Novel Protein Interactions of G Protein-Coupled Receptor Kinase 5 (GRK5) Searched with Yeast Two-Hybrid System

BACKGROUND AND OBJECTIVES: G protein-coupled receptors were considered to be the only natural substrates of G protein-coupled receptor kinases (GRKs). However, it was recently demonstrated that GRKs can also bind to other signal molecules. The purpose of this study was to investigate new molecules that might interact with the GRK5 using a yeast two-hybrid system to screen the cDNA library. MATERIALS AND MEDTHODS: For the yeast two-hybrid system, the "bait" was constructed to generate a LexA-GRK5 fusion protein in the EGY48 yeast strain. Rat library cDNA was inserted into the "prey". The first step in the library screening was performed by a galactose dependent leucine orthotrophism. For the second step screening, a beta-galactosidase dependent discoloration of colonies was used. Sequencing and searching of the gene bank was undertaken to characterize the clones. RESULTS: We screened a total of 1.3X10 6 clones from the cDNA library. On the first screening, 162 clones were identified by leucine orhotrophism. Another 54 clones were identified on the second screening by beta-galactosidase activation. Seven clones were selected by PCR and restriction patterns. Sequencing of seven molecules revealed that four of the clones were emerin fragments, with 2 of the remaining 3 clones being: an ID2 protein and a mitochondrial cytochrome c oxidase subunit II, with the last one remaining an unknown molecule. For the emerin fragments, their interactions with the GRK5 were confirmed by immunoprecipitation. CONCLUSION: We describe the novel protein-protein interactions of the GRK5, specifically, with three molecules. At first, these proteins may modulate the activation of the GRK5 through this specific protein-protein interaction desensitizing the beta-adrenergic receptors. Conversely, the localization of these molecules inside the cell indicates a potential new physiological role for the GRK5.

Effect of Hypercholesterolemia and Age on Endothelial Function in Women

BACKGROUND AND OBJECTIVES: It is known that both hypercholesterolemia and aging cause endothelial dysfunction, but which has the greater effect is less well known. A useful, non-invasive method for the evaluation of endothelial function is flow-mediated dilation (FMD) of the brachial artery (BA) employing high-resolution ultrasound. This study was performed to evaluate the effect of serum cholesterol and age on endothelial function in women utilising the above method. SUBJECTS AND METHODS: 21 of the 37 women in this study (mean age 46+/-15 years, range 24-72), had hypercholesterolemia (>or=200 mg/dL) and 16 had normocholesterolemia (or=50 years (r=-0.54, p<0.05). Serum cholesterol levels were not significantly related to the EID, regardless of age. In contrast, age was not related to EDD or EID in either normocholesterolemic or hypercholesterolemic subjects. CONCLUSION: These results suggest hypercholesterolemia has a greater effect than age in impairing the endothelial function in women.

Influence of the Angiotensin II AT1 Receptor Antagonist on Reperfusion Injury in Rat Myocardial Ischemia Model

BACKGROUND AND OBJECTIVES: The protective effect of angiotensin converting enzyme (ACE) inhibitor against ischemia/reperfusion injury has been demonstrated in animal models, however the effect of AT1 receptor antagonist is contradictory. The present study was designed to investigate the myocardial protective effects of the AT1 receptor antagonist irbesartan during myocardial ischemia/reperfusion in vivo. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to a 45-minute left coronary artery ligation followed by a 2-hour re-perfusion. An inert vehicle (group I:n=14) or irbesartan (50 mg/kg/day:group II, n=12) was administered for 3 days before coronary occlusion. The ratio of the myocardial infarct area to the ischemic area at risk was assessed through triphenyltetrazolium chloride staining. Apoptosis was evaluated by analyzing DNA fragmentation and TdT-mediated dUDP nick end labeling staining. Western blot analysis was performed for MAP Kinases (ERK1/2 and p38) and Bcl-2 and Bax. RESULTS: The ratio of the infarct area to the ischemic area at risk of group II was smaller than that of group I (42.6+/-2.7% vs. 64.1+/-4.6%;p<0.005). Agarose gel electrophoresis revealed discrete DNA laddering in the ischemic zone of group I, however DNA ladder formation was attenuated in group II. The expressions of ERK1 MAPK and Bcl-2 were increased in the ischemic area of group II compared to that of group I. CONCLUSION: AT1 receptor antagonist was effective in reducing myocardial reperfusion injury in vivo. This effect can at least be partially attributed to the attenuation of cardiomyocyte apoptosis, and this anti-apoptotic effect appears to be related to the increased expression of Bcl-2 and alterations in MAP kinase signaling.

Sublingual Nitrate-Augmented Redistribution in Thallium-201 Myocardial Perfusion SPECT Compared with Repeated Injection to Detect Viable Myocardium

BACKGROUND: To assess the myocardial perfusion state after myocardial infarction, Tl-201 SPECT (Thallium-201 Single Photon Emission Computed Tomography) with a repeated "booster" injection before the acquisition of delayed redistribution image is more sensitive and more effective than conventional 4 hour redistribution image. However, this protocol has several disadvantages such as patient inconvenience, additional Tl-201 dose and compromised quantitative analysis. In this study, we compared 4 hour nitrate-augmented redistribution protocol with standard 24 hour delayed redistribution protocol with reinjection to evaluate the usefulness of sublingual nitrate to augment myocardial perfusion and the effectiveness of myocardial assessment for each protocol. METHODS: In 20 myocardial infarction patients, stress-redistribution Tl-201 SPECT was performed. Immediately after resting redistribution image was taken, each patient was administered 0.6 mg of nitroglycerin sublingually without additional Tl-201 and nitrate-augmented SPECT was taken after 30 minutes. Each patient then returned the next day and was injected with a booster dose of Tl-201 30 minutes before the delayed redistribution SPECT acquisition. For the analysis of SPECT study, the myocardium was divided into 22 segments, and the perfusion to each segment was scored on a four-point scale by consensus. An overall cardiac perfusion score was derived by summing the perfusion score for each segment. RESULTS: Reduced stress perfusion was identified in 258 segment among total 440 segments: 61 (23.6%) had improved perfusion after rest redistribution; 145 (56.2%) had improved perfusion after nitrate-augmented redistribution; 140 (54.2%) had improved perfusion after 24 hour delayed redistribution after Tl-201 reinjection. The cardiac perfusion score after stress was 38.2+/-13.1. The score increased to 41.5+/-13.1 after rest redistribution. The perfusion score were improved to 46.3+/-10.4 (p< or =0.05 vs. rest redistribution) after nitrate augmentation. The cardiac perfusion score, 46.2+/-10.8, did not improve further after delayed redistribution. CONCLUSION: Tl-201 SPECT with sublingual nitrate-augmented redistribution is as same or better than 24-hour delayed redistribution with reinjection to detect viable myocardium. Therefore, Tl-201 SPECT with sublingual nitrate-augmented redistribution has economic and time sparing advantage over traditional 24 hour delayed redistribution with reinjection.

Association between betaARK1 Level of Circulating Mononuclear Leukocytes and Left Ventrcular mass in Non-treated Hypertensive Patients

BACKGROUND: Beta-adrenergic receptor Kinase 1(betaARK1) is a serine/threonine kinase attached, which inhibits the coupling of beta-adrenergic receptor with G-protein. Myocardial betaARK1 level is usually elevated in heart failure and hypertrophy, but it is not known whether the circulating betaARK1 level is related with the degree of cardiac hypertrophy. This study was performed to evaluate the association of the betaARK1 level in circulating mononuclear leukocytes(MNL) in untreated hypertension with left ventricular mass in hypertensive patients. Method: Nineteen non-treated hypertensive patients were included for this study. High blood pressure was confirmed when systolic BP is over 150 mmHg or diastoli BP is over 95 mmHg. Echocardiography was performed to evaluate the degree of hypertrophy by measuring the left ventricular mass index(LVMI) and relative wall thickness(RWT), and test the LV function by measuring the ejection fraction(EF) according to ASE guideline. At the same time, blood was collected from each patient and MNL were isolated by gradient centrifuge with Ficoll-400. Total RNA was purified from MNL and semi-quantitative RT-PCR was performed. After reverse transcription, PCR was done with primers for human betaARK1 and GAPDH as external control. betaARK1 levels were expressed by ratio to GAPDH level and estimated the relations with clinical and Echocardiographic parameters. Result: We studied confirmed 19 hypertensive patients(10 men and 9 women, mean age of 50.6 years). Echocardiographically measured indices(mean+/-SD) were as follows; LVMI(137.3+/-30.6g/m2), PWT(0.53+/-0.09) and EF(54.6+/-8.5%). Ratio of betaARK1 levels to GAPDH was from 0.10 to 0.96 (0.62+/-0.25). betaARK1 levels were correlated with LVMI(correlation coefficient: r=.502, p=.029) and RWT(r=.627, p=.004). But Systolic BP(r=0.009, p=.93), diastolic BP(r=.07, p=.85) or EF(r=.045, p=.84) were not related to level of betaARK1. CONCLUSIONS: The betaARK1 level of circulating MNL was correlated well with the degree of the cardiac hypertrophy estimated by LVMI and RWT. This data suggests that activation of sympatho-adrenal system would exert a major role in developing cardiac hypertrophy and we can expect the decreased responsiveness to catecholamine in the heart of hypertensive patients. betaARK1 in circulating MNL might be used as a predictor or marker for LV hypertrophy in hypertensive patients.

A Case of Pure Unroofed Coronary Sinus without Persistent Left Superior Vena Cava

We report a case of 51-year-old woman with pure unroofed coronary sinus without persistent left superior vena cava and other cardiac anomaly. She presented with dyspnea on exertion during several years. Her chest film showed prominent cardiomegaly and dilated hilar vessels. Cardiac rhythm was atrial fibrillation. Transthoracic echocardiography demonstrated the enlarged coronary sinus with defect toward left atrium on parasternal long axis view and significant flow from coronary sinus into right atrium on subxyphoid view, and its other findings were dilated right ventricle and right atrium, paradoxical septal motion, moderate tricuspid regurgitation and mild mitral regurgitation, which were mimicking of large secundum atrial septal defect. Radionuclide cardioangiography and cardiac catheterization showed the existence of significant shunt. There was no evidence of persistent left superior vena cava on chest CT. Closure of Coronary sinus opening was done. Thereafter her symptoms of congestive heart failure were much improved. We think that careful examination of 2-D echocardiography can be valuable tool for diagnosis of unroofed coronary sinus in adult patient.

Effect of 17beta-estradiol on the Contraction to Endothelin-1 in Porcine Coronary Artery / 대한내과학회지

OBJECTIVES: It is widely accepted that estrogen has favorable effects on cardiovascular diseases, especially in the postmenopausal women. Endothelin-1(ET-I), released from the vascular endothelium, is a 21-amino acid peptide with strong vasoconstrictor activity. However, the effect of estrogen on the vasoconstriction to ET-1 has not been extensively studied. METHODS: To investigate the effect of estrogen (175beta-estradiol) on the vascular contraction to ET-1, porcine coronary artery(PCA) rings were suspended in organ chambers(37 degrees C, 95% O2/5% CO2) for measurement of isometric tension change. Endothelium was removed mechanically if necessary. In acute experiments, vascular rings were preincubated for 15minutes with 3different concentrations of 170beta-estradiol(10(-6), 10(-5), 10(-4)M) and concentration-contraction curves to cumulative doses of ET-1 were constructed. In the experiments after a longer exposure to 17beta-estradiol, the vessels with endothelium were exposed in the 5% CO2 incubator to 3different concentrations of 17beta-estradiol(10(-9), 10(-8), 10(-7)M) for 44-50 hours, and then concentrationcontraction curves to ET-1 were obtained. RESULTS: Incubation for 15minutes with 170beta-estradiol(10(-4)M) inhibited ET-1-induced contraction in the vessels with endothelium(area under the curve and maximal contraction, p<0.05 compared with control). This effect persisted regardless of the sex and the presence or absence of the endotheliurn. Incubation of the vessels far a longer time with 170beta-estradiol(44-50 hours) resulted in the inhibition of maximal contraction to ET-1(p<0.05) by a lower concentration of 175beta-estradiol(10(-7)M) than in acute experiments in male PCA rings, but an enhanced contraction to ET-1(area under the curve; p<0.05) by 10M of 175beta-estradiol was observed in female PCA rings. CONCLUSION: Short-time incubation with 17Pbeta-estradiol has an inhibitory effect on the contraction to ET-1 in PCA rings. This effect is independent of the presence of the endothelium and the sex of the pigs. A longer incubation with 17beta-estradiol results in a similar inhibitory effect on male(but not female) PCA rings, suggesting that a sex-related difference may exist concerning the effect of 17beta-estradiol on ET-1-induced contraction.

Office Blood Pressure is Higher than Home Blood Pressure, and Digital Electronic Sphygmomanometer is Useful for Self-Monitoring of Blood Pressure in Hypertensive Patients

BACKGROUND: To investigate the possibie difference, if any, between office blood pressure(BP) and home BP may be important in the diagnosis and treatment of hypersensive patients. This report deails the difference between the two BP's and the usefulness of digital electronic sphygmomanometer(DES) for self-monitoring of home BP. METHODS: The BP's of 14 patients with essential hypertension were measured with mercury sphygmomanometers at outpatient department by physician and with DES at home(twice a day) by the patients. Patients were followed up every 2 weeks for 4 weeks and previous 2 weeks' average home BP's were compared with the office BP's of each 2 weeks' end. RESULTS: There was a significant difference between office and home BP(both systolic and diastolic) ; office average BP(151/95mmHg) was higher than home average BP(136/86mmHg). CONCLUSION: Caution may be needed in the interpretation of office BP unless it is measured several times after adequate rest.

Assessment of Rapid Atrial Pacing in the Diagnosis of Coronary Artery Disease

The sensitivity and specificity of ST segment change on ECG for detection of coronary artery disease(CAD) by pacing stress test were assessed. Among 28 cases with chest pain(mean age 52, M/F : 21/7), 10 patients had normal coronary angiographic finding(Group I), and 18 had coronary artery disease(Group II). Pacing stress test showed high specificity(100%), but low sensitivity(61%) for the diagnosis of CAD. Especially in patients with 1 vessel disease, the sensitivity was only 50%, and positive results were not attained unless there was at least 90% or more stenosis in any of the major branches(LAD, RCA or LCX). But in patients with multivessel disease, the sensitivity was much higher(83%). Lateral(V4-6) or inferior leads(2, 3, aVF) showed ischemic ST segment depression most commonly. Therefore one of the inferior lead and V5 may be a minimum requirement for monitoring pacing-induced ST segment changes. Time constant during isovolumic relaxation showed statistically significant prolongation after pacing only in CAD patient group, suggesting pacing-induced impairment of early left ventricular relaxation.