Effect of Antihypertensive Drugs on Uric Acid Metabolism in Patients with Hypertension: Cross-Sectional Cohort Study.

Background: Hypertension is a common complication in patients with gout and/or hyperuricemia. Besides, hyperuricemia is a risk factor of gout as well as ischemic heart disease in hypertensive patients. Moreover, the risk of gout is modified by antihypertensive drugs. However, it remains unclear how antihypertensive agents affect uric acid metabolism. Purpose: In the present study, we investigated the uric acid metabolism in treated hypertensive patients to find out whether any of them would influence serum levels of uric acid. Patients and methods: 751 hypertensive patients (313 men and 438 women) under antihypertensive treatment were selected. Blood pressure (BP), serum uric acid (SUA) and serum creatinine (Scr) were measured and evaluated statistically. Results: In patients treated with diuretics, beta-blockers and/or alpha-1 blockers SUA levels were significantly higher than in patients who were not taking these drugs. Besides, the estimated glomerular filtration rate (eGFR) in patients treated with diuretics, beta-blockers and/or alpha-1 blockers was negatively correlated with SUA level. There were gender differences in the effects of beta-blockers and alpha-1 blockers. Multiple regression analysis indicated that both diuretics and beta-blockers significantly contributed to hyperuricemia in patients with medication for hypertension. Conclusion: Diuretics, beta-blockers and alpha-1 blockers reduced glomerular filtration rate and raised SUA levels. Calcium channel blockers, ACE inhibitors and angiotensin receptor blockers, including losartan, did not increase SUA levels.

Effects of Uric Acid on the NO Production of HUVECs and its Restoration by Urate Lowering Agents.

BACKGROUND: Although urate impaired the endothelial function, its underlying mechanism remains unknown. We hypothesized that urate impaired nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) via activation of uric acid transporters (UATs). PURPOSE AND METHOD: In the present study, we studied effects of urate on NO production and eNOS protein expression in HUVEC cells in the presence and absence of urate lowering agents using molecular biological and biochemical assays. RESULTS: HUVECs expressed the 4 kinds of UATs, URATv1, ABCG2, MRP4 and MCT9. Exposure to urate at 7 mg/dl for 24 h significantly reduced production of NO. Pretreatment with benzbromarone, losartan or irbesartan normalized NO production. The same exposure resulted in dephosphorylation of endothelial NO synthase (eNOS) in HUVECs. Again pretreatment with benzbromarone, losartan or irbesartan abolished this effect. CONCLUSION: Urate reduced NO production by impaired phosphorylation of eNOS in HUVEC via activation of UATs, which could be normalized by urate lowering agents.

Carvedilol Suppresses Apoptosis and Ion Channel Remodelling of HL-1 Cardiac Myocytes Expressing E334K cMyBPC.

BACKGROUND: Besides its antiarrhythmic action, carvedilol has an activity to suppress cardiac tissue damage. However, it is unknown whether it has any effect on cellular apoptosis and ion channel remodelling. PURPOSE: To know whether carvedilol has any effect on apoptosis and ion channel remodeling of HL-1 cells expressing E334K MyBPC, and comparing it with bisoprolol. METHOD: We examined effects of carvedilol and bisoprolol on the levels of pro- and anti-apoptotic proteins and ion channels as well as apoptosis of HL-1 cells transfected with E334K MyBPC using Western blot and flow cytometry. RESULTS: Carvedilol decreased the protein levels of p53, Bax and cytochrome c and increased that of Bcl-2 in HL-1 cells expressing E334K MyBPC. Bisoprolol failed to affect the protein levels. Both carvedilol and bisoprolol increased the protein levels of Cav1.2 but not that of Nav1.5. Carvedilol was stronger than bisoprolol at decreasing the number of annexin-V positive cells in HL-1 cells expressing E334K MyBPC. CONCLUSION: Carvedilol suppressed apoptosis of HL-1 cells expressing E334K MyBPC through modification of pro- and anti-apoptotic proteins, whose was associated with an increase of Cav 1.2 protein expression.

Simultaneous treatment with azelnidipine and olmesartan inhibits apoptosis of Hl-1 cardiac myocytes expressing E334k cMyBPC.

BACKGROUND: Apoptosis appears to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM). We have previously reported 3 HCM patients carrying the E334K MYBPC3, and that heterologous expression of E334K cMyBPC in cultured cells induced apoptosis. The purpose of this study was to identify pharmacological agents that would inhibit apoptosis in HL-1 cardiomyocytes expressing E334K cMyBPC. METHODS AND RESULTS: E334K cMyBPC expression in cells increased levels of pro-apoptosis (p53, Bax and cytochrome c) and decreased levels of anti-apoptosis (Bcl-2 and Bcl-XL). While the beta blocker carvedilol (1 µM) normalized the level of p53 and Bcl-2 and the calcium channel blocker (CCB) bepridil (0.5 µM) normalized that of Bcl-2, both the CCB azelnidipine (1 µM) and the angiotensin receptor blocker (ARB) olmesartan (10 µM) normalized those of p53, Bax, cytochrome c, and Bcl-XL. Among those proteins, cytochrome c was the one which showed the highest degree of change. Both azelnidipine (0.1 µM) and olmesartan (1 µM) reduced the level of cytochrome c by 40.2 ± 4.3% and 31.3 ± 5.1%, respectively. The CCB amlodipine and the ARB valsartan reduced it only by 19.1 ± 2.1% and 20.1 ± 5.2%, respectively. Flow cytometric analysis and annexin V staining showed that treatment of cells with azelnidipine (0.1 µM) plus olmesartan (0.3 µM) or that with amlodipine (0.1 µM) plus valsartan (0.3 µM) reduced the number of apoptotic cells by 35.8 ± 10.5% and 18.4 ± 3.2%, respectively. CONCLUSION: Azelnidipine plus olmesartan or amlodipine plus valsartan inhibited apoptosis of HL-1 cells expressing E334K cMyBPC, and the former combination was more effective than the latter.

A vasodilating ß1 blocker celiprolol inhibits muscular release of uric acid precursor in patients with essential hypertension.

Although nonvasodilating ß1 blockers increase the levels of uric acid in serum, it is not known whether vasodilating ß1 blockers have a similar effect. In the present study, we evaluated the effect of celiprolol on the release of hypoxanthine, a uric acid precursor, from muscles after an exercise. We used the semi-ischemic forearm test to examine the release of lactate (ΔLAC), ammonia (ΔAmm), and hypoxanthine (ΔHX) before and 4, 10, and 60 min after an exercise in 18 hypertensive patients as well as 4 normotensive subjects. Before celiprolol treatment, all the levels of ΔHX and ΔAmm, and ΔLAC were increased by semi-ischemic exercise in hypertensive patients, and the increases were remarkably larger than those in normotensive subjects. Celiprolol decreased both systolic and diastolic pressure. It also decreased the levels of ΔHX and ΔAmm without changes in ΔLAC after an exercise. These findings also were confirmed by summation of each metabolite (ΣΔMetabolites). Celiprolol caused a marginal decrease of serum uric acid, but the difference was not statistically significant. On the other hand, nonvasodilating ß1 blockers did not suppress the levels of ΔHX and ΔAmm, whereas they significantly increased ΔLAC after an exercise. Celiprolol improved energy metabolism in skeletal muscles. It suppressed HX production and consequently did not adversely affect serum uric acid levels.

Transcriptional activation of the anchoring protein SAP97 by heat shock factor (HSF)-1 stabilizes K(v) 1.5 channels in HL-1 cells.

BACKGROUND AND PURPOSE: The expression of voltage-dependent K(+) channels (K(v) ) 1.5 is regulated by members of the heat shock protein (Hsp) family. We examined whether the heat shock transcription factor 1 (HSF-1) and its inducer geranylgeranylacetone (GGA) could affect the expression of K(v) 1.5 channels and its anchoring protein, synapse associated protein 97 (SAP97). EXPERIMENTAL APPROACH: Transfected mouse atrial cardiomyocytes (HL-1 cells) and COS7 cells were subjected to luciferase reporter gene assay and whole-cell patch clamp. Protein and mRNA extracts were subjected to Western blot and quantitative real-time polymerase chain reaction. KEY RESULTS: Heat shock of HL-1 cells induced expression of Hsp70, HSF-1, SAP97 and K(v) 1.5 proteins. These effects were reproduced by wild-type HSF-1. Both heat shock and expression of HSF-1, but not the R71G mutant, increased the SAP97 mRNA level. Small interfering RNA (siRNA) against SAP97 abolished HSF-1-induced increase of K(v) 1.5 and SAP97 proteins. A luciferase reporter gene assay revealed that the SAP97 promoter region (from -919 to -740) that contains heat shock elements (HSEs) was required for this induction. Suppression of SIRT1 function either by nicotinamide or siRNA decreased the level of SAP97 mRNA. SIRT1 activation by resveratrol had opposing effects. A treatment of the cells with GGA increased the level of SAP97 mRNA, K(v) 1.5 proteins and I(Kur) current, which could be modified with either resveratrol or nicotinamide. CONCLUSIONS AND IMPLICATIONS: HSF-1 induced transcription of SAP97 through SIRT1-dependent interaction with HSEs; the increase in SAP97 resulted in stabilization of K(v)1.5 channels. These effects were mimicked by GGA.

Age and origin of the G774A mutation in SLC22A12 causing renal hypouricemia in Japanese.

Renal hypouricemia is an inherited disorder characterized by impaired tubular uric acid transport. Impairment of the function of URAT1, the main transporter for the reabsorption of uric acid at the apical membrane of the renal tubules, causes renal hypouricemia. The G774A mutation in the SLC22A12 gene encoding URAT1 predominates in Japanese renal hypouricemia. From data on linkage disequilibrium between the G774 locus and the 13 markers flanking it (12 single nucleotide polymorphisms and 1 dinucleotide insertion/deletion locus), we here estimate the age of this mutation at approximately 6820 years [95% confidence interval (CI) 1860-11,760 years; median = 2460 years]. This indicates that the origin of the G774A mutation dates back from between the time when the Jomon people predominated in Japan and the time when the Yayoi people started to migrate to Japan from the Korean peninsula. These data are consistent with a recent finding that this G774A mutation was also predominant in Koreans with hypouricemia and indicate that the mutation originated on the Asian continent. Thus, this mutation found in Japanese patients was originally brought by immigrant(s) from the continent and thereafter expanded in the Japanese population either by founder effects or by genetic drift (or both).

Fragment-specific actions of parathyroid hormone in isolated perfused rat hearts.

Different regions within the parathyroid hormone (PTH) molecule are known to have different biological activities. In the heart, the physiological actions of the intact PTH molecule are known as positive chronotropy and coronary vasodilatation. However, it is unclear which region of the PTH exerts which physiological action in the heart. Therefore, to clarify this point, we examined the hemodynamic effect of intact PTH(1-84) and selected PTH analogs, namely, PTH(1-34), PTH(2-34), [Nle8, 18Tyr34]PTH(3-34), PTH(4-34), [Tyr34]PTH(7-34), and PTH(13-34) in isolated perfused rat hearts. Both PTH(1-84) and PTH(1-34) significantly increased heart rate and decreased coronary perfusion pressure. In contrast, neither PTH(2-34) nor [Nle8,18Tyr34]PTH(3-34) increased heart rate, but they did decrease coronary perfusion pressure. Peptides further truncated at the amino terminus, PTH(4-34), [Tyr34]PTH(7-34), and PTH(13-34), had no effect on hemodynamics. Furthermore, the protein kinase A inhibitor H89, but not the protein kinase C inhibitor H7, attenuated the hemodynamic effects of PTH(1-34) or PTH(2-34), while it prevented those of [Nle8,18Tyr34]PTH(3-34). These results clearly demonstrate that the first amino acid of PTH is essential for its chronotropic property whereas the first 3 amino acids of PTH are involved in its coronary vasodilatory action. Furthermore, protein kinase A, but not protein kinase C, appears to be involved in the chronotropic and coronary vasodilatory actions of PTH.

Signaling pathway and chronotropic action of parathyroid hormone in isolated perfused rat heart.

Parathyroid hormone (PTH) activates both adenylyl cyclase and phospholipase C via the PTH-1 receptor. We previously reported that PTH increased heart rate and that this effect was mediated via the pacemaker current (I f ). However, it has been reported that PTH exerts its chronotropic effect via an interaction with adrenergic receptors or via L-type calcium channels. Thus, the objective of the study was to elucidate the exact mechanism of the chronotropic effect of PTH. We tested whether its chronotropic effects could be abolished by inhibitors of the following systems in isolated perfused rat hearts: alpha-adrenergic (prazosin); beta-adrenergic (propranolol); angiotensin II (CV11974); endothelin-1 (TAK044); calcium channel (verapamil); adenylyl cyclase (miconazole); phospholipase C (U73122) or I f (CsCl). In addition, we measured the cyclic adenosine monophosphate level of the heart after PTH administration. Whereas prazosin, propranolol, CV11974, TAK044, verapamil, and U73122 did not inhibit the chronotropic effect of PTH, CsCl or miconazole suppressed it significantly. PTH increased the cyclic adenosine monophosphate level of the atrium but not the left ventricle. These results indicate that the chronotropic actions of PTH are mediated via selective activation of adenylyl cyclase to increase the I f current.

Ca(2+)-sensitizing effect is involved in the positive inotropic effect of troglitazone.

1. Troglitazone, an insulin sensitizing agent, has a direct positive inotropic effect. However, the mechanism of this effect remains unclear. Thus, we examined the inotropic effect of troglitazone while focusing on intracellular Ca2+ handling. 2. Troglitazone significantly increased peak isovolumic left ventricular pressure (LVP(max)), peak rate of rise of LVP (dP/dt(max)), peak rate of fall of LVP (dP/dt(min)) in isolated rat hearts perfused at a constant coronary flow and heart rate. This inotropic effect of troglitazone was not inhibited by pretreatment with carbachol (muscarine receptor agonist), H89 (protein kinase A inhibitor), U73122 (phospholipase C inhibitor), H7 (protein kinase C inhibitor), verapamil (L-type Ca2+ channel antagonist), thapsigargin (Ca(2+)-adenosine triphosphatase inhibitor) or ryanodine (ryanodine receptor opener). 3. Radioimmunoassay showed that the cyclic adenosine monophosphate concentration in the left ventricle was not increased by troglitazone. 4. Whole-cell patch clamp analysis revealed that troglitazone had no effect on inward Ca2+ currents in cardiomyocytes. 5. In fura-2 loaded perfused rat hearts, troglitazone exerted its positive inotropic effect without increasing Ca2+ concentration. 6. These results suggest that neither the inward Ca2+ currents nor Ca2+ handling in the sarcoplasmic reticulum was involved in the inotropic effect of troglitazone. Furthermore, troglitazone exerted its positive inotropic effect without affecting the intracellular concentration of Ca2+. 7. In conclusion, the positive inotropic effect of troglitazone is mediated by a sensitization of Ca2+.

Altered purine nucleotide degradation during exercise in patients with essential hypertension.

Purine degradation occurs during strenuous muscle exercise and plasma levels of hypoxanthine (HX), purine degradation intermediate, increase. Purine nucleotide degradation has not been investigated in patients with essential hypertension (HTN). The present study determined whether purine nucleotide degradation is altered in patients with HTN. Cardiopulmonary exercise test was performed with serial measurements in blood lactate and plasma HX in 24 patients (14 men and 10 women) with essential HTN (World Health Organization [WHO] class I to II; mean age, 57.7 +/- 2.1 years) and 24 age-, sex-matched normal subjects. Exercise was terminated either by severe fatigue or excess blood pressure increase. Peak work rate (WR) (normal v HTN, 151 +/- 10 v 135 +/- 8 W, not significant [NS]) was not different, but peak oxygen uptake (peak Vo(2), 26.3 +/- 1.5 v 22.2 +/- 0.9 mL/min/kg, P <.05) and anaerobic threshold were lower in patients with HTN. Resting levels of blood lactate and plasma HX were similar, but the increment from rest to peak exercise (Delta) for lactate (Delta lactate: 4.4 +/- 0.4 v 3.4 +/- 0.4 mmol/L, P <.05) and for HX (Delta HX, 15.9 +/- 2.2 v 9.1 +/- 1.1 micromol/L, P <.05) were significantly smaller in patients with HTN. When normalized by the peak WR, Delta HX/peak WR (0.105 +/- 0.013 v 0.069 +/- 0.007 micromol/L/W, P <.05) was significantly lower in patients with HTN. Patients with HTN exhibited reduced HX response to exercise with impaired exercise capacity. The exercise-induced changes in plasma HX were smaller in patients with HT when normalized with peak WR. These results suggest that the purine nucleotide degradation is reduced in patients with HTN.

Mitochondrial DNA deletion associated with the reduction of adenine nucleotides in human atrium and atrial fibrillation.

BACKGROUND: Structural changes in the number, size, and shape of mitochondria (mt) have been observed in the atrial muscles of patients with atrial fibrillation (AF) and of animals with rapid atrial pacing, however, it is not known whether the mitochondrial function is impaired in human atrium with AF. MATERIALS AND METHODS: We determined adenine nucleotides concentrations and mtDNA deletions in 26 human right atria obtained at the time of cardiac surgery, using HPLC and PCR amplification, and studied the relationship between mtDNA deletions and clinical manifestations, the haemodynamic parameters of the patients and adenine nucleotide concentrations in their atrium. RESULTS: The age and the prevalence of AF were significantly higher in the patients with a mtDNA deletion of 7.4 kb than in those without a deletion; there were no significant differences regarding haemodynamic parameters between the two groups. The concentrations of ATP, ADP, AMP and total adenine nucleotides in the right atrium were significantly lower in the patients with mtDNA deletions than the patients without a deletion. In a gender- and diseased-matched population, the mtDNA deletion was still significantly associated with age and a decreased concentration of adenine nucleotides in the atrium. Using quantitative PCR analysis, the proportion of mtDNA deletion to normal mtDNA of the atrium, was estimated to be 0.3-2% in four cases. CONCLUSION: These results suggest that the deletion of mtDNA associated with ageing or AF can lead to a bioenergetic deficiency due to an impaired ATP synthesis in the human atrium; however, no conclusion can be made whether mtDNA deletion were the result or the cause of an impaired ATP synthesis, ageing, hemodynamic deterioration, or AF.

The release of the substrate for xanthine oxidase in hypertensive patients was suppressed by angiotensin converting enzyme inhibitors and alpha1-blockers.

OBJECTIVE: Hyperuricemia is associated with the vascular injury of hypertension, and purine oxidation may play a pivotal role in this association, but the pathophysiology is not fully understood. We tested the hypothesis that in hypertensive patients, the excess amount of the purine metabolite, hypoxanthine, derived from skeletal muscles, would be oxidized by xanthine oxidase, leading to myogenic hyperuricemia as well as to impaired vascular resistance caused by oxygen radicals. METHODS: We investigated the production of hypoxanthione, the precursor of uric acid and substrate for xanthine oxidase, in hypertensive patients and found that skeletal muscles produced hypoxanthine in excess. We used the semi-ischemic forearm test to examine the release of hypoxanthine (deltaHX), ammonium (deltaAmm) and lactate (deltaLAC) from skeletal muscles in essential hypertensive patients before (UHT: n = 88) and after treatment with antihypertensive agents (THT: n = 37) in comparison to normotensive subjects (NT: n = 14). RESULTS: deltaHX, as well as deltaAmm and deltaLAC, were significantly higher in UHT and THT (P< 0.01) than in NT. This release of deltaHX from exercising skeletal muscles correlated significantly with the elevation of lactate in NT, UHT and THT (y = 0.209 + 0.031x; R2 = 0.222, n = 139: P < 0.01). Administration of doxazosin (n = 4), bevantolol (n = 5) and alacepil (n = 8) for 1 month significantly suppressed the ratio of percentage changes in deltaHX by -38.4 +/- 55.3%, -51.3 +/- 47.3% and -76.3 +/- 52.2%, respectively (P< 0.05) but losartan (n = 3), atenolol (n = 7) and manidipine (n = 10) did not reduce the ratio of changes; on the contrary, they increased it in deltaHX by +188.2 +/- 331%, +96.2 +/- 192.2% and +42.6 +/- 137.3%, respectively. The elevation of deltaHX after exercise correlated significantly with the serum concentration of uric acid at rest in untreated hypertensive patients (y = 0.194 - 0.255x; R2 = 0.185, n = 30: P < 0.05). The prevalence of reduction of both deltaHX and serum uric acid was significantly higher in the patients treated with alacepril, bevantolol and doxazosin (67%: P < 0.02) than in the patients treated with losartan, atenolol and manidipine (12%). CONCLUSIONS: It is concluded that the skeletal muscles of hypertensive patients released deltaHX in excess by activation of muscle-type adenosine monophosphate (AMP) deaminase, depending on the degree of hypoxia. The modification of deltaHX by angiotensin-converting enzyme inhibitors and alpha1-blockers influenced the level of serum uric acid, suggesting that the skeletal muscles may be an important source of uric acid as well as of the substrate of xanthine oxidase in hypertension.

Enhanced activity of the purine nucleotide cycle of the exercising muscle in patients with hyperthyroidism.

Myopathy frequently develops in patients with hyperthyroidism, but its precise mechanism is not clearly understood. In this study we focused on the purine nucleotide cycle, which contributes to ATP balance in skeletal muscles. To investigate purine metabolism in muscles, we measured metabolites related to the purine nucleotide cycle using the semiischemic forearm test. We examined the following four groups: patients with untreated thyrotoxic Graves' disease (untreated group), patients with Graves' disease treated with methimazole (treated group), patients in remission (remission group), and healthy volunteers (control group). To trace the glycolytic process, we measured glycolytic metabolites (lactate and pyruvate) as well as purine metabolites (ammonia and hypoxanthine). In the untreated group, the levels of lactate, pyruvate, and ammonia released were remarkably higher than those in the control group. Hypoxanthine release also increased in the untreated group, but the difference among the patient groups was not statistically significant. The accelerated purine catabolism did not improve after 3 months of treatment with methimazole, but it was completely normalized in the remission group. This indicated that long-term maintenance of thyroid function was necessary for purine catabolism to recover. We presume that an unbalanced ATP supply or conversion of muscle fiber type may account for the acceleration of the purine nucleotide cycle under thyrotoxicosis. Such acceleration of the purine nucleotide cycle is thought to be in part a protective mechanism against a rapid collapse of the ATP energy balance in exercising muscles of patients with hyperthyroidism.

Augmented response in plasma brain natriuretic peptide to dynamic exercise in patients with left ventricular dysfunction and congestive heart failure.

OBJECTIVES: We have previously demonstrated that patients with symptomatic congestive heart failure (CHF), but not with asymptomatic left ventricular dysfunction (LVD), have augmented plasma atrial natriuretic peptide (ANP) response to exercise. Plasma brain natriuretic peptide (BNP) response to exercise is less extensively studied. The aim of this study was to determine whether responses of plasma BNP during exercise normalized for exercise workload are altered in patients with LVD and CHF. SUBJECTS AND METHODS: Twenty-nine patients with LVD, 32 patients with CHF (NYHA classes II-III) and 27 age-matched control subjects were studied. Ventilatory, plasma ANP and BNP responses were assessed during symptom-limited cardiopulmonary exercise testing. Plasma natriuretic peptide levels were measured at rest and immediately after peak exercise. The increment in plasma BNP was divided by the increment in oxygen uptake (VO2) from rest to peak exercise, and this ratio [BNP exercise ratio: (peak BNP - rest BNP)/(peak VO2 - rest VO2)] was compared amongst the three groups. RESULTS: Peak VO2 (Control, LVD and CHF: 28.2 +/- 1.7, 21.1 +/- 1.8, 16.2 +/- 0.6 ml, min(-1) kg(-1), respectively), anaerobic threshold and peak workload became smaller as heart failure worsened. Resting and peak plasma ANP levels were significantly higher only in CHF, whilst resting and peak plasma BNP levels displayed a significant and continuous increase from normal subjects to LVD and CHF. The ANP exercise ratio (1.25 +/- 0.36, 2.61 +/- 0.57, 7.72 +/- 1.65, ANOVA P = 0.0002) was significantly higher only in patients with CHF, whilst the BNP exercise ratio (0.35 +/- 0.10, 2.60 +/- 0.69, 4.98 +/- 0.97, ANOVA P = 0.0001) was significantly higher in patients with LVD and became progressively higher in patients with CHF. CONCLUSIONS: These data showed that the BNP exercise ratio, an exercise plasma BNP response normalized with exercise workload, was augmented in patients with LVD, and became progressively higher in CHF, suggesting that an augmented exercise BNP ratio exists early in the course of developing CHF.

Block of sodium channels by divalent mercury: role of specific cysteinyl residues in the P-loop region.

Divalent mercury (Hg(2+)) blocked human skeletal Na(+) channels (hSkM1) in a stable dose-dependent manner (K(d) = 0.96 microM) in the absence of reducing agent. Dithiothreitol (DTT) significantly prevented Hg(2+) block of hSkM1, and Hg(2+) block was also readily reversed by DTT. Both thimerosal and 2,2'-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg(2+) block of hSkM1. Y401C+E758C rat skeletal muscle Na(+) channels (mu1) that form a disulfide bond spontaneously between two cysteines at the 401 and 758 positions showed a significantly lower sensitivity to Hg(2+) (K(d) = 18 microM). However, Y401C+E758C mu1 after reduction with DTT had a significantly higher sensitivity to Hg(2+) (K(d) = 0.36 microM) than wild-type hSkM1. Mutants C753Amu1 (K(d) = 8.47 microM) or C1521A mu1 (K(d) = 8.63 microM) exhibited significantly lower sensitivity to Hg(2+) than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region may play an important role in the Hg(2+) block of the hSkM1 isoform. The heart Na(+) channel (hH1) was significantly more sensitive to low-dose Hg(2+) (K(d) = 0.43 microM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance (K(d) = 1.12 microM) to Hg(2+) than did wild-type hH1. In summary, Hg(2+) probably inhibits the muscle Na(+) channels at more than one cysteinyl residue in the Na(+) channel P-loop region. Hg(2+) exhibits a lower K(d) value (<1. 23 microM) for inhibition by forming a sulfur-Hg-sulfur bridge, as compared to reaction at a single cysteinyl residue with a higher K(d) value (>8.47 microM) by forming sulfur-Hg(+) covalently. The heart Na(+) channel isoform with more than two cysteinyl residues in the P-loop region exhibits an extremely high sensitivity (K(d) < 0. 43 microM) to Hg(+), accounting for heart-specific high sensitivity to the divalent mercury.

Ammonia response to constant exercise: differences to the lactate response.

1. We evaluated the plasma ammonia response to constant exercise at different intensities. Ten healthy male volunteers were asked to perform constant exercise for 15 min at five different intensities: 80, 90, 100, 110 and 120% of their ventilatory threshold (VT). Blood concentrations of lactate, ammonia and hypoxanthine were measured during and after exercise. 2. The concentration of lactate increased continuously during exercise intensities equivalent to 100, 110 and 120% VT. Plasma ammonia began to increase at 6 min exercise and continued increasing during exercise at all five exercise intensities. Plasma hypoxanthine levels also increased continuously during exercise at all exercise intensities; however, they peaked at 5-10 min after exercise. The response of plasma ammonia and hypoxanthine increased with increasing intensities of exercise. 3. While the extent of the increase in lactate levels during exercise at 100, 110 and 120% VT was significantly higher than that at 80% VT, only the increase in ammonia and hypoxanthine levels at 120% VT were significantly higher than those at 80% VT. 4. In conclusion, the plasma ammonia response to constant exercise differed to the lactate and ammonia responses to short-term exhaustive exercise.

Acute hemodynamic effects of insulin-sensitizing agents in isolated perfused rat hearts.

Troglitazone has direct effects on the hemodynamics of the heart. We investigated the effects of other insulin-sensitizing agents (rosiglitazone, pioglitazone and JTT-501 (4-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]benzyl]-3, 5-isoxazolidinedione)) on the hemodynamics of the heart using isolated perfused rat hearts. Rosiglitazone significantly decreased heart rate and coronary perfusion pressure, and increased peak isovolumic left ventricular pressure, peak rate of rise of left ventricular pressure and peak rate of fall of left ventricular pressure. The effects of rosiglitazone, however, were milder than those of troglitazone. Neither pioglitazone nor JTT-501 had any effect on the heart. D-alpha-tocopherol, a structural component of troglitazone, did not exert any effect on the heart. The coronary vasorelaxant effect of troglitazone and rosiglitazone was significantly suppressed by indomethacin, but not by N(omega)-nitro-L-arginine methyl ester. In conclusion, only rosiglitazone, as well as troglitazone, exerted positive inotropic, positive lusitropic, negative chronotropic, and coronary vasorelaxant effects on the heart. The coronary vasorelaxant effect of troglitazone and rosiglitazone was mediated by prostaglandin production.

Cardiac and plasma catecholamine responses to exercise in patients with type 2 diabetes: prognostic implications for cardiac-cerebrovascular events.

BACKGROUND: Patients with diabetes mellitus have an altered exercise plasma catecholamine response, which may be related to the abnormal sympathoadrenal function and autonomic neuropathy. Presence of autonomic neuropathy is associated with poor prognosis, but relationship between exercise plasma catecholamine and prognosis has not been investigated. This study determined if altered plasma catecholamine response to exercise was associated with cardiac-cerebrovascular events. METHODS: Forty patients with type 2 diabetes without apparent macrovascular complications and 30 control subjects performed treadmill exercise with serial measurements of plasma norepinephrine and epinephrine. Clinical, exercise, and catecholaminergic variables considered relevant to the cardiac-cerebrovascular events were examined by Cox regression model. Analysis of 24-hour heart rate variability was performed in a subgroup of patients. RESULTS: During 7.2 years, 8 patients, but no control subjects, had events (3 myocardial and 5 cerebral infarctions). Compared with Event(-) patients, Event(+) patients had: (1) orthostatic hypotension; (2) lower peak exercise heart rate; (3) lower plasma norepinephrine immediately after exercise; and (4) lower plasma epinephrine at peak exercise. High frequency components in heart rate variability analysis were diminished in Event(+) patients. Multivariate analysis showed that peak heart rate (P = 0.04) and plasma epinephrine at peak exercise (P = 0.03) were independent predictors of subsequent events. CONCLUSIONS: These data suggest that chronotropic incompetence and lower plasma epinephrine response to exercise are associated with high risk of cardiac-cerebrovascular events in patients with type 2 diabetes.

Fibrillin gene (FBN1) mutations in Japanese patients with Marfan syndrome.

Marfan syndrome (MFS; MIM #154700) is a connective tissue disorder characterized by cardiovascular, skeletal, and ocular abnormalities. The fibrillin-1 gene (FBN1; MIM no. 134797) on chromosome 15 was revealed to be the cause of Marfan syndrome. To date over 137 types of FBN1 mutations have been reported. In this study, two novel mutations and a recurrent de-novo mutation were identified in patients with MFS by means of single-strand conformational polymorphism (SSCP) analysis. The two novel mutations are a 4-bp deletion at nucleotide 2820-2823 and a G-to-T transversion at nucleotide 1421 (C474F), located on exon 23 and exon 11, respectively. A previously reported mutation at the splicing donor site of intron 2 (IVS2 G + 1A), which is predicted to cause exon skipping, was identified in a sporadic patient with classical MFS.