Concentration-dependent effects of insulin on Ca2+ influx in vascular smooth muscle cells of normotensive and spontaneously hypertensive rats

The levels of CD26 expression, their capacity to induce protein tyrosine phosphorylation and their functional implication in natural killer (NK) cytolysis have been studied. It was found that only a small fraction (12-15%) of peripheral NK cells expresses CD26 compared with the high expression (99%) found in NK clones. The protein tyrosine phosphorylation mediated by means of CD26 activation was studied in NK cells treated with the anti-CD26 MoAb 134-2C2, and two new proteins of 50 and 21 kDa appeared phosphorylated in tyrosine residues. To study the influence of CD26 antigen in NK lysis, we analysed the lytic capacity of NK cells stimulated with different anti-CD26 MoAbs or after separation into CD26+ and CD26- subsets and using K562 as target cells. Under these conditions, no differences were found in the chromium release by the target cells. Redirected lysis through CD16 was also measured by arming the effector cells (CD26+ and CD26-) with anti-CD16 antibody and using K562 as target cells. It was found that CD26- cells showed significantly less CD16-dependent lysis than CD26+ cells. These results indicate that CD26 is related to the CD16-dependent lysis but not to NK cytolysis which may be caused by mediation of protein tyrosine phosphorylation.     

Different calcium storage pools in vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats

OBJECTIVE: To evaluate whether the distribution of intracellular free calcium may be impaired in primary hypertension. DESIGN: Cytosolic free calcium and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR). METHODS: The concentrations of intracellular and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats aged 6 months from the Münster strain (SHR) and from age-matched normotensive Wistar-Kyoto (WKY) rats. Vascular smooth muscle cells were grown on coverslips, and fluorescence measurements of the intracellular calcium concentration were performed using fura-2. The different effects of thapsigargin, a selective Ca-ATPase inhibitor, and of angiotensin II (Ang II) on the calcium storage pools were investigated. RESULTS: In the absence of external calcium thapsigargin produced a dose-dependent transient increase in the concentration of intracellular calcium in vascular smooth muscle cells. The thapsigargin-induced maximum peak increase in the concentration of intracellular calcium was not significantly different in SHR and WKY rats. After depletion of the thapsigargin-sensitive calcium pools the addition of 100 nmol/l Ang II produced a rise in the concentration of intracellular calcium in vascular smooth muscle cells from SHR and WKY rats. Using vascular smooth muscle cells from the SHR the Ang II-induced increase in the concentration of intracellular calcium was not significantly different in the presence and absence of thapsigargin, indicating that the calcium pools depleted by thapsigargin and Ang II do not overlap significantly in vascular smooth muscle cells from SHR. In contrast, in the WKY rats the response to Ang II was significantly diminished after depletion of the thapsigargin-sensitive pool. When Ang II and thapsigargin were administered in the reverse order, i.e. Ang II before thapsigargin, the thapsigargin response was diminished in the WKY rats but not in the SHR. CONCLUSION: SHR differ from WKY rats in having vascular smooth muscle cells that contain thapsigargin-sensitive calcium storage pools that are distinct from the Ang II-sensitive calcium pools.     

Transmembrane Ca2+ gradient-mediated phosphatidylcholine modulating sarcoplasmic reticulum C(a2+)-ATPase

The sarcoplasmic reticulum (SR) C(a2+)-ATPase was purified and reconstituted into the sealed phospholipids vesicles with or without transmembrane Ca2+ gradient. The role of phospholipids, especially phosphatidylcholine (PC), in the modulation of C(a2+)-ATPase by transmembrane Ca2+ gradient was investigated. The results are as follows. (i) Incubated with phospholipids, the enzyme activity of the delipidated C(a2+)-ATPase is inhibited by Ca2+ and the highest inhibition is observed in the presence of PC. (ii) When there exists a transmembrane Ca2+ gradient (higher Ca2+ concentration inside vesicles, 1,000 mumol/L:50 mumol/L, similar to the physiological condition), the inhibition of C(a2+)-ATPase by transmembrane Ca2+ gradient can be only observed in the vesicles containing PC:PE, but not in those containing PS:PE or PG:PE. The highest inhibition is obtained at a 50:50 molar ratio of PC:PE (iii) By comparing the effects of PC differing in acyl chains, higher inhibition of C(a2+)-ATPase is observed in vesicles containing DPPC:PE and DOPC:PE, while no inhibition in DMPC:PE vesicles (iv) If the transmembrane Ca2+ gradient is in the inverse direction, the enzyme activity of C(a2+)-ATPase is inhibited whenever reconstituted with acidic or neutral phospholipids.     

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum

Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase.     

Enhanced inhibition by melatonin of alpha-adrenoceptor-induced aortic contraction and inositol phosphate production in vascular smooth muscle cells from spontaneously hypertensive rats

PURPOSE: To theoretically derive and empirically validate the relationship between the actual thick intraocular lens and the thin lens equivalent. METHODS: Included in the study were 12 consecutive adult patients ranging in age from 54 to 84 years (mean +/- SD, 73.5 +/- 9.4 years) with best-corrected visual acuity better than 20/40 in each eye. Each patient had bilateral intraocular lens implants of the same style, placed in the same location (bag or sulcus) by the same surgeon. Preoperatively, axial length, keratometry, refraction, and vertex distance were measured. Postoperatively, keratometry, refraction, vertex distance, and the distance from the vertex of the cornea to the anterior vertex of the intraocular lens (AV(PC1)) were measured. Alternatively, the distance (AV(PC1)) was then back-calculated from the vergence formula used for intraocular lens power calculations. RESULTS: The average (+/-SD) of the absolute difference in the two methods was 0.23 +/- 0.18 mm, which would translate to approximately 0.46 diopters. There was no statistical difference between the measured and calculated values; the Pearson product-moment correlation coefficient from linear regression was 0.85 (r2 = .72, F = 56). The average intereye difference was -0.030 mm (SD, 0.141 mm; SEM, 0.043 mm) using the measurement method and +0.124 mm (SD, 0.412 mm; SEM, 0.124 mm) using the calculation method. CONCLUSION: The relationship between the actual thick intraocular lens and the thin lens equivalent has been determined theoretically and demonstrated empirically. This validation provides the manufacturer and surgeon additional confidence and utility for lens constants used in intraocular lens power calculations.     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca2+-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca2+-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca2+. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca2+-activated K+ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca2+ influx was triggered through the Na+/Ca2+ exchanger and the other Ca2+ entry pathways were blocked. Thapsigargin, another specific SR Ca2+-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca2+-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca2+ influx presumably via nifedipine-sensitive Ca2+ channels. This study suggests the important role of SR Ca2+-ATPase in the modulation of the muscle tone and the function of SR as a "buffer barrier" to Ca2+ entry in the cat gastric fundus smooth muscle.     

Inhibition of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase by nitric oxide

The effects of nitric oxide on the activities of thapsigargin-sensitive sarcoplasmic reticulum Ca2+-ATPase (SERCA) and Ca2+ uptake by sarcoplasmic reticulum (SR) membranes prepared from white skeletal muscle of rabbit femoral muscle were studied. Pretreatment of the SR preparations with nitric oxide at concentrations of up to 250 microM for 1 min decreased the SERCA activity concentration dependently, and also decreased their Ca2+ uptake. Both these effects of nitric oxide were reversible. Inhibitors of guanylyl cyclase and protein kinase G (PKG) had no significant effect on the nitric oxide-induced inhibitions of SERCA and Ca2+ uptake. Moreover, dithiothreitol did not reverse the inhibitory effects of nitric oxide on SERCA and Ca2+ uptake. These findings suggest that nitric oxide inhibits SERCA, mainly SERCA 1, of rabbit femoral skeletal muscle by an action independent of the cyclic GMP-PKG system or oxidation of thiols, and probably by a direct action on SERCA protein.     

Irreversible inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport by 4-OH-2,3-trans-nonenal

4-OH-2,3-trans-nonenal (HNE), a major aldehydic lipid peroxidation product, has been shown to cause cellular toxicities and has been linked to a number of pathophysiological processes including atherogenesis. Specifically, in vitro exposure of erythrocyte plasma membrane preparations to HNE resulted in the inhibition of membrane transport function and integrity. To characterize the nature of the inhibitory effects of HNE on plasma membrane regulatory mechanisms, we investigated its effects on substrate and calmodulin (CaM) stimulation on erythrocyte Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities. Concentration-effect relationship analysis in erythrocyte membrane "ghosts" and inside-out vesicles (IOVs) yielded purely noncompetitive kinetics for Ca2+, ATP, and CaM activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport. Reductions of Vmax from direct addition of 0.1 mM HNE to the assay incubation mixtures ranged from 23 to 41%. Similarly, pretreatment with HNE of both membrane ghosts and IOVs resulted in a concentration-dependent inactivation of ATPase and transport activities without changes in affinity for Ca2+, ATP, or CaM. Conversely, pretreatment of CaM itself did not impair its ability to stimulate (Ca2+ + Mg2+)-ATPase activity threefold. Moreover, HNE-pretreated membranes exhibited unaltered acetylcholinesterase activity compared to sham-pretreated membranes. Together, these results suggest that HNE may structurally, and thus irreversibly, modify one or more functionally important sites on the transport protein itself.     

The oxidative inactivation of sarcoplasmic reticulum Ca(2+)-ATPase by peroxynitrite

The oxidative inactivation of rabbit skeletal muscle Ca(2+)-ATPase in sarcoplasmic reticulum (SR) vesicles by peroxynitrite (ONOO-) was investigated. The exposure of SR vesicles (10 mg/ml protein) to low peroxynitrite concentrations ( < or = 0.2 mM) resulted in a decrease of Ca(2+)-ATPase activity primarily through oxidation of sulfhydryl groups. Most of this deactivation (ca.70%) could be chemically reversed by subsequent reduction of the enzyme with either dithiothreitol (DTT) or sodium borohydride (NaBH4), indicating that free cysteine groups were oxidized to disulfides. The initial presence of 5 mM glutathione failed to protect the SR Ca(2+)-ATPase activity. However, as long as peroxynitrite concentrations were kept < or = 0.45 mM, the efficacy of DTT to reverse Ca(2+)-ATPase inactivation was enhanced for reaction mixtures which initially contained 5 mM glutathione. At least part of the disulfides were formed intermolecularly since gel electrophoresis revealed protein aggregation which could be reduced under reducing conditions. The application of higher peroxynitrite concentrations ( > or = 0.45 mM) resulted in Ca(2+)-ATPase inactivation which could not be restored by exposure of the modified protein to reducing agents. On the other hand, treatment of modified protein with NaBH4 recovered all SR protein thiols. This result indicates that possibly the oxidation of other amino acids contributes to enzyme inactivation, corroborated by amino acid analysis which revealed some additional targets for peroxynitrite or peroxynitrite-induced processes such as Met, Lys, Phe, Thr, Ser, Leu and Tyr. Tyr oxidation was confirmed by a significant lower sensitivity of oxidized SR proteins to the Lowry assay. However, neither bityrosine nor nitrotyrosine were formed in significant yields, as monitored by fluorescence spectroscopy and immunodetection, respectively. The Ca(2+)-ATPase of SR is involved in cellular Ca(2+)-homeostasis. Thus, peroxynitrite mediated oxidation of the Ca(2+)-ATPase might significantly contribute to the loss of Ca(2+)-homeostasis observed under biological conditions of oxidative stress.     

Effect of Paeonia lactiflora on platelet cytosolic free calcium and erythrocyte membrane Ca(2+)-Mg(2+)-ATPase activity in hyperlipid rabbits

The effect of Paeonia lactiflora (PL) on platelet cytosolic free calcium and erythrocyte membrane Ca(2+)-Mg(2+)-ATPase activity in hyperlipid rabbits were observed. Results showed the level of platelet cytosolic free calcium in the PL group (276.25 +/- 27.00 nmol/L) was significantly lower than that in the cholesterol group (390.88 +/- 70.00 nmol/L), P < 0.01, the basal and calmodulin-stimulated activities of erythocyte membrane Ca(2+)-Mg(2+)-ATP ase in PL group (0.79 +/- 0.05 mumol.pi-1.mg-1.h-1 and 1.34 +/- 0.10 mumol.pi-1.mg-1.h-1) were higher than that in the cholesterol group (0.65 +/- 0.09 mumol.pi-1.mg-1.h-1 and 1.04 +/- 0.13 mumol.pi-1.mg-1.h-1).     

Intracellular alkalinization by NH4Cl increases cytosolic Ca2+ level and tension in the rat aortic smooth muscle

Intracellular pH (pHi) is elucidated to be an important regulator of various cell functions, but the role of pHi in smooth muscle contraction remains to be clarified. The purpose of the present study is to examine the effects of cell alkalinization by exposure to NH4Cl on cytosolic Ca2+ level ([Ca2+]i) and on muscle tone. We attempted simultaneous measurements of both [Ca2+]i and contractile force in rat isolated thoracic aorta from which the endothelium was removed. NH4Cl (10-80 mM) increased both [Ca2+]i and muscle tone in the presence of external Ca2+. These responses were reproducible. The removal of Ca2+ from the nutrient solution partially inhibited the rise in [Ca2+]i and the smooth muscle contraction induced by NH4Cl. In addition, the Ca2+ channel blocker verapamil also partially attenuated the responses to NH4Cl. The NH4Cl-induced responses were gradually reduced as NH4Cl was repeatedly added in a Ca(2+)-free solution. Norepinephrine (NE, 1 microM) induced a transient increase in [Ca2+]i and sustained contraction in the absence of external Ca2+, and the subsequent application of NE had little effect on [Ca2+]i. After internal Ca2+ stores were depleted by exposure to NE, the subsequent application of NH4Cl induced increases in [Ca2+]i and tension of the aorta in a Ca(2+)-free solution. These results suggest that NH4Cl mainly evokes Ca2+ release from the internal Ca2+ stores that are not linked with adrenergic alpha-receptor and causes Ca2+ influx through voltage-dependent Ca2+ channels in the vascular smooth muscle.     

Biochemical identification of transmembrane segments of the Ca(2+)-ATPase of sarcoplasmic reticulum

The transmembrane segments of sarcoplasmic reticulum Ca(2+)-ATPase were determined by trypsinization of cytoplasmic side-out intact sarcoplasmic reticulum vesicles. The membrane portion of tryptic digest comprising the transmembrane fragments, joined by the intravesicular segments, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after labeling with fluorescein 5-maleimide in the presence of sodium dodecyl sulfate. In this way, seven fluorescent bands of tryptic fragments below 11 kDa were observed which were derived from 4 pairs of membrane spanning segments and one hydrophobic sequence at the C-terminal end. Two peptides of 10.8 and 10.6 kDa had the identical N-terminal sequence beginning at Glu826, representing the transmembrane segments M7 and M8 and their connecting loop. A band at 8.1 kDa contained one peptide beginning at Tyr36 (M1/loop/M2). A 7.7-kDa peptide starting at Leu253 (M3/loop/M4) and a 7.3-kDa peptide beginning at Ala752 (M5/loop/M6) were also observed. A band at 6.7 kDa contained two peptides, one beginning at Ser48 (M1/loop/M2) and another beginning at Tyr763 (M5/loop/M6). In addition, a 4-kDa peptide beginning at Met925 was observed. The size of this peptide did not allow for a complete pair of transmembrane segments, but this peptide could have been derived from trypsinolysis between the last pair of membrane spanning segments. These data therefore provide biochemical evidence for at least 8 transmembrane segments and perhaps two more at the C-terminal end of the enzyme.     

Control of heat produced during ATP hydrolysis by the sarcoplasmic reticulum Ca(2+)-ATPase in the absence of a Ca2+ gradient

The amount of heat produced during the hydrolysis of ATP by the sarcoplasmic reticulum Ca(2+)-ATPase was found to vary depending on the Ca2+ concentration in the medium. When the CaCl2 concentration is raised from 0.1 to 2.0 mM a part of the energy derived from ATP hydrolysis is not dissipated as heat but it is used by the enzyme to resenthesize a small fraction of the ATP previously cleaved. Thus, Ca2+ seems to regulate the ATPase in such a way as to vary the fraction of energy derived from ATP hydrolysis which is converted into heat and that which is conserved as chemical energy.     

ETA and ETB receptors on vascular smooth muscle cells from mesenteric vessels of spontaneously hypertensive rats

1. To investigate the contribution of ETA and ETB receptors, calcium responses to the ETB agonist, IRL-1620, to endothelin-1 (ET-1) and to the ETA antagonist, BQ-123, were examined in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric vessels of 3, 9 and 17 week old spontaneously hypertensive rats (SHR), Wistar and Wistar-Kyoto (WKY) rats using Fura-2 methodology. 2. IRL-1620 (10(-7) mol/L) and ET-1 (10(-9) mol/L) increased [Ca2+]i in all strains and ages. Responses to ET-1 and IRL-1620 were blunted in 17 week SHR. BQ-123 significantly reduced ET-1-stimulated [Ca2+]i. In endothelium-denuded mesenteric vessels, ET-1 and IRL-1620 induced significant [Ca2+]i responses. 3. Binding of ET-1 was significantly lower in mesenteric artery membranes from 17 week SHR compared to controls. 4. Thus, ETA and ETB receptors are present in rat mesenteric VSMC. In adult SHR, a reduced density of ET receptors results in decreased responses to IRL-1620 and to ET-1.     

Characterization of endothelium-dependent vasodilation and vasoconstriction in coronary arteries from spontaneously hypertensive rats

Coronary artery disease often occurs in patients with hypertension. The present study was designed to evaluate coronary vascular function in isolated coronary arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats and to determine the effect of antihypertensive treatment on coronary vascular responsiveness. Male SHR and WKY rats (12 to 14 weeks old) were divided into control and hydralazine-treated (120 mg/L drinking water for 10 days) groups. After 10 days, arterial pressure and heart rate were recorded while rats were conscious and unrestrained. Left ventricular coronary arteries (200 to 300 microns diameter) were isolated and intraluminal diameter was continuously recorded while vessels were maintained at a constant intraluminal pressure of 40 mm Hg. Relaxation of coronary arteries to both acetylcholine and nitroprusside was slightly, but significantly, enhanced in vessels from SHR compared to WKY rats. The enhanced relaxation was a specific effect, since isoproterenol induced similar relaxation in coronary arteries from SHR and WKY rats. Contraction to phenylephrine, but not endothelin-1, was augmented in coronary arteries from SHR compared to WKY rats. Treatment with hydralazine significantly lowered arterial pressure in SHR and WKY rats, but did not alter the enhanced contraction to phenylephrine or the enhanced relaxation to acetylcholine and nitroprusside in coronary arteris from SHR. These results indicate that coronary arteries of 12 to 14 week-old SHR do not have impaired endothelium-dependent relaxation, but to exhibit enhanced alpha-adrenoceptor-mediated contraction that is not reduced by lowering arterial pressure.     

Effect of magnesium on calcium responses to vasopressin in vascular smooth muscle cells of spontaneously hypertensive rats

PURPOSE: We discuss conventional intestinal cystoplasty and show how concern about potential complications has led to an interest in alternative methods for cystoplasty. Techniques such as gastrocystoplasty, ureterocystoplasty, vesicomyomectomy (autoaugmentation), seromuscular augmentation, alloplastic replacement and bioprosthetic materials are reviewed. Laboratory and clinical results of these techniques are examined critically to compare advantages, disadvantages and potential applications. MATERIALS AND METHODS: Computer searches of available medical data bases were used to generate a list of relevant publications, including original contributions and review articles, which were then reviewed, compared and summarized. RESULTS: Augmentation cystoplasty is used routinely for treatment of reduced bladder compliance and capacity secondary to infectious, inflammatory, neurogenic and congenital disorders. Sigmoidocystoplasty and ileocystoplasty have become standard techniques but there is renewed interest in alternative techniques due to the relatively high morbidity of intestinal cystoplasty. Alternative techniques have been described to avoid inclusion of intestinal mucosa in the urinary tract while creating a compliant bladder of adequate capacity. These techniques include gastrocystoplasty, vesicomyotomy, seromuscular augmentation, various alloplastic or biodegradable scaffolds and in vitro culture with subsequent grafting of autologous urothelium. Although encouraging animal and human results have been reported, each technique is associated with its own limitations and disadvantages. CONCLUSIONS: While intestinal cystoplasty remains the standard, several alternative techniques show promise. At present only gastrocystoplasty, ureterocystoplasty and seromuscular augmentation should be considered clinically useful.     

Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase

In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.     

Coronary hemodynamics in aging spontaneously hypertensive and normotensive Wistar-Kyoto rats

Conventionally, the hamstring:quadriceps strength ratio is calculated by dividing the maximal knee flexor (hamstring) moment by the maximal knee extensor (quadriceps) moment measured at identical angular velocity and contraction mode. The agonist-antagonist strength relationship for knee extension and flexion may, however, be better described by the more functional ratios of eccentric hamstring to concentric quadriceps moments (extension), and concentric hamstring to eccentric quadriceps moments (flexion). We compared functional and conventional isokinetic hamstring: quadriceps strength ratios and examined their relation to knee joint angle and joint angular velocity. Peak and angle-specific (50 degrees, 40 degrees, and 30 degrees of knee flexion) moments were determined during maximal concentric and eccentric muscle contractions (10 degrees to 90 degrees of motion; 30 and 240 deg/sec). Across movement speeds and contraction modes the functional ratios for different moments varied between 0.3 and 1.0 (peak and 50 degrees), 0.4 and 1.1 (40 degrees), and 0.4 and 1.4 (30 degrees). In contrast, conventional hamstring:quadriceps ratios were 0.5 to 0.6 based on peak and 50 degrees moments, 0.6 to 0.7 based on 40 degrees moment, and 0.6 to 0.8 based on 30 degrees moment. The functional hamstring:quadriceps ratio for fast knee extension yielded a 1:1 relationship, which increased with extended knee joint position, indicating a significant capacity of the hamstring muscles to provide dynamic knee joint stability in these conditions. The evaluation of knee joint function by use of isokinetic dynamometry should comprise data on functional and conventional hamstring:quadriceps ratios as well as data on absolute muscle strength.     

Single L-type calcium channels in smooth muscle cells from resistance arteries of spontaneously hypertensive rats

The amplitude of the whole-cell L-type Ca2+ channel current recorded from vascular smooth muscle cells is reportedly greater in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). However, no study has examined properties of single Ca2+ channels in arterial cells from these strains. To further test the hypothesis that activation of L-type Ca2+ channels in arterial smooth muscle cells would be enhanced in SHR, we recorded single Ca2+ channel currents in resistance mesenteric artery cells from SHR and WKY (8 to 9 weeks of age) using a cell-attached patch clamp technique. With 50 mmol/L Ba2+ in the recording pipette, the depolarizing pulse from a holding potential of -40 mV evoked the single L-type Ca2+ channel current. Opening of the single channels was more frequent in cells from SHR than from WKY. Single-channel conductance (20 pS) and open time (1 ms at 0 mV) did not differ in the two strains. The results suggest that an increased amplitude of the whole-cell current can be attributed to the enhanced opening of single Ca2+ channels in the arterial smooth muscle cells from SHR compared with WKY.