FoxO1 signaling plays a pivotal role in the cardiac telomere biology responses to calorie restriction.

This study examined whether the forkhead transcription factors of O group 1 (FoxO1) might be involved in telomere biology during calorie restriction (CR). We used FoxO1-knockout heterozygous mice (FoxO1(+/-)) and wild-type mice (WT) as a control. Both WT and FoxO1(+/-) were subjected to ad libitum (AL) feeding or 30% CR compared to AL for 20 weeks from 15 weeks of age. The heart-to-body weight ratio, blood glucose, and serum lipid profiles were not different among all groups of mice at the end of the study. Telomere size was significantly lower in the FoxO1(+/-)-AL than the WT-AL, and telomere attrition was not observed in either WT-CR or FoxO1(+/-)-CR. Telomerase activity was elevated in the heart and liver of WT-CR, but not in those of FoxO1(+/-)-CR. The phosphorylation of Akt was inhibited and Sirt 1 was activated in heart tissues of WT-CR and FoxO1(+/-)-CR. However, the ratio of conjugated to cytosolic light chain 3 increased and the level of p62 decreased in WT-CR, but not in FoxO1(+/-)-CR. A marker of oxidative DNA damage, 8-OhdG, was significantly lower in WT-CR only. The level of MnSOD and eNOS increased, and the level of cleaved caspase-3 decreased in WT-CR, but not FoxO1(+/-)-CR. Echocardiography showed that the left ventricular end-diastolic and systolic dimensions were significantly lower in WT-CR or FoxO1(+/-)-CR than WT-AL or FoxO1(+/-)-AL, respectively. The present studies suggest that FoxO1 plays beneficial roles by inducing genes involved in telomerase activity, as well as anti-oxidant, autophagic, and anti-apoptotic genes under conditions of CR, and suggest that FoxO1 signaling may be an important mediator of metabolic equilibrium during CR.

Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme.

This study aimed to examine whether an elevated activity of heme oxygenase (HO)-1 in the tissue attenuates endothelial cell-leukocyte interactions microvessels in vivo. When rats were pretreated with an intraperitoneal injection of hemin, an HO-1 inducer, mesenteric tissues, including their microvessels, displayed a marked induction of HO-1 concurrent with an increase in plasma concentrations of bilirubin-IXalpha, the product of HO-catalyzed degradation of protoheme IX. In these rats, oxidative stress such as superfusion with H(2)O(2) and ischemia-reperfusion of the tissues neither induced rolling nor exhibited adherent responses of leukocytes in venules. In contrast, the oxidative stresses evoked marked rolling and adhesion of leukocytes in the control rats without HO-1 induction. The HO-1 induction also downregulated leukocyte adhesion elicited by other pro-oxidant stimuli such as N(omega)-nitro-L-arginine methyl ester. The decreases in the oxidant-elicited leukocyte adhesive responses under HO-1-inducing conditions were restored by perfusion with zinc protoporphyrin-IX, an HO inhibitor, but not with copper protoporphyrin-IX, which did not inhibit the enzyme. Furthermore, the effects of zinc protoporphyrin-IX were repressed by superfusion with bilirubin or biliverdin at the micromolar level, but not by the same concentration of carbon monoxide, another product of HO. These results indicate that induction of the HO-1 activity serves as a potential stratagem to prevent oxidant-induced microvascular leukocyte adhesion through the action of bilirubin, a product of HO reaction.

Role of oligomeric interactions in the cooperativity of crayfish hemocyanin.

To examine the effect of interactions between the hexameric units in the dodecameric hemocyanin, crayfish (Procambarus clarki) hemocyanin was partially dissociated. Gel-filtration yielded fractions containing the undissociated dodecamer and a mixture of the hexamer and heptamer (referred to as 'half-dodecamer'). Their O2 equilibria were compared and were analyzed by curve fitting of cooperativity models. The partial dissociation of the dodecamer significantly lowered the cooperativity but little affected the O2 affinities. The O2 equilibrium of the half-dodecamer could be described by the two-state allosteric model of Monod-Wyman-Changeux on the assumption that the cooperative unit is composed of six subunits. We tested the applicability of allosteric models for the dodecamer that deal with the interactions between the hexamers. To reduce the arbitrariness of the fitting, we presumed that some parameters are equal to the two-state model parameters for the half-dodecamer. It was found that the 'nesting model', which assumes allosteric equilibria at two different hierarchical levels, could be applied successfully to the dodecamer.

Effect of dietary omega-3 eicosapentaenoic acid supplements on cholesteryl ester transfer from HDL in cholesterol-fed rabbits.

We investigated the reactivities of cholesteryl ester transfer protein (CETP) in Japanese white rabbits fed either a low-cholesterol diet containing 0.1% cholesterol (Control group) or a diet containing 0.1% cholesterol plus 17.5% omega-3 eicosapentaenoic acid (omega-3 20:5, EPA) of 4.5% (w/w) total lipid (EPA group) for 6 weeks. The plasma total and LDL cholesterol levels and aortic cholesterol content were all significantly higher in the EPA group than in the control group. The aortic cholesterol content significantly correlated with LDL cholesterol (r = 0.81). HDL cholesterol levels tended to be lower in the EPA group compared with control group, which was not statistically significant. The plasma VLDL cholesterol levels did not differ significantly between the groups. In addition, no significant differences were observed in the plasma CETP activity or lecithin:cholesterol acyltransferase (LCAT) activity between the groups. However, the cholesteryl ester (CE) mass transfer from fractionated HDL in the EPA group to excess VLDL and/or LDL as acceptors by purified CETP increased significantly compared with the control group, even if the acceptors were fractionated from either the EPA or the control group. Fatty acid analyses of CE showed that the omega-3 18:3, 20:4 or omega-3 20:5 fatty acid acyl groups in CE of HDL were significantly more transferred to apo B-containing lipoproteins compared with the 14:0,16:0, 18:0, 18:1 or 18:2 fatty acid acyl groups in CE of HDL during the incubation period. The amount of CE in HDL containing omega-3 18:3 and omega-3 20:5 fatty acid acyl groups was greater, while the amount of CE containing 18:2 fatty acid acyl groups was smaller in the EPA group than in the control group. These results show that although CETP itself did not change, the transfer of CE in HDL to apo B-containing lipoproteins by CETP increased in the rabbits fed a diet containing EPA as the HDL is modified by the diet, which may partly explain why atherogenicity was thus found to progress in the rabbits fed a cholesterol plus EPA diet.

Kinetic studies on the removal of extracellular tert-butyl hydroperoxide by cultured fibroblasts.

In fibroblasts toxic hydroperoxides are removed mainly by GSH peroxidase. The reaction depends on NADPH, since GSSG must be reduced by GSSG reductase for recycling. In this work we have studied the kinetics of tert-butyl hydroperoxide (tBH) removal by cultured fibroblasts in relation to the GSSG reduction. The rate of the reaction showed biphasic dependence on tBH concentration. About a third of the reaction was saturated below 10 microM tBH, while the rest of the reaction showed less steep dependence, reaching a plateau at 200 microM tBH. The latter reaction is thought to be due to GSH peroxidase, and the concentration dependence could be explained on the basis of reaction kinetics of GSH peroxidase and GSSG reductase. The maximum rate of tBH removal was estimated as 40-50 nmol tBH/min/mg of protein, while the glutathione reductase activity is the solubilized cell was 33.0 +/- 3.5 nmol GSSG/min/mg of protein. It was concluded that, under the oxidative stress as in the present experiments, the step catalyzed by GSSG reductase is rate-limiting in the reaction sequence.

Kinetics of hydrogen peroxide elimination by human umbilical vein endothelial cells in culture.

H2O2 is a key substance in the oxidative stress. To evaluate the antioxidant activity of intact human umbilical vein endothelial cells (HUVEC), we measured the H2O2 removal rate by the cell in monolayer culture at various H2O2 concentrations (1-300 microM). It was shown that the removal reaction can be divided into two kinetically different reactions: reaction 1 apparently following the Michaelis-Menten kinetics and reaction 2 following the first-order kinetics. Reaction 1, which was diminished by treatment with diethyl maleate, could be attributed to GPx. Reaction 2, which was inhibited by aminotriazole, was principally attributable to catalase, though non-enzymatic reactions may contribute to it partially. Furthermore, we have constructed a mathematical model for the H2O2 elimination including the pentose phosphate pathway enzymes, GSSG reductase and GSH peroxidase. On the basis of the known kinetics and observed activities of the enzymes, the model could reproduce well the observed concentration dependence of the H2O2 removal rate. It was suggested from the simulation study that GSSG reductase is more important than G6PD in determining the rate of the NADPH-dependent H2O2 elimination.

Stimulation of Ca2+-pump in rat heart sarcolemma by phosphatidylethanolamine N-methylation.

Incubation of purified cardiac sarcolemmal vesicles (SL) in the presence of S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation of phosphatidylethanolamine (PE), increased the Ca2+-stimulated ATPase and ATP-dependent Ca2+ accumulation activities. Quantitative analysis of the methylated phospholipids revealed that maximal increase of Ca2+-pump activities was associated with predominant synthesis and intramembranal accumulation of phosphatidyl-N,N-dimethylethanolamine. The stimulation of SL Ca2+-pump activities was prevented by inhibitors of PE N-methylation such as S-adenosyl-L-homocysteine and methyl acetimidate hydrochloride. The results suggest a possible role of PE N-methylation in the regulation of Ca2+-transport across the heart SL membrane.

Chronic antisense therapy for angiotensinogen on cardiac hypertrophy in spontaneously hypertensive rats.

OBJECTIVE: We examined the effect of the suppression of plasma angiotensinogen (AGT) by the intravenous injection of antisense oligodeoxynucleotides (ODNs) against AGT targeted to the liver on cardiac remodeling in spontaneously hypertensive rats (SHR). The ODNs against rat AGT were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for regulating liver gene expression. METHODS: Male SHR (n = 18), and age-matched male Wistar-Kyoto rats (WKY, n = 6) were used for this study. At 10 weeks of age, the SHR were divided into three groups (each group n = 6), and the systolic blood pressure (SBP) did not significantly change among them. The control SHR and WKY groups received saline, the sense SHR group was injected with the sense ODNs complex and the antisense SHR group was injected with the antisense ODNs complex, from 10 to 20 weeks of age. ASOR-poly(L)lysine-ODNs complex was injected via the tail veins twice a week. RESULTS: At the end of the treatment, a reduction of hepatic AGT mRNA, cardiac angiotensin II type 1 receptor mRNA and the plasma AGT concentration was only observed in the antisense-injected SHR but not in the other groups of SHR and WKY. This antisense therapy did not significantly change the mRNA expression for angiotensin converting enzyme, angiotensin II type 2 receptor and AGT in the left ventricle (LV) among all three groups. Although the plasma angiotensin II (Ang II) concentration significantly decreased to the level of WKY after the antisense therapy, the SBP, LV to body weight ratio and % collagen volume fraction also showed a reduction, however, these findings were still larger than in the WKY than in either the sense-injected SHR or control SHR. CONCLUSION: The plasma AGT is considered to play a role in the development of cardiac hypertrophy in SHR, but it has not a complete effects on cardiac remodeling even if the plasma Ang II levels are inhibited because of an insufficient suppression of hypertension.

Status of myocardial antioxidants in ischemia-reperfusion injury.

BACKGROUND: Myocardial ischemia-reperfusion represents a clinically relevant problem associated with thrombolysis, angioplasty and coronary bypass surgery. Injury of myocardium due to ischemia-reperfusion includes cardiac contractile dysfunction, arrhythmias as well as irreversible myocyte damage. These changes are considered to be the consequence of imbalance between the formation of oxidants and the availability of endogenous antioxidants in the heart. OBSERVATIONS: An increase in the formation of reactive oxygen species during ischemia-reperfusion and the adverse effects of oxyradicals on myocardium have now been well established by both direct and indirect measurements. Although several experimental studies as well as clinical trials have demonstrated the cardioprotective effects of antioxidants, some studies have failed to substantiate the results. Nonetheless, it is becoming evident that some of the endogenous antioxidants such as glutathione peroxidase, superoxide dismutase, and catalase act as a primary defense mechanism whereas the others including vitamin E may play a secondary role for attenuating the ischemia-reperfusion injury. The importance of various endogenous antioxidants in suppressing oxidative stress is evident from the depression in their activities and the inhibition of cardiac alterations which they produce during ischemia-reperfusion injury. The effects of an antioxidant thiol containing compound, N-acetylcysteine, and ischemic preconditioning were shown to be similar in preventing changes in the ischemic-reperfused hearts. CONCLUSIONS: The available evidence support the role of oxidative stress in ischemia-reperfusion injury and emphasize the importance of antioxidant mechanisms in cardioprotection.

Molecular mechanism of angiotensin II type I and type II receptors in cardiac hypertrophy of spontaneously hypertensive rats.

We administered angiotensin (Ang) II receptor type 1 (AT1) blockade (losartan; 10 or 40 mg/kg per day), type II receptor (AT2) blockades (PD123319; 100 mg/kg per day), or angiotensin-converting enzyme (ACE) inhibitor (enalapril; 30 mg/kg per day) to spontaneously hypertensive rats (SHR) from 10 to 20 weeks of age. At the end of the treatment, high doses of losartan and enalapril significantly reduced the arterial systolic blood pressure compared with the untreated SHR to the level of WKY rats. But low doses of losartan and PD123319 were without effect. High doses of losartan and enalapril also significantly reduced both the left ventricular (LV) weight and the ratio of LV to body weight compared with the untreated SHR, which were still larger than that of WKY rats. However, the collagen concentration of SHR treated with high doses of losartan or enalapril was completely reduced to the level of WKY rats. Using reverse transcription polymerase chain reaction, we examined the mRNA expression for ACE, AT1, and AT2 in experimental animals. The enhanced AT1 mRNA expression was significantly decreased in the SHR treated with a high dose of losartan or PD123319 compared with the untreated SHR. The level of ACE mRNA was also decreased in the SHR treated with a high dose of losartan or enalapril. The level of AT2 mRNA was not significantly different between the Wistar-Kyoto rats and the SHR; however, this expression was decreased significantly after the treatment with a high dose of losartan or PD123319. These results indicate that AT1 receptor and ACE, but not AT2 receptor, play a crucial role in the remodeling of matrix tissue but a smaller role in the development of the hypertrophy of LV myocyte in SHR and that the LV/body weight changes do not fully account for the complete suppression of hypertension.

Intravenous injection with antisense oligodeoxynucleotides against angiotensinogen decreases blood pressure in spontaneously hypertensive rats.

In the renin-angiotensin system, renin is known to cleave angiotensinogen to generate angiotensin I, which is the precursor of angiotensin II. Angiotensin II is a vasoactive peptide that plays an important role in blood pressure. On the other hand, the liver is the major organ responsible for the production of angiotensinogen in spontaneously hypertensive rats (SHR). To test the hypothesis that a reduction of angiotensinogen mRNA in the liver by antisense oligodeoxynucleotides (ODNs) may affect both plasma angiotensinogen and angiotensin II levels, as well as blood pressure, we intravenously injected antisense ODNs against rat angiotensinogen coupled to asialoglycoprotein carrier molecules, which serve as an important regulator of liver gene expression, into SHR via the tail vein. The SHR used in the present study were studied at 20 weeks of age and were fed a standard diet throughout the experiment. Plasma angiotensinogen, angiotensin II concentrations, and blood pressure all decreased from the next day until up to 5 days after the injection of antisense ODNs. These concentrations thereafter returned to baseline by 7 days after injection. A reduction in the level of hepatic angiotensinogen mRNA was also observed from the day after injection until 5 days after injection with antisense ODNs. However, in the SHR injected with sense ODNs, plasma angiotensinogen, angiotensin II concentrations, and blood pressure, as well as hepatic angiotensinogen mRNA, did not significantly change throughout the experimental period. Although the exact role of angiotensinogen in hypertension still remains to be clarified, these findings showed that intravenous injection with antisense ODNs against angiotensinogen coupled to asialoglycoprotein carrier molecules targeted to the liver could thus inhibit plasma angiotensinogen levels and, as a result, induce a decrease in blood pressure in SHR.

Interaction of mRNAs for angiotensin II type 1 and type 2 receptors to vascular remodeling in spontaneously hypertensive rats.

We administered angiotensin II (Ang II) receptor type 1 (AT1) blockade (losartan, 40 mg x kg-1 x d-1), type II receptor (AT2) blockade (PD123319, 100 mg x kg-1 x d-1), or angiotensin-converting enzyme (ACE) inhibitor (enalapril, 30 mg x kg-1 x d-1) to spontaneously hypertensive rats (SHR) from 10 to 20 weeks of age. Control SHR and Wister-Kyoto rats (WKY) received a placebo for the same period. At the end of treatment, losartan and enalapril were both found to have significantly reduced the arterial systolic blood pressure and the collagen concentration to the level of WKY, whereas PD123319 had no effect. Enalapril and PD123319 significantly reduced the media cross-sectional area of the aorta in comparison to that of untreated SHR, which was still larger than that of the WKY; however, losartan did not change it. Using reverse transcription-polymerase chain reaction, we next examined the mRNA expressions for ACE, AT1 receptor, and AT2 receptor in experimental animals. We observed significantly enhanced mRNA expression for AT1 and AT2 receptors and ACE in untreated SHR compared with WKY. The AT1 mRNA level was also significantly decreased in the SHR treated with either losartan or enalapril, whereas the AT2 mRNA level was significantly decreased in the SHR treated with either PD123319 or enalapril in comparison to untreated SHR. The level of ACE mRNA was significantly decreased only in the SHR treated with enalapril. These results indicate that AT1 receptor, but not AT2 receptor, plays a crucial role in the remodeling of matrix tissue, while AT2 receptor may play a role in the development of hypertrophy of smooth muscle in aorta in SHR, and that the reduction of hypertrophy of smooth muscle does not fully account for the suppression of hypertension.

The effects of renin-angiotensin system inhibition on aortic cholesterol content in cholesterol-fed rabbits.

To investigate how the renin-angiotensin system (RAS) might be involved in cholesterol-induced atherosclerosis, we studied the effects of a nonsulhydryl angiotensin converting enzyme (ACE) inhibitor, enalapril, and an angiotensin II receptor antagonist, E-4177, in cholesterol fed rabbits. Japanese white rabbits were randomly divided into four groups with the following dietary regimens: group A (n = 8) received a standard diet; group B (n = 8) had a 0.5% cholesterol diet; group C (n = 8) had a 0.5% cholesterol diet plus enalapril (10 mg/kg/day, p.o.); group D (n = 8) received a 0.5% cholesterol diet plus E-4177 (20 mg/kg/day, p.o.) and were fed these diets for 5 weeks. Enalapril or E-4177 had no significant effect on either the total plasma or the high density lipoprotein (HDL) cholesterol concentrations. However, the aortic cholesterol content in groups C and D was equally significantly less than that in group B. The plasma and aortic ACE activities were significantly reduced only in group C compared with those in the other groups. The aortic ACE mRNA and AT1 mRNA levels were assessed by a reverse transcription polymerase chain reaction (RT-PCR). The aortic ACE mRNA level was only significantly less in group C than in any of the other groups. The aortic AT1 mRNA level increased significantly in group B compared with that in group A and was significantly and equally reduced in both groups C and D compared with that in group B. These data indicate that angiotensin II rather than ACE may therefore be related to aortic cholesterol content. It follows therefore that the inhibition of angiotensin II by either ACE inhibitor or angiotensin II (type 1) receptor antagonist may play a role in prevention of atherosclerosis.

Secretion of prebeta HDL increases with the suppression of cholesteryl ester transfer protein in Hep G2 cells.

Prebeta HDL are small, protein rich lipoproteins that are predominantly composed of apo A-I, without apo A-II. Prebeta HDL are secreted from the liver as nascent HDL and/or are produced in the incubated plasma by cholesteryl ester transfer protein (CETP). However, the role of CETP in the secretion of HDL from the liver has yet to be determined. In the present study, we examined the effect of the suppression of hepatic CETP by antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the secretion of apo A-I using a Hep G2 cell culture. The ODNs against CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for the regulation of liver gene expression. Hep G2 cells were cultured in DMEM supplemented with 10 FBS. After 2 days, the medium was changed to DMEM with EGF and the cells were divided into three groups. The control group received saline, while the sense group was mixed with the sense ODNs complex and the antisense group was mixed with the antisense ODNs complex, respectively, for 2 days. Both the hepatic CETP mRNA and the CETP mass in the medium in the antisense group decreased significantly more than in the sense and the control groups (CETP mass: 1.697 + /- 0.410 ng/mg cell protein vs. 2.367 + /- 0.22 and 2.360 + /- 0.139, n = 3 in each determination). In contrast, both the hepatic apo A-I mRNA and the apo A-I mass in the medium in the antisense group were significantly higher than those in the sense and the control groups (apo A-I mass; 1.877 + /- 0.215 micro/mg cell protein vs. 1.213 + /- 0.282 and 1.097 + /- 0.144, n = 3 in each determination). The increase in apo A-I was mainly due to the increase in prebeta apo A-I. These findings may partly explain why HDL and apo A-I increase in patients with CETP deficiency, while also indicating the possibility that the original level of prebeta HDL is sufficient in such patients.

Reduction of plasma angiotensin II to normal levels by antisense oligodeoxynucleotides against liver angiotensinogen cannot completely attenuate vascular remodeling in spontaneously hypertensive rats.

OBJECTIVE: The exact role of angiotensinogen (AGT) in vascular remodeling has yet to be determined. In the present study, we examined the effects of reducing plasma AGT by intravenous injections with antisense oligodeoxynucleotides (ODNs) against AGT targeted to the liver on vascular remodeling in spontaneously hypertensive rats (SHRs). DESIGN AND METHODS: The ODNs against rat AGT were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for regulating liver gene expression. Male SHRs (n = 18) and age-matched male Wistar- Kyoto (WKY) rats (n = 4) were used for this study. All animals were fed a standard rat diet throughout the experiment At 10 weeks of age, the SHRs were divided into three groups (n = 6); systolic blood pressure (SBP) was similar in each group. The control group received saline, the sense group was injected with the sense ODN complex and the antisense group was injected with the antisense ODN complex. WKY rats were fed for the same period of time. The ASOR-poly(L)lysine-ODN complex was injected into the tail veins twice a week. RESULTS: At the end of the treatment, a reduction in AGT mRNA levels in the liver and plasma AGT was observed only in the animals injected with antisense ODNs. Antisense ODNs significantly reduced the plasma angiotensin II (Ang II) concentrations to levels similar to those observed in WKY rats. Antisense ODNs significantly reduced the SBP (180.7 +/- 4.4 mmHg) and media cross-sectional areas of the aorta (1.11 +/- 0.02 mm2), which were still larger than those seen in WKY rats (140.3 +/- 2.1 mmHg, 0.84 +/- 0.02 mm2), compared with the SHRs injected with sense ODNs (225.2 +/- 4.4 mmHg, 1.24 +/- 0.02 mm2) and control SHRs (223.7 +/- 4.8 mmHg, 1.25 +/- 0.02 mm2). The aortic angiotensin-converting enzyme (ACE) activity and collagen concentrations, which were significantly higher than those seen in WKY rats, did not significantly change among the SHR groups. The aortic AGT, ACE, angiotensin II type 1 (AT1) receptor and angiotensin II type 2 (AT2) receptor mRNA also did not significantly change among the SHR groups. CONCLUSION: On the basis of these findings, plasma AGT is thus considered to play a role in the development of hypertrophy of smooth muscle in the aorta of SHRs, it is thought to have only a slight effect, however, on the remodeling of the matrix tissue when the suppression of hypertension is insufficient.

Modulation of adrenergic receptors during regression of cardiac hypertrophy.

OBJECTIVE: To determine whether alpha 1- or beta-adrenergic receptors are altered during regression of cardiac hypertrophy produced by antihypertensive agents. DESIGN AND METHODS: Cardiac hypertrophy was induced in rats by aortic banding. After 6 weeks banding the rats were treated with an angiotensin converting enzyme (ACE) inhibitor (enalapril), an alpha 1-adrenergic antagonist (bunazosin) or a beta-adrenergic antagonist (propranolol) for 6 weeks to induce regression. The numbers of alpha 1- and beta-adrenergic receptors, haemodynamics, tissue noradrenaline content and tissue ACE activity were measured. RESULTS: Regression of cardiac hypertrophy occurred after treatment of aortic banded rats with a high dose of enalapril, bunazosin or propranolol, and was accompanied by a reduction in systolic blood pressure. The number of alpha 1- or beta-adrenergic receptors was unchanged by propranolol treatment, but the number of alpha 1-adrenergic receptors was increased in the hearts of rats treated with bunazosin. A low dose of enalapril (3 mg/kg body weight) caused regression of hypertrophy without a concomitant reduction in blood pressure, and decreased the number of alpha 1-adrenergic receptors. The dissociation constants for alpha 1- and beta-adrenergic receptors were not different among the experimental groups, and the positive derivatives of left ventricular pressure was unaltered in rats treated with a low dose of enalapril but was reduced by the other drugs. CONCLUSION: Of the three drugs tested, only the low dose of enalapril affected adrenergic receptors during regression of cardiac hypertrophy, causing a decrease in alpha 1-adrenergic receptor number without a reduction in blood pressure. This effect may be explained by non-haemodynamic actions of the ACE inhibitor enalapril, probably by modulation of peripheral sympathetic activity.

The characteristics of erythrocyte Na+ transport systems in normal pregnancy and pregnancy-induced hypertension.

OBJECTIVE: To assess the role Na plays in the pathogenesis of pregnancy-induced hypertension (PIH). METHODS: We assessed Na and K content, the maximum number of ouabain binding sites, Na(+)-Li+ countertransport and Na(+)-K+ cotransport in erythrocytes from women with untreated PIH, normal pregnant women and healthy non-pregnant women. RESULTS: In normal pregnancy, the Na content of erythrocytes decreased, accompanied by the activation of Na excretion systems. In women with PIH, the Na content of erythrocytes and the Na(+)-K+ cotransport activity significantly increased, whilst erythrocyte K content and the maximum number of ouabain binding sites significantly decreased, compared with observations in normal pregnancy. In both normal pregnancy and PIH, there were no differences in Na(+)-Li+ countertransport. CONCLUSIONS: These results suggest that the increase of erythrocyte Na content in women with PIH may be contributed to by a reduction in the number of ouabain binding sites, whilst Na(+)-K+ cotransport and Na(+)-Li+ countertransport may compensate for this effect in women with PIH.

Hemocyanin from Tachypleus gigas. I. Oxygen-binding properties.

Hemocyanin was prepared from an Asian horseshoe crab, Tachypleus gigas. The hemocyanin was found to be similar to Limulus hemocyanin in the size of native molecules (48-mer) and dissociation under nonphysiological conditions. It also showed the reverse Bohr effect. The O2 affinity of the dissociated monomer was higher than that of the native molecule. Equilibrium O2 binding to T. gigas hemocyanin was studied with special attention to the effect of inorganic ions. Neutral salts decreased the O2 affinity of the associated hemocyanin. In the presence of CaCl2 the strength of the effect was in the order of Na+ greater than Cs+ not equal to K+ for the series of chlorides, and Br- not equal to Cl- greater than SO4(2-) for the series of Na+ salts. A high concentration of CaCl2 (50-500 mM) considerably increased the Hill coefficient. The O2 binding data obtained under various ionic conditions were analyzed by model fitting. The two-state concerted model could be fitted to the data, if the ligand affinity of the states was allowed to vary. Statistical tests of the fitting showed that the hexameric structure can be regarded as the functional unit under physiological conditions.

Hemocyanin from Tachypleus gigas. II. Cooperative interactions of the subunits.

Six subunits (I to VI) were isolated from hemocyanin of an Asian horseshoe crab, Tachypleus gigas, by anion exchange chromatography of the dissociated hemocyanin. The subunit preparations were nearly homogeneous as judged by alkaline electrophoresis, but they still showed the presence of isoproteins in isoelectric focusing. The subunits were reassembled (in 10 mM CaCl2 at pH 7.5) and tested for restoration of the cooperativity in O2 binding. The reassembly of the subunits gave equilibrium mixtures of the monomer and hexamer with small amounts of larger molecules. Homogeneous and heterogeneous hexamers were prepared by reassembling a single kind or two kinds of subunits, followed by isolation of the hexamer fraction by gel filtration. Among the homohexamers, only the subunit V hexamer showed cooperativity in O2 binding with the Hill coefficient of 1.6. Among the heterohexamers the subunit I/V hybrid was most noteworthy, showing a Hill coefficient (1.7) higher than that of any other heterohexamer examined. It was concluded that there are specific interactions between the subunits I and V. It is suggested that their interactions are important for the cooperativity in the native hemocyanin.

Oxygenation-linked changes in the ultraviolet absorption and circular dichroism spectra of spiny lobster hemocyanin.

As an approach to elucidate the mechanism of the protein structure change in the cooperative ligand binding, the UV difference and CD spectra of aromatic residues in Panulirus japonicus (spiny lobster) hemocyanin were examined. The native hemocyanin showed an O2-induced narrow-banded change in the absorption spectrum around 290 nm, which was not affected by pH in the range of 7.5 to 9.5. When the native hexameric protein was stripped of divalent cations with EDTA (at pH 7.5), the magnitude of the narrow-banded difference was reduced to about half, whereas it was almost completely abolished on dissociation into subunits (stripped at pH 9.5). The magnitude of the absorption change was found to be proportional to the degree of O2 saturation in the native and stripped hemocyanins. It was inferred that the spectral difference reflects a tertiary structure change directly linked to the oxygenation, though it depends greatly on the subunit association. Panulirus hemocyanin showed negative CD bands in the region of 260 to 300 nm, the intensities of which were considerably reduced by oxygenation and also by dissociation into subunits.