoids with non-labeled atrazine and 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine were carried out, to elucidate whether benzylamino-1,3,5-triazines use the same binding niche as atrazine. [(14)C]-Atrazine and [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine bound to wild-type thylakoids were replaced by non-labeled 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine and non-labeled atrazine, respectively. The above two replacements showed mutual competition. To clarify further whether benzylamino-1,3,5-triazines bind at the D1-protein to amino acid residue(s) different from atrazine or not, experiments to replace [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines bound to atrazine-resistant Chenopodium thylakoids by non-labeled atrazine, 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU and DNOC were carried out. Although the bound [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine was difficult to be replaced even with high concentrations of atrazine, [(14)C]-labeled 1,3,5-triazine was competitively replaced by non-labeled 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU or DNOC. Thus, 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides are considered to bind to the same niche at the D1 protein as atrazine, but use amino acid residue(s) different from those involved with atrazine binding.

Journal Article↗

PubMed2003

Physiologic assessment of left anterior descending coronary artery stenosis by quantitative intravenous myocardial contrast echocardiography in humans: comparison with exercise single-photon emission computed tomography.

OBJECTIVE: To clarify the potential of quantitative intravenous myocardial contrast echocardiography (MCE) for physiologic assessment of the left anterior descending artery (LAD) stenosis. METHODS: We studied 38 patients with suspected coronary artery disease. MCE was performed by continuous infusion of Levovist and intermittent ultrasonic exposure. Images were obtained from the apical four-chamber view at rest and after dipyridamole infusion. The background-subtracted intensity versus pulsing interval plots were fitted to an exponential function,Y=A(1 e-ss), to obtain the plateau level (A) and rate of rise (ss) of background-subtracted intensity both at rest and after dipyridamole infusion. We compared the results with those of exercise thallium-201 single-photon emission computed tomography (SPECT). RESULTS: Of the 38 patients, 18 patients exhibited redistribution in the LAD territories with SPECT (group A), although 20 did not (group B). The ss reserve (DIP/rest) in group A was significantly lower than those in group B (0.8 +/- 0.5 versus 2.0 +/- 1.1, P < 0.001), while the A reserve did not differ between the two groups (1.2 +/- 0.6 versus 1.0 +/- 0.5, P = NS). The ss reserve <1.1, which was the optimal cutoff value, provided sensitivity of 79% and specificity of 84% for the presence of redistribution in SPECT. CONCLUSIONS: Quantitative intravenous MCE allows us to estimate physiologic severity of the LAD stenosis in the clinical setting.

Contrast Media↗

PubMed2003

Noninvasive assessment of myocardial ischemia in the left ventricular inferior regions by coronary flow reserve measurement using transthoracic Doppler echocardiography.

OBJECTIVE: The purpose of this study was to evaluate the potential of noninvasive measurement of coronary flow reserve (CFR) by transthoracic Doppler echocardiography (TTDE) for the assessment of myocardial ischemia in the left ventricular (LV) inferior regions. BACKGROUND: Although coronary flow assessment by TTDE has been determined for the assessment of perfusion abnormality in the LV anterior regions, the usefulness of this method has not been well investigated in the LV inferior regions. METHODS: We studied 50 patients (43 men; mean age 60 +/- 9 years) with suggested coronary artery disease. CFR in the posterodescending coronary artery (PDA) was calculated as a ratio of hyperemic to basal peak (peak CFR) and mean (mean CFR) flow velocities in the PDA, which were measured by TTDE. CFR values were compared with the results of exercise 201-thallium single photon emission computed tomography. RESULTS: CFR was successfully measured in 43 of 50 patients (86%). Mean and peak CFR < 2 were shown in 10 of 12 patients with abnormal perfusion in the LV inferior regions, whereas CFR > or = 2 were shown in 30 of 31 patients with normal perfusion. Thus, CFR < 2 in the PDA by TTDE had a sensitivity of 83% and a specificity of 97% for the assessment of perfusion abnormality in the LV inferior regions by 201-thallium single photon emission computed tomography. CONCLUSIONS: CFR in the PDA measured by TTDE provides data equivalent to those obtained by 201-thallium single photon emission computed tomography for myocardial ischemia in the LV inferior regions.

Aged↗

PubMed2003

Rapid and accurate noninvasive assessment of global left ventricular systolic function using biplane advanced automated contour tracking method.

BACKGROUND: The advanced automated contour tracking (AACT) method has been newly developed for automated detection of the left ventricular endocardial boundary. Left ventricular ejection fraction (LVEF) may be estimated by applying the AACT method to 2 orthogonal planes of patients even when regional wall-motion abnormalities exist. The purpose of this study was to examine the reliability of the biplane AACT method in the measurement of LVEF in patients with suggested ischemic heart disease with use of quantitative gated single photon emission computed tomography (QGS) as a reference standard. METHODS: The study population consisted of 47 consecutive patients with suggested ischemic heart disease. All patients underwent 2-dimensional echocardiography and QGS. Biplane LVEF from apical 4- and 2-chamber views was measured offline by the AACT method using disk summation method. The accuracy of the AACT method for LVEF measurement was determined in comparison with QGS. RESULTS: In 41 (29 with and 12 without regional wall-motion abnormalities) of 47 patients (87%), automated tracing of the endocardial border was adequately achieved with the AACT method. LVEF measured by the AACT method correlated well with that measured by QGS (y = 0.97x +2.4, r = 0.91). The mean difference between AACT and QGS was 0.6 +/- 5.5% (mean +/- SD). The mean time required for analysis of 1 set of images during 1 cardiac cycle by the AACT method was much shorter than that required by manual tracing method (7 +/- 1 vs 37 +/- 4 seconds, P <.0001). CONCLUSION: The biplane AACT method provides accurate and quick measurement of LVEF in patients even with regional wall-motion abnormalities.

Adult↗

PubMed2003

Clinical and imaging features of autoimmune pancreatitis with focal pancreatic swelling or mass formation: comparison with so-called tumor-forming pancreatitis and pancreatic carcinoma.

OBJECTIVES: Autoimmune pancreatitis (AIP) with a mass formation or swollen pancreas located in one or two segments of the gland (focal type AIP) has been reported. The aims of this study were to elucidate the relationship of the disease entity between this focal variant and so-called tumor-forming pancreatitis (TFP) and to describe the clinical and imaging features discriminating focal AIP from pancreatic carcinoma (Pca). METHODS: The clinical, radiologic, and pathologic profiles of nine patients with focal AIP were reviewed retrospectively and compared with those of 11 patients with alcohol-induced TFP and 80 patients with Pca. RESULTS: The patients with focal AIP were predominantly older (mean age 64.7 +/- 13.6 yr, range 28-78 yr), male, and presenting with obstructive jaundice or focal pancreatic enlargement accompanied by mild abdominal symptoms. In comparison, the patients with alcohol-induced TFP who were mostly middle-aged (mean age 50.1 +/- 7.95 yr, range 39-62 yr), male, and often had attacks of pancreatitis associated with findings of CT scans showing pseudocysts or peripancreatic effusion. Focal AIP usually demonstrated no abnormalities on pancreatograms downstream from the stricture or obstruction and often presented few contrast-filled side branches in the area of main pancreatic duct (MPD) stenosis. These characteristics were similar to the imaging features of Pca. Significant factors differentiating focal AIP from Pca were lower serum levels of CA19-9, homogeneous delayed enhancement evident in dynamic CT scans, and ERCP findings exhibiting a longer stenosed MPD and a thinner MPD upstream from the stricture. CONCLUSIONS: Focal AIP is associated with clinical and radiologic features that are different from those of alcohol-induced TFP. In TFP there are two causative factors, namely, AIP and alcohol-induced chronic pancreatitis. Differential diagnosis of focal AIP from Pca seems to be possible in many cases by evaluating imaging findings such as dynamic CT and ERCP, although focal AIP sometimes shows clinical and radiologic features similar to those of Pca.

Adult↗

PubMed2003

Pitavastatin inhibits upregulation of intermediate conductance calcium-activated potassium channels and coronary arteriolar remodeling induced by long-term blockade of nitric oxide synthesis.

UNLABELLED: We have reported that intermediate conductance Ca(2+)-activated K(+) channels (ImK) showed augmented expression in angiotensin II (AII) type 1 receptor-dependent manner in post-ischemic rat heart. ImK has tyrosine phosphorylation sequence in the C-terminus and motifs for NFkappaB and AP1 in the promoter. While statin inhibits AII-mediated vascular remodeling via anti-inflammatory effect independent of cholesterol lowering. To test the possible effect of statin on expression of ImK, Wistar-Kyoto rats received L-nitro-arginine methyl ester (LNAME: 1 mg/ml in drinking water) for 4 weeks in group L. While in L+P group, rats received both LNAME and pitavastatin (PTV: 1 mg/kg/day in drinking water). Temporal profile of ImK mRNA was examined by RT-PCR using specific primers for ImK. RESULTS: Long-term LNAME administration produced significant hypertension and resulted in marked microvascular remodeling characterized by medial thickening and perivascular fibrosis of coronary arterioles (100-200 microm in diameter). RT-PCR revealed significant up-regulation of ImK mRNA with two distinct peaks in L group in the early phase (days 3-7) and the late phase (4 weeks). PTV partially inhibited a rise in systolic blood pressure, but completely abolished the first peak of ImK upregulation (0.76 +/- 0.04 vs. 3.96 +/- 1.43 folds at day 7, p < 0.001). Co-treatments with PTV also significantly inhibited medial thickening and perivascular fibrosis. These findings indicate that statin inhibits microvascular remodeling induced by chronic inhibition of NO synthesis through the action independent of cholesterol lowering.

Animals↗

PubMed2003

TNF-alpha rapidly antagonizes the beta-adrenergic responses of the chloride current in guinea-pig ventricular myocytes.

The purpose of this study was to test the hypothesis that tumor necrosis factor-alpha (TNF-alpha) rapidly antagonizes the beta-adrenergic responses of the chloride current and to clarify the intracellular mechanisms responsible for the anti-adrenergic action. The whole-cell patch-clamp technique was used to monitor the anti-adrenergic effects of TNF-alpha on the cAMP-dependent chloride current (I(Cl)) recorded from isolated guinea-pig ventricular myocytes. Ramp pulses (+/-120 mV; dv/dt = +/-0.4 V/s) were applied from the holding potential of -40 mV. TNF-alpha rapidly (<15 min) inhibited the isoproterenol (Iso, 0.1 micromol/L)-induced I(Cl) in a concentration-dependent manner (30-1,000 U/ml, IC (50) = 144 U/ml, n=30). The inhibitory action of TNF-alpha was also observed when I(Cl) had been previously stimulated by 1 micromol/L forskolin (n=5). Prior exposure of myocytes to 5 microg/ml pertussis toxin (PTX) hardly affected the anti-adrenergic action of TNF-alpha (n=4). However, when I(Cl) was induced by both 8-bromo-cAMP (100 micromol/L) and isobutylmethylxanthine (0.1 mmol/L), TNF-alpha (1,000 U/ml) failed to decrease I(Cl) amplitude (n=5). Prior exposure of myocytes to 5 mg/ml pertussis toxin (PTX) hardly affected the anti-adrenergic action of TNF-alpha (n=4). Furthermore, despite of the presence of nitro-L-arginine methyl ester (0.1 mmol/L), a nitric oxide synthase (NOS) inhibitor, TNF-alpha reversed the Iso-induced increase in I(Cl) (n=5). These results suggest that TNF-alpha rapidly antagonizes the beta-adrenergic responses of I(Cl) by reducing cAMP concentration. This anti-adrenergic action is mediated by neither the PTX-sensitive G proteins regulatory pathway nor constitutive NOS activation.

Adrenergic beta-Agonists↗

PubMed2002

Reversible secondary pseudohypoaldosteronism due to pyelonephritis.

We report a 5-week-old boy who developed severe hyponatremia and hyperkalemia secondary to acute pyelonephritis. The patient presented with non-specific signs, including poor appetite, failure to thrive, and dehydration. An endocrinological evaluation led to a diagnosis of pseudohypoaldosteronism. The patient had phimosis, but no congenital urinary tract malformations. Outflow obstruction secondary to the phimosis appears to have caused pyelonephritis, and renal inflammation decreased responsiveness to aldosterone transiently.

Acute Disease↗

PubMed2002

Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells.

We have compared activation by heat of TRPV4 channels, heterogeneously expressed in HEK293 cells, and endogenous channels in mouse aorta endothelium (MAEC). Increasing the temperature above 25 degrees C activated currents and increased [Ca(2+)](i) in HEK293 cells transfected with TRPV4 and in MAEC. When compared with activation of TRPV4 currents by the selective ligand 4alphaPDD (alpha-phorbol 12,13-didecanoate), heat-activated currents in both systems showed the typical biophysical properties of currents through TRPV4, including their single channel conductance. Deletion of the three N-terminal ankyrin binding domains of TRPV4 abolished current activation cells by heat in HEK293. In inside-out patches, TRPV4 could not be activated by heat but still responded to the ligand 4alphaPDD. In MAEC, the same channel is activated under identical conditions as in the HEK expression system. Our data indicate that TRPV4 is a functional temperature-sensing channel in native endothelium, that is likely involved in temperature-dependent Ca(2+) signaling. The failure to activate TRPV4 channels by heat in inside-out patches, which responded to 4alphaPDD, may indicate that heat activation depends on the presence of an endogenous ligand, which is missing in inside-out patches.

Animals↗

PubMed2002

The intracellular tyrosine residues of the ATP-gated P2X(1) ion channel are essential for its function.

The four highly conserved intracellular tyrosine residues of the P2X(1) ion channel were mutated into phenylalanine. Simultaneous electrophysiological and calcium measurements in transfected human embryonic kidney (HEK 293) cells indicated that Y362F and Y370F mutants were non-functional, despite their proper plasma membrane expression. The Y16F and Y363F mutants retained 2.2% and 26% of the wild-type P2X(1) activity, respectively. However, no tyrosine phosphorylation was detected on Western blots of P2X(1) immunoprecipitates derived either from HEK 293 cell lysates or from human platelets, expressing P2X(1) endogenously. Thus, Y16, Y362, Y363 and Y370 are required for the appropriate three-dimensional structure and function of the intracellular P2X(1) domains.

Adenosine Triphosphate↗

PubMed2002

Molecular determinants of permeation through the cation channel TRPV4.

We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca(2+) permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp(682) but not Asp(672) strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met(680), which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca(2+) permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys(675), had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.

Amino Acid Sequence↗

PubMed2002

Change in coronary flow reserve on transthoracic Doppler echocardiography after a single high-fat meal in young healthy men.

BACKGROUND: High-fat meals and elevated triglyceride levels are associated with cardiovascular disease. In recent studies of brachial artery vasoactivity, a single high-fat meal reduced endothelial function in young healthy men. It is unknown whether coronary microcirculation is affected after high-fat meals. OBJECTIVE: To evaluate change in coronary flow reserve after a single high-fat meal. DESIGN: Controlled interventional study. SETTING: University hospitals. PATIENTS: 15 young healthy men (mean age [+/-SD], 29 +/- 4 years). INTERVENTION: Coronary flow reserve was determined by using transthoracic Doppler echocardiography before and after consumption of a high-fat meal in all 15 men and before and after consumption of a low-fat meal in 5 of 15 men. MEASUREMENTS: Coronary flow reserve, lipid levels, and hemodynamic characteristics. RESULTS: In all men, triglyceride levels increased significantly from baseline 5 hours after the high-fat meal, from 1.1 mmol/L to 2.8 mmol/L (100 mg/dL to 250 mg/dL) (P < 0.001). Average coronary flow reserve was 4.02 before and 3.30 5 hours after the high-fat meal (decrease, 18% [95% CI, 13% to 23%]). In the 5 men who received both meals, mean coronary flow reserve decreased by 0.79 after the high-fat meal and increased by 0.07 after the low-fat meal (difference, -0.86 [CI, -1.36 to -0.37]; P = 0.03). Mean triglyceride levels increased by 1.6 mmol/L (140 mg/dL) after the high-fat meal and 0.1 mmol/L (10 mg/dL) after the low-fat meal (difference, 1.5 mmol/L [CI, 0.3 to 2.7 mmol/L], 130 mg/dL [CI, 23 to 236 mg/dL]; P = 0.03). CONCLUSIONS: Coronary flow reserve decreased after a single high-fat meal in young healthy men. High-fat meals may be detrimental to coronary microcirculation.

Adult↗

PubMed2002

ATP and nitric oxide modulate a Ca(2+)-activated non-selective cation current in macrovascular endothelial cells.

We have studied the properties of a non-selective cation current (NSC(Ca)) in macrovascular endothelial cells derived from human umbilical vein (EA cells) that is activated by an increase of intracellular Ca(2+) concentration, [Ca(2+)](i). Current-voltage relationships are linear and the kinetics of the current is time-independent. Current-[Ca(2+)](i) relationships were fitted to a Ca(2+) binding site model with a concentration for half-maximal activation of 417 +/- 76 nM, a Hill coefficient of 2.3 +/- 0.8 and a maximum current of -23.9 +/- 2.7 pA/pF at -50 mV. The Ca(2+)-activated channel is more permeable to Na(+) than for Cs(+) ( P(Cs)/ P(Na)=0.58, n=7), but virtually impermeable to Ca(2+). Current activation was transient if ATP was omitted from the pipette solution. The maximal currents at 300 and 500 nM [Ca(2+)](i) were smaller than in the absence of ATP, but were not significantly different at 2 microM. The intracellular Ca(2+) concentration for half-maximal activation of the Ca(2+)-activated current was shifted to 811 +/- 12 nM in the absence of ATP. Substitution of ATP by the non-hydrolysable ATP analogue adenylylimidodiphosphate (AMP-PNP) did not affect current activation. Sodium nitroprusside (SNP) decreased NSC(Ca) in a concentration-dependent manner. The nitric oxide (NO) donors S-nitroso- N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) also inhibited NSC(Ca). In contrast, nitro- L-arginine (NLA), which inhibits all NO-synthases, potentiated NSC(Ca), whereas superoxide dismutase (SOD), which inhibits the breakdown of NO, inhibited NSC(Ca). It is concluded that the Ca(2+)-activated non-selective action channel in EA cells is modulated by the metabolic state of the cell and by NO.

Adenosine Triphosphate↗

PubMed2002

Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives.

We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. 4alpha-Phorbol 12,13-didecanoate (4alpha-PDD) induced an increase in intracellular Ca(2+) concentration, [Ca(2+)](i), in 1321N1 cells stably transfected with human VRL-2 (hVRL-2.1321N1) or HEK-293 cells transiently transfected with murine TRP12, but not in nontransfected or mock-transfected cells. Concomitantly with the increase in [Ca(2+)](i), 4alpha-PDD activated an outwardly rectifying cation channel with an Eisenman IV permeation sequence for monovalent cations that is Ca(2+)-permeable with P(Ca)/P(Na) = 5.8. Phorbol 12-myristate 13-acetate also induced an increase in [Ca(2+)](i) but was approximately 50 times less effective than 4alpha-PDD. EC(50) for Ca(2+) increase and current activation was nearly identical (pEC(50) approximately 6.7). Similar effects were observed in freshly isolated mouse aorta endothelial cells which express TRP12 endogenously. By using 4alpha-PDD as a tool to stimulate TRP12, we showed that activation of this channel is modulated by [Ca(2+)](i); an increase in [Ca(2+)](i) inhibits the channel with an IC(50) of 406 nm. Ruthenium Red at a concentration of 1 microm completely blocks inward currents at -80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.

Animals↗

PubMed2002

L-nitro-arginine inhibits increase in endothelin binding sites induced by ischemia and reperfusion.

We have demonstrated that ischemia and reperfusion promoted augmented contractile response to endothelin-1 (ET) in coronary arteries in the presence of polymorphonuclear leukocytes (PMN). It has been also reported that ischemia and reperfusion increase ET binding sites in cardiac membrane in isolated rat heart perfused by blood cell-free system. To determine the role of PMN and L-arginine to nitric oxide (NO) pathway in these phenomena, isolated perfused rabbit hearts were subjected to 30 min of global ischemia followed by 30 min of reflow in the absence or presence of PMN and 10(-5)M of L-nitro-arginine (LNA). PMN was prepared with Percoll density gradients from peritoneal exudate elicited by glycogen. PMN activated with 10(-6)M of phorbol myristate acetate or their supernatant were infused into the coronary perfusion circuit after 5 min of reflow. LNA was added to perfusate also after reflow. The effect of superoxide dismutase (SOD: 50 IU/ml) was also determined. After the end of protocols, membrane fraction was isolated from the hearts for (125)I-ET-1 binding assay. ET-1 binding (Bmax) showed a significant increase by ischemia and reperfusion (P<0.01 vs control). That was markedly augmented with addition of activated PMN or their supernatant (both P<0.01), but abolished either by LNA or SOD (P<0.01 and P<0.05, respectively). These results indicate that increase in ET-receptor by ischemia and reperfusion is mediated by free radicals generated via L-arginine to NO pathway.

Animals↗